Lorenz Adlung - Cell and Molecular Biology For Non-Biologists - A Short Introduction Into Key Biological Concepts-Springer (2023)

Lorenz
Adlung
Cell and
Molecular Biology
for Non-Biologists
A short introduction into key biological
concepts
Cell and Molecular Biology
for Non-Biologists
Lorenz Adlung
Cell and Molecular Biology

for Non-Biologists
A Short Introduction into Key Biological
Concepts
Lorenz Adlung
University Medical Center Hamburg-
Eppendorf
Hamburg, Germany
ISBN 978-3-662-65355-5 ISBN 978-3-662-65357-9 (eBook)

https://doi.org/10.1007/978-3-662-65357-9
© Springer-Verlag GmbH Germany, part of Springer Nature 2022

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Preface
This book reflects the diversity of Modern Biology with its plethora of emerging
disciplines. It is dedicated to all non-biologists who introduce their knowledge and
expertise to the field thereby enriching the scientific landscape in biology. Experts
from all over the life sciences are needed to answer current questions in today’s
biology and medicine [99]. Facing the challenge to combat a disease as complex
as cancer demands interdisciplinary collaboration.
Mathematics, computer sciences, physics and engineering became the vital core
of disciplines such as systems biology and synthetic biology. A vivid exchange is
a prerequisite for synergies and mutual benefits in the biological as well as the
non-biological spheres. A major hurdle to overcome is language. Computational
modelling allows a new understanding of non-intuitive biological phenomena.
Concepts and formalisms are introduced for systematic scrutinization of biological
systems and rational hypothesis testing. However, nomenclature and semantics are
often very different among the fields, which leads to misconceptions that hamper
communication and thus scientific progress. Once a common ground is discovered
with a coherent language, findings can be shared to the full extent. This approach
would enable an entirely new perspective on living entities that could then be
described mechanistically and quantitatively.
This book addresses the fundamental principles of Modern Biology exempli-
fied by the latest research results. The combination of both is described with a
vocabulary that requires no pre-existing knowledge. Schemes are depicted in sim-
ple shapes boiled down to central motifs that could be re-drawn in any sketchbook.
Many concepts were borrowed from foreign disciplines to highlight similarities
and possible linking points for career changers. Wherever possible, open-access
versions of original literature are cited. This book is intended as a brief introduc-
tion to Modern Biology for interested readers from other disciplines of natural
sciences. It is recommended especially to people with a background in mathemat-
ics, computer sciences, physics or engineering, who are planning to study or work
in biomedical life sciences. Please feel kindly encouraged to further foster a true
interdisciplinary debate that facilitates scientific progress in Modern Biology.
Hamburg Lorenz Adlung
v
Contents
1 Cell Architecture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Cells and Their Biochemical Composition . . . . . . . . . . . . . . . . . . . 2
1.2 The Cell’s Skeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.1 Actin Filaments Form Flexible Meshes. . . . . . . . . . . . . . . . 5
1.2.2 Intermediate Filaments for Robustness. . . . . . . . . . . . . . . . 5
1.2.3 Microtubules as Tracks for Intracellular Transportation. . . 6
1.2.4 Motor Proteins to Position Cellular Cargo. . . . . . . . . . . . . . 7
1.3 Organelles of a Cell. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.4 Cell-To-Cell Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4.1 Occluding Junctions as Diffusion Barriers . . . . . . . . . . . . . 9
1.4.2 Anchoring Junctions for Adhesion. . . . . . . . . . . . . . . . . . . . 9
1.4.3 Gap Junctions for Communication . . . . . . . . . . . . . . . . . . . 9
2 DNA & RNA & Associated Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.1 Structural Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2 Organization of Genetic Information. . . . . . . . . . . . . . . . . . . . . . . . 14
2.2.1 Gene Elements, Histones and Chromatin . . . . . . . . . . . . . . 14
2.2.2 Chromosomes, Genes and Heredity. . . . . . . . . . . . . . . . . . . 15
2.3 The Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.4 DNA Replication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4.1 Molecular Components Involved in DNA Replication . . . . 19
2.4.2 DNA Recombination to Secure Integrity and Diversity . . . 21
3 Transcription and Translation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1 The Genome is Read and Transcribed. . . . . . . . . . . . . . . . . . . . . . . 26
3.1.1 Start the Transcription. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.1.2 Quality Control During Transcription. . . . . . . . . . . . . . . . . 28
3.2 Processing of Transcripts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2.1 Stop the Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2.2 Splice the Transcript. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.3 Transcripts are Translated into Polypeptides. . . . . . . . . . . . . . . . . . 30
3.3.1 Initiation of Translation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3.2 Ribosomal Structure and Function. . . . . . . . . . . . . . . . . . . . 32
3.3.3 Protein Folding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
vii
viii Contents
3.4 Measuring Cellular Content. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.4.1 DNA Sequence Variants. . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.4.2 Omics Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4 Regulation of Gene Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.1 Transcriptional Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.2 Regulatory RNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.3 Protein Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.3.1 Post-Translational Modifications. . . . . . . . . . . . . . . . . . . . . 42
4.3.2 Protein Decay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.4 Epigenetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.4.1 Mechanisms of Epigenetic Regulation . . . . . . . . . . . . . . . . 45
4.4.2 Causes and Consequences of Epigenetic Regulation. . . . . . 48
5 Membranes and Intracellular Transport. . . . . . . . . . . . . . . . . . . . . . . 49
5.1 Composition and Assembly of Cellular Membranes. . . . . . . . . . . . 50
5.2 Targeted Protein Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5.2.1 Protein Transport through Nuclear Pores. . . . . . . . . . . . . . . 52
5.2.2 Unfolding of Proteins for Mitochondria
and Chloroplasts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
5.2.3 Co-Translational Import into the Endoplasmatic
Reticulum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.3 Vesicular Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.3.1 Glycosylation of Proteins and Quality Control. . . . . . . . . . 55
5.3.2 Exo- and Endocytosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
6 Signalling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.1 Molecular Switches between On- and Off-State. . . . . . . . . . . . . . . 62
6.2 Molecules and Physicochemical Properties as Input
from the Sender. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6.3 Receiving Cues via Cellular Receptors. . . . . . . . . . . . . . . . . . . . . . 64
6.3.1 Ion Channels Maintain the Flow of Ions and Thus
Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6.3.2 G-Protein Coupled Receptors Enable Switching
Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6.3.3 Enzyme-Coupled Receptors Regulate Complex
Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6.4 Signal Conversion in Protein Networks. . . . . . . . . . . . . . . . . . . . . . 66
6.4.1 Second Messengers to Spread Information Rapidly . . . . . . 66
6.4.2 Kinase Cascades for Signal Relay. . . . . . . . . . . . . . . . . . . . 67
6.4.3 Transcription Factors Control Gene Expression . . . . . . . . . 67
6.5 Characteristic Motifs and Behaviours. . . . . . . . . . . . . . . . . . . . . . . 68
6.5.1 Signal-Response Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.5.2 Time-Course Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
6.5.3 Complex Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Contents ix
6.6 Emergent Properties as Output . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

6.7 Signal Input, Processing and Output Happens on 
Various Levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
7 Cell Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
7.1 Cell-Cycle Model Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
7.1.1 Cellular Contexts to Study the Cell Cycle. . . . . . . . . . . . . . 77
7.1.2 Discovery of Core Cell-Cycle Components. . . . . . . . . . . . . 78
7.2 Checkpoints Regulating the Cell Cycle. . . . . . . . . . . . . . . . . . . . . . 79
7.2.1 The Restriction Point Mediated by Rb and E2F . . . . . . . . . 79
7.2.2 The DNA-Damage Response. . . . . . . . . . . . . . . . . . . . . . . . 81
7.2.3 Hyperproliferative Stress. . . . . . . . . . . . . . . . . . . . . . . . . . . 82
7.3 Conceptual Views on Cell-Cycle Phenomena. . . . . . . . . . . . . . . . . 83
7.3.1 Cell Growth as Biomass Acquisition. . . . . . . . . . . . . . . . . . 83
7.3.2 Cell Division as Biomass Distribution. . . . . . . . . . . . . . . . . 84
7.3.3 Find a Balance: Divide et cresce!. . . . . . . . . . . . . . . . . . . . . 85
7.3.4 A Molecular Cell-Cycle Set-up for Oscillations. . . . . . . . . 86
7.3.5 The Cell-Cycle Programme. . . . . . . . . . . . . . . . . . . . . . . . . 86
8 Immunology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
8.1 Components of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . 90
8.1.1 Sites and Non-Cellular Substances
of Immune Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
8.1.2 The Hematopoiesis Tree and Its Branches. . . . . . . . . . . . . . 91
8.2 Innate Immunity as First Line of Host Defence. . . . . . . . . . . . . . . . 94
8.2.1 Inflammation and the Complement System. . . . . . . . . . . . . 94
8.2.2 Pattern Recognition: Know Your Enemy. . . . . . . . . . . . . . . 95
8.3 Adaptive Immunity as Ability to Strike Back . . . . . . . . . . . . . . . . . 96
8.3.1 Clonal Selection of Special Forces . . . . . . . . . . . . . . . . . . . 96
8.3.2 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
8.3.3 Regulation of T Cells for Killing, Help, Memory
and Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
8.3.4 (Auto-)immune Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
9 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
9.1 Different Types of Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
9.1.1 Phenotypic Tumour Development. . . . . . . . . . . . . . . . . . . . 105
9.1.2 Causes of Cancer Development. . . . . . . . . . . . . . . . . . . . . . 106
9.2 Pro et Contra Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
9.2.1 Immortality and Programmed Cell Death. . . . . . . . . . . . . . 108
9.2.2 Molecular Complexity Underlying Cancer. . . . . . . . . . . . . 109
9.2.3 Hallmarks of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
9.3 Combat Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
9.3.1 Current Therapeutic Approaches. . . . . . . . . . . . . . . . . . . . . 113
9.3.2 the Future of Personalized Medicine. . . . . . . . . . . . . . . . . . 113
x Contents
Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Acronyms
4E-BP 4E-binding protein

ADP adenosine diphosphate
AKT thymoma viral proto-oncogene
APC anaphase-promoting complex
APCs antigen-presenting cells
ATM adenine-thymine mutated
ATP adenosine triphosphate
ATR ATM- and Rad3-related
Bcl2 B cell lymphoma 2
BCR B cell receptor
BRCA1 breast cancer type 1 susceptibility protein
cAMP cyclic adenosine monophosphate
CD clusters of differentiation
cdc cell division cycle
CDK cyclin-dependent kinase
CFTR cystic fibrosis transmembrane conductance regulator
Chk checkpoint kinase
CIS cytokine-inducible SH2 domain-containing protein
CTD C-terminal domain
CTLA-4 cytotoxic T-lymphocyte-associated protein 4
DAMPs damage-associated molecular patterns
DNA deoxyribonucleic acid
DNMT DNA methyltransferase
DUSP dual-specificity phosphatase
eIF-4E eukaryotic initiation factor 4E
EMT epithelial-mesenchymal transition
Epo erythropoietin
EpoR erythropoietin receptor
ER endoplasmatic reticulum
ERAD endoplasmatic reticulum-associated protein degradation
ERK extracellular signal-regulated kinase
GAP GTPase-activating protein
GDP guanosine diphosphate
GEF guanine nucleotide exchange factor
xi
xii Acronyms
GFP green fluorescent protein

GLUT1 glucose transporter 1
GTP guanosine triphosphate
HAT histone acetyltransferase
HDAC histone deacetylase
HLA human leukocyte antigen
HSC hematopoietic stem cell
IFN interferon
InsP3 inositol 3,4,5 trisphoshpate
JAK2 Janus kinase 2
LDL low-density lipoprotein
MAPK mitogen-activated protein kinase
MAPKK mitogen-activated protein kinase kinase
MAPKKK mitogen-activated protein kinase kinase kinase
MBD methyl-CpG-binding domain
MC1R melanocortin 1 receptor
Mdm2 Mouse double minute 2 homolog
MEK mitogen/extracellular signal-regulated kinase
MET mesenchymal-epithelial transition
MHC major histocompatibility complex
MPF mitosis-promoting factor
MRN Mre11, Rad50, Nbs1
mRNA messenger ribonucleic acid
mTOR mammalian target of rapamycin
NF2 Neurofibromin 2
NFκB nuclear factor kappa-light-chain-enhancer of activated B cells
ORI origin of replication
OST oligosaccharyl transferase
PAMPs pathogen-associated molecular patterns
PET positron emission tomography
PI3K phosphoinositide 3-kinase
PIP2 phosphatidylinositol 3,4 bisphoshpate
PIP3 phosphatidylinositol 3,4,5 trisphoshpate
PRRs pattern-recognition receptors
PTEN phosphatase and tensin homolog
Rb retinoblastoma
RNA ribonucleic acid
RPA replication protein A
rRNA ribosomal ribonucleic acid
SCF Skp, Cullin, F-box containing
SNARE soluble N-ethylamine sensitive factor attachment protein receptor
SNP single-nucleotide polymorphism
SOCS3 suppressor of cytokine signalling 3
SRP signal recognition particle
SSB single-strand binding protein
Acronyms xiii
STAT5 signal transducer and activator of transcription 5

TBP TATA box binding protein
TCR T cell receptor
TGF-β transforming growth factor beta
TLRs Toll-like receptors
TNF tumor necrosis factor
TRAIL TNF-related apoptosis-inducing ligand
tRNA transfer ribonucleic acid
TSS transcription start site
UPR unfolded protein response
UV ultra-violet
Cell Architecture
1
Contents
1.1 Cells and Their Biochemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 The Cell’s Skeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Organelles of a Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.4 Cell-To-Cell Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Introduction
What is a cell? A cell in biology is the fundamental unit of life [7]. All living enti-
ties are built from cells. Life in its essence means self-maintenance and replication.
And cells are nothing more than self-maintaining, replicating droplets. Replication
is performed by a sequence of biochemical reactions. To provide a separate com-
partment for those reactions to occur, cells are enclosed by a membrane surrounding
its aqueous inner solutions. To fulfil their function of self-maintenance and repli-
cation, different structures evolved inside the cells. Ensembles of molecules form
a machinery that builds up, maintains and replicates cellular contents. For replica-
tion, a blueprint of the cell has to be delivered as a construction plan for each newly
produced cell. This information is stored on deoxyribonucleic acid (DNA), a long
macromolecule. In sum, cells can be considered as a membrane-enclosed piece of
DNA in an aqueous solution (Fig. 1.1). Beside membrane and DNA, additional parts
exist to allow more sophisticated structures and thus more specialized functions to
be carried out inside the cells.
Our human body consists of a skeleton and organs. Likewise, the cellular body
contains a “cytoskeleton” and “organelles”. The structural elements of a cell are its
building blocks and thus the bricks of cellular architecture. Diversity and complexity
of the structures vary depending on the cell’s specialized function. While some cells
© Springer-Verlag GmbH Germany, part of Springer Nature 2022 1

L. Adlung, Cell and Molecular Biology for Non-Biologists,
https://doi.org/10.1007/978-3-662-65357-9_1
2 1 Cell Architecture
Fig. 1.1 A cell as central unit of life consists in its essence of a membrane that contains DNA as
information storage in aqueous solution
remained rather simple like our red blood cells, others became sophisticated biore-
actors full of specialized structural elements and derived functions like brain cells.
From unicellular organism to higher life forms, the structure of biological entities is
always tightly connected to its function. The emerging diversity that discriminates
different species lays the foundation for life with all its variety.
1.1 Cells and Their Biochemical Composition
The components of living cells are diverse, but the majority of molecules belong to
one of the four main categories, which are:
• carbohydrates (“sugars”, “carbs”),

• lipids (“fats”),
• proteins and
• nucleic acids.
These macromolecules are assembled from building blocks: monosaccharides, fatty

acids, amino acids and nucleotides (Table 1.1). These core molecules account for a
large fraction of cellular mass. In fact, the cellular body should be disregarded as an
aqueous solution because it is rather a dense gel-like mixture crowded with proteins
made by ribosomes (see Sect. 1.3) [62]. Therefore, the term cytoplasm implies the
correct constitution of the cellular body rather than the term cytosol.
Table 1.1 Monomers as building blocks for functionally distinct molecular assemblies
Bricks Macromolecules Function
Monosaccharides Sugars Energy source, regulatory units
Fatty acids Fats Membranes, energy storage
Amino acids Proteins Cell architecture, enzymes
Nucleotides Nucleic acids Information storage,
regulatory units
1.1 Cells and Their Biochemical Composition 3
Sugars are a main source of energy. Glucose plays a central role. It can be broken
down for the release of energy, or it can be lined up in long chains for storage. Chains
of monosaccharides can also be plugged onto the other molecule classes forming e.g.
glycolipids or glycoproteins. Glycoproteins are also an integral part of the bacterial
cell wall and the extracellular matrix in tissues of higher organisms.
Lipids consist of fatty acids and similar biomolecules such as waxes and vitamins.
Fatty acids are important for membranes of cells (see Chap. 5) and their subunits
(“organelles”). Breaking down fatty acids yields even more energy per mass unit
than glucose. Lipids are stored in cytoplasmic droplets. Mostly, three fatty acids are
connected to a glycerol molecule forming triacylglycerol, the main component of
lipid droplets.
Proteins exert ubiquitous functions from shaping the cell’s interior (see Sect. 1.2)
to catalysing biochemical reactions as enzymes. The functional diversity originates
from the modular structure. Proteins are long chains formed from 20 amino acids
that serve as building blocks allowing for combinatorial complexity. Polymers of
hundreds of amino acids fold into a 3D structure of the mature protein. Proteins are
encoded by the sequence of nucleic acids in the genetic code (see Sect. 2.3).
Nucleic acids exist in two forms: ribonucleic acid (RNA) and deoxyribonucleic
acid (DNA). The latter one is chemically more stable and used for long-term storage
of hereditary information in the genetic code, which will be explained in the next
chapter (see Chap. 2). RNA is less stable and carries short-term instructions as mes-
senger molecule or regulatory subunit of protein machineries and cellular organelles
(ribosomes).
As all these macromolecules are modularly formed from subunits, size scales
are important to bear in mind (Table 1.2). Modularity is an efficient feature emerging
from the evolution of life allowing almost unlimited complexity with a predefined set
of building blocks that can be easily maintained and sustainably produced. However,
it is not sufficient to know the mere sequence of subunits that form a protein or an
RNA chain. The 3D structure of a molecule is essential for the exertion of its specific
function. Structure and function are intimately related in biology. Enzymes can only
catalyse biochemical reactions because they fit their substrates like “lock and key”.
A certain shape always carries a certain property, otherwise, it would be a waste of
resources to maintain it in the course of evolution.
Built from these macromolecules at different scales, two kinds of cells can be
discriminated.
1. Prokaryotic cells, derived from the Greek words “pro” and “karyon” meaning
“before” and “kernel”, refer to cells without an information-storing compartment.
In prokaryotes, DNA as storage of heritable properties is not encapsulated by a
membrane in a so-called nucleus. Instead, the DNA is loosely arranged within the
Table 1.2 Size scales of cellular components. 1 nm = 10−9 m, 1 µm = 10−6 m

Size 0.1 nm = 1 Å 20 nm 1.5 µm 20 µm
Component Atom Molecule Prokaryotic cell Eukaryotic cell
cell throughout the entire aqueous solution called cytoplasm. Many cell-derived
terms are commonly used with the prefix “cyto”. Despite their rather simple
and ancient structure (Fig. 1.1), prokaryotes show high diversity and abundance.
Diversity originates from their ability to adapt to biospheres such as undersea vol-
canoes or our human guts. Abundance results from their ability to double quickly,
like some gut bacteria within 20 min under optimal conditions. Robustness is pro-
vided by additional layers supporting the cellular membrane thereby creating a
cell wall. Bacteria, the most prominent domain within prokaryotes, e.g. grows
and evolves rapidly leading to the spread of disease and antibiotic resistance.
2. Eurkaryotic cells with the Greek prefix “eu” for “good” indicates that they do
have a nucleus. Besides, they also contain many other specialized compartments
called organelles (see Sect. 1.3). Some of these are derived from ingested, highly
adapted bacteria that could produce energy from light or oxygen. Since mainte-
nance of such organelles is time-consuming, eukaryotes grow slower but they are
also bigger than the ≈ 1.5 µm unicellular prokaryotes. Eukaryotes can exist as
single cells, e.g. yeast, or live as multi-cellular assemblies creating all our body
tissues.
1.2 The Cell’s Skeleton
The cells of our body differ widely in their shape and thus in their function. The
diversity of cellular structures can only be maintained by an underlying network
of particles that comprise the cytoskeleton. These structures manifest tight cellular
connections, e.g. the outer layer of our body: the skin. Unlike the skeleton of our
body, the cellular skeleton, however, is a highly dynamic structure that can adapt in
space and time to steer cellular re-organization, for instance, during the cell cycle
(see Chap. 7).
The machinery of the cell’s skeleton is based on three different types of protein
filaments.
1. Actin filaments
– ø: 5–9 nm, “micro filaments”
– two intertwined helix polymers of actin, flexible (i.e. dynamic) meshes
2. Intermediate filaments
– ø: 10 nm, heterogeneous
– rope-like structure, stable
3. Microtubules
– ø: 25 nm, basis (i.e. source) is centrosome
– tube of Tubulin molecules, long, straight and compact
1.2 The Cell’s Skeleton 5
Fig. 1.2 An actin filament of ≈ 7 nm diameter is dynamically assembled from actin monomers.
Actin-ATP molecules bind more strongly to each other. Upon ATP hydrolysis, actin-ADP molecules
fall apart. Net dis-/assembly at the minus/plus end leads to a cyclic process called treadmilling
1.2.1 Actin Filaments Form Flexible Meshes
Actin filaments with a diameter of ≈ 7 nm are chains of actin monomers. Actin

monomers exist in an ATP-bound state and an ADP-bound state. If ATP is bound to
actin, actin-ATP molecules start to form filaments. When ATP is hydrolysed to ADP,
the actin-ADP molecules fall apart from the filament, because the binding strength
between actin-ATP molecules is stronger than between actin-ADP molecules. The
exchange of ADP with ATP can only occur in free actin-ADP monomers. The
dynamic equilibrium of actin filament assembly and disassembly depends on the
availability of free actin monomers and the velocity of the ATP hydrolysis in actin-
ATP molecules. At one end of the actin filament, ATP hydrolysis is faster than
addition of monomers. This end shrinks and is called the minus end. At the plus end
on the other side, the rate of newly added monomers is faster than ATP hydrolysis,
which results in net growth of the filament in this direction. This polar structure
of plus end and minus end emerging from these reactions is known as treadmilling
(Fig. 1.2).
Fifty per cent of all actin molecules in a cell exist as monomers. To prevent these
free molecules from spontaneous polymerization, they are bound to thymosin and
profilin. Dynamic dis-/assembly of actin filaments is further guided by additional
capping and cross-linking proteins that stabilize actin filaments. Actin filaments are
an integral part of contractile bundles in the cytoplasm that connect different spots of
the plasma membrane thereby giving the cell its characteristic shape. Dynamic de-
/formation of actin filaments is required in filopodia, which are cellular feet to move
forward. These aspects highlight the important structural and dynamic properties of
actin filaments in cellular physiology.
1.2.2 Intermediate Filaments for Robustness
These filaments are called “intermediate” because, when they were first discovered
in smooth muscles, the 10 nm diameter was between the diameter of thin actin fila-
ments (see Sect. 1.2.1) and thick myosin filaments (see Sect. 1.2.4). The intermediate
filaments form “rope teams”, which are the toughest and most physically endurable
cellular elements. They not only form a network throughout the cytoplasm but also
support the nuclear envelope and cell-cell contacts called desmosomes (see Sect. 1.4).
The structure of intermediate filaments reminds of a rope with many long strands
that are twisted together. Each of these single ropes is an α-helical fibril with an N-
terminal head region and a C-terminal tail region that spans a total length of 48 nm.
Head and tail regions are heterogeneous and determine interactions with cytoplasmic
proteins. Each of two ropes is twined together to form a pair. Two of these dimers are
staggered to form a tetramer. Eight of these tetramers in turn can be packed together
to form a non-covalently bound strand that assembles the final intermediate filament.
The main purpose of intermediate filaments is to make the cells more robust
against mechanical stress such as squeezing or tearing. There are four major classes
of intermediate filaments.
• Keratin filaments characterize the different thin tissue layers on the outer and
inner surfaces of our body, called epithelia. The cells of each epithelium have an
individual mixture of keratin filaments that span the cellular sheet horizontally
through desmosomes (see Sect. 1.4.2).
• Vimentin and related filaments are mainly found in connective tissue, muscle cells
and glial cells of the nervous system. Vimentin structures can be reinforced or
cross-linked by accessory proteins such as plectin.
• Neurofilaments, as the name implies, are an integral part of nerve cells. Mutations
that cause disruption of neurofilaments lead to neurodegenerative disorders.
• Nuclear lamina builds up the nuclear envelope. Tetramers of lamina can be quickly
assembled and disassembled during cell cycle (see Chap. 7).
1.2.3 Microtubules as Tracks for Intracellular Transportation
Hollow tubes with a diameter of ≈ 25 nm are called microtubules. These tubes are
built from subunits that exist as α-tubulin and β-tubulin forming heterodimers. These
pairs of α- and β-tubulin exhibit a polarity with β-tubulin always pointing to the plus
end and α-tubulin indicating the minus end in the opposite direction. The microtubule
is dis-/assembled at the minus/plus end by dis-/assembly of tubulin. A chain of tubulin
heterodimers forms a protofilament, and 13 protofilaments compose the wall of the
tube surrounding the lumen (Fig. 1.3).
Microtubules form tracks from the cell’s centre to the periphery for intracellular
transport. The centre from which they grow out is called centrosome consisting of a
pair of centrioles. This structure contains γ -tubulin that forms a ring thereby serving
as a starting and anchoring point for the minus end of microtubules. The switching
between the assembly and the disassembly of microtubules is known as dynamic
instability (growing back and forth). Capping proteins prevent microtubules from
Fig. 1.3 A microtubule is a hollow tube of ≈ 25 nm diameter that is built from heterodimers of
α- and β-tubulins. Long chains of these dimers form a protofilament, and 13 of these surround the
lumen of a microtubule that exhibits a polar orientation from the minus to the plus end
1.3 Organelles of a Cell 7
shrinking at the plus end. Such selective stabilization can polarize a cell if capping
proteins are provided at restricted areas, e.g. a particular site at the cell membrane.
Microtubules can form cilia, 0.25 µm thin hair-like structures that sweep mucous (see
Sect. 8.2) in our respiratory tract, and flagella, that propel sperm cells towards the
egg. A ubiquitous function of microtubules is the formation of the mitotic spindle
(see Chap. 7) and organization of the cell interior by allowing transport processes
catalysed by motor proteins.
1.2.4 Motor Proteins to Position Cellular Cargo
One of the best-studied processes involving the cell’s skeleton and motor proteins is
muscle contraction involving actin filaments and myosin molecules. Myosin motor
proteins exist as two subfamilies. Myosin-I is present in all cell types for vesicu-
lar transport (see Sect. 5.3). Myosin-II is only present in muscle cells. Each of the
transport processes is actin-dependent and uses ATP as a source of energy. Most
important for the cellular architecture are transport processes along microtubules.
The two most important families of motor proteins for the transport of cargo
inside the cell are dyneins and kinesins. They both consist of two globular heads
capable of tubulin binding and a tail domain specifically carrying the cargo. Motor
proteins move forward by conformational changes. The energy for these movements
is obtained by ATP hydrolysis. Dyneins move towards the minus end of microtubules
at the cell’s centre, whereas kinesins move towards the plus end of microtubules at
the cell’s periphery. The Golgi apparatus is positioned by dyneins around the nucleus,
while the endoplasmatic reticulum (ER) is stretched throughout the cell by kinesins.
The function of these cellular organs will be explained in the subsequent section.
1.3 Organelles of a Cell
Like our body, a eukaryotic cell contains organs, which are sites within the organism
carrying out specific tasks. Cellular organs are called organelles and are introduced
in the following.
• The nucleus is the central compartment of every eukaryotic cell because it stores
DNA. The hereditary information is segregated from the cytoplasm by two nuclear
membranes. Pores within the envelope allow the shuttling of molecules between
the two cellular compartments. Most cells only contain a single nucleus. However,
some cell types, such as liver cells, are binucleated and thus contain two nuclei.
• The mitochondrion is also surrounded by two membrane layers. The inner mem-
brane is highly folded to increase the surface for the oxidation of molecules to
produce energy in the form of adenosine triphosphate (ATP). In this process, oxy-
gen is consumed and carbon dioxide is produced. A cell usually contains several
mitochondria to fuel all cellular processes with ATP. Mitochondria are referred
to as cellular power plants, which make the cell breathe. Mitochondria contain
their own DNA and divide to reproduce independently of the cell. Due to the
two membranes, their size, function and separate genetic information, mitochon-
dria are believed to be derived from prokaryotes, which were engulfed by other
cells creating a symbiotic relationship. Symbiosis means that two organisms live
together with mutual benefits.
• The ribosome is the protein synthesis machinery where messenger transcripts of
the genetic code are translated into polypeptide chains to form functional proteins
(see Sect. 3.3.2).
• A chloroplast is similarly organized as a mitochondrion. It is surrounded by two
membranes, contains its own DNA, divides to reproduce and is derived from
procaryotes. Chloroplasts are parts of plant cells and algae. They harness the
energy from sunlight to build up sugar molecules. Therefore they contain stacks
of membranes full of the green pigment chlorophyll. A side product of sugar man-
ufacturing is oxygen. Sugars and oxygen can both be used then by mitochondria
to produce energy.
• The endoplasmatic reticulum (ER) is a maze of interconnected compartments
surrounded by a single membrane. The ER is the site where most membrane
components and molecules destined for export from the cell are made.
• The Golgi apparatus (named after its discoverer Camillo Golgi) is an intricate
network for sorting and barcoding of molecules for export from the cell or intra-
cellular shipping (see Sect. 5.3.1).
• Cargo is transported from one cellular compartment to another in vesicles. In a
special membrane-enclosed vesicle called lysosome, digestion of cellular compo-
nents takes place. Breaking down molecules in a separate compartment is impor-
tant for the release of nutrients, the recycling of building blocks or the excretion of
unwanted substances. The peroxisome is another specialized membrane-enclosed
vesicle to keep the cell from reactions involving hydrogen peroxide or other toxic
substances. Some cell types are defined by their specific vesicles, e.g. neutrophils
of the immune system.
A useful representation of these organelles can be found under https://smart.servier.

com/.
1.4 Cell-To-Cell Contacts
Unicellular organisms are well equipped with their organelles to maintain life. In
multi-cellular ensembles, cells have to interact and communicate with each other for
vital functions of cellular layers. In tissues, cell-to-cell contacts serve as both, stabi-
lizing elements and channels for information exchange. As they join cells, they are
called “junctions”. Three different classes of cellular junctions can be discriminated
that will be introduced in the following.
1.4 Cell-To-Cell Contacts 9
1.4.1 Occluding Junctions as Diffusion Barriers
Occluding junctions, as the name implies, close up cellular layers. An epithelium is

a sheet of cells that are all horizontally connected to separate outer medium from
underlying cells. The border has to be leak-proof, e.g. for the fluids that lubricate the
inner gut lining (see Sect. 8.1.1). The sealing is achieved by tight junctions, which
adhere neighbouring cells so that aqueous solutions cannot diffuse between them. The
two most important components of tight junctions are the transmembrane-proteins
occludin and claudin. The function of tightly joined epithelial cells as a border implies
a structural feature. Epithelial cells are polarized with their top side being different
from their bottom side. In the gut, the apical (i.e. upper) side of the epithelial cells
mainly contains the tight junctions plus microvilli, which form a cellular brush to
increase the surface for uptake of nutrients. Other cells of this epithelium contain
many vesicles on their apical side for the production of lubricious substances that
flush the inner surface of the gut. The cytoskeleton of the basal (i.e. lower) side of
these epithelial cells is connected to an extracellular matrix forming the basal lamina.
1.4.2 Anchoring Junctions for Adhesion
While tight junctions create a diffusion barrier at the apical side of the cells, anchor-
ing junctions hold together the entire epithelium by mechanically attaching the
cells with each other and with the basal lamina. Two classes of anchoring junctions
can be discriminated: adherens junctions and desmosomes. Both rely on cadherin
molecules, which can bind to one another. A cadherin molecule connects to the
skeleton of one cell, spans the plasma membrane and connects extracellularly in
the presence of Ca2+ ions to a cadherin molecule of a neighbouring cell. The name
cadherin is derived from “calcium-dependent adhesion”. Adherens junctions and
desmosomes rely on different types of cadherin molecules but their main difference
originates from the component of the cytoskeleton they are bound to. Adherens junc-
tions are connected to bundles of actin filaments, whereas desmosomes are linked
with keratin molecules. The actin filaments of adheren junctions connect all cells
along the epithelium thereby forming an adhesion belt on the apical side capable of
contractions. Keratin filaments in desmosomes tightly anchor cells to one another.
If keratin is connected to the basal lamina, the junction is made by integrins not
cadherins and the junction is called hemidesmosome.
1.4.3 Gap Junctions for Communication
Tight junctions and anchoring junctions keep the cells of an epithelium together, but
gap junctions form tunnels that connect the cytoplasms of neighbouring cells by
bridging the plasma membrane. So-called connexon channels of each six connexin
subunits span the cellular membrane and align to connexon channels of adjacent cells.
The pore through two connexon tubes allows direct exchange of soluble molecules
between cells and thus couples them metabolically. The flux of electrically charged
ions through gap junctions becomes important for instance in the transmission of
neuronal signals in the retina [107].
Plant cells do only have one type of cell-to-cell contact, namely plasmodesmata,
which penetrate the otherwise stabilizing cell walls and allow communication.
Summary
• Cells are mainly composed of carbohydrates, lipids, proteins and nucleic acids.
• The cellular skeleton contains three types of protein filaments: Actins, inter-
mediate filaments and microtubules.
• The compartments of a cell are its organelles for specific reactions and func-
tions.
• Cell-to-cell contacts allow both, tight closure of cellular layers and exchange
between individual cells.
DNA & RNA & Associated Proteins
2
Contents
2.1 Structural Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2 Organization of Genetic Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 The Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.4 DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Introduction
The architecture of the cell needs a blueprint. Information on molecular components
has to be encoded in the cell. It is written on long chains of nucleic acids. The
discovery of deoxyribonucleic acid (DNA) as hereditary material, its structure and
its maintenance represent central cornerstones for modern biology that paved the
way for deciphering the human genome in 2001, and nowadays, high-throughput
sequencing of genomes from bacteria, yeast, worms and fruit flies to cancer patients
[41]. DNA sequence information has certain properties that allow being passed on
from generation to generation. First, DNA is modular. Just a few simple building
blocks make up long chains of information with sections called gene, from the Greek
term génos meaning origin or offspring.
The long DNA chains are mostly wound up and condensed in chromosomes that
define our sex and store all genetic information to be passed on to the next generation.
The sequence of the hereditary material constitutes the genetic code, which is written
as the language for the programme of life. But all the encoded information were
useless if it could not be propagated. The molecular structure allows for the second
important feature: DNA can be replicated. Building blocks are fused with a stretch of
DNA complementary to a template strand thereby copy-pasting genetic information.
If no errors occur within the process, the generated sister molecules are a duplicate
of the original DNA.
The modularity and replication of DNA are translated to proteins that exert cer-
tain functions in cells. An intermediate product of protein production from the DNA
blueprint is ribonucleic acid (RNA) molecules (DNA → RNA → protein). RNA

https://doi.org/10.1007/978-3-662-65357-9_2
12 2 DNA & RNA & Associated Proteins
molecules share modular features of DNA. Besides being a DNA transcript for pro-
teins, RNA has the ability to directly fulfil regulatory tasks. RNA molecules can either
facilitate protein production as structural components of protein synthesis machinery
or block this process by specific binding to RNA transcripts. These diverse features
fuelled speculation that RNA could have been the central molecule for the origin
of life [90]. However, the emergence of DNA and RNA is not mutually exclusive,
because they share a common modular sequence structure (Fig. 2.1), which forms
the virtual basis for all processes of modern biology.
2.1 Structural Elements
Most of the cell’s biochemical components exhibit modularity because the assem-
bly of complex structures from a small set of building blocks allows diversity of
products and sustainable management of resources (see Sect. 1.1). The same holds
true for DNA and RNA, which are made up from sugars that are linked to bases and
connected by phosphate bridges (Fig. 2.1). The main difference between DNA and
RNA arises from the type of sugar molecule that is used, which is implied by the
name. For DNA, it is a Desoxyribose while for RNA, it is a Ribose. These sugars
are pentoses because they contain five carbon atoms. DNA is more stable than RNA
because the desoxyribose contains only a hydrogen atom at the 2’ carbon, whereas
the ribose exhibits a hydroxyl group that is much more prone to hydrolysis. DNA
and RNA also differ with respect to their nitrogenous bases. There are five different
nucleobases, and each one can be connected to the 1’ carbon of the sugar residue in
the macromolecule. Adenine, cytosine and guanine are part of both, DNA and RNA.
Uracil is just incorporated into RNA, while thymine is solely part of DNA. Adenine
and guanine belong to the purine bases. Cytosine, thymine and uracil belong to the
pyrimidine bases. Sugar and base together are called nucleoside. If a phosphate
group is added at the 5’ carbon of the nucleoside’s sugar, a nucleotide is formed.
Depending on the sugar and the base that are connected, the nucleosides and respec-
tive nucleotides are called as given in Table 2.1.
The phosphate group that is added to the nucleoside for the formation of a
nucleotide is derived from phosphoric acid, which loses its protons at physiological
pH and is therefore negatively charged. The phosphate from phosphoric acid makes
the nucleotide with its nucleobase a nucleic acid, too. The prefix “nucle-” originates
from the fact that the material was first isolated from nuclei of white blood cells
[108]. Nucleotides are the building blocks of DNA and RNA. Long stretches are
created by phosphate-ester linkages. The phosphate group of a single nucleotide is
basei basei+1 basei+2 basei+3
sugar P sugar P sugar P sugar P
Fig. 2.1 Modular structure of DNA and RNA. Sugar molecules are linked via phosphate bridges
and connected to different bases
2.1 Structural Elements 13
Table 2.1 Composition, names and abbreviations for building blocks of DNA and RNA
Base Nucleoside Nucleotide (Abbreviation)
Purine
Adenine Adenosine Adenosine 5’-monophosphate (5’-AMP)
Deoxyadenosine Deoxyadenosine 5’-monophosphate (5’-dAMP)
Guanine Guanosine Guanosine 5’-monophosphate (5’-GMP)
Deoxyguanosine Deoxyguanosine 5’-monophosphate (5’-dGMP)
Pyrimidine
Cytosine Cytidine Cytidine 5’-monophosphate (5’-CMP)
Deoxycytidine Deoxycytidine 5’-monophosphate (5’-dCMP)
Thymine Deoxythymidine Deoxythymindine 5’-monophosphate (5’-dTMP)
Uracil Uridine Uridine 5’-monophosphate (5’-UMP)
always added to 5’ carbon of the sugar and can form a phosphodiester with the 3’
hydroxyl group of another nucleotide.
When the frequency of nucleosides in isolated DNA was analysed, adenosine (A)
occurred always in similar amounts as thymidine (T) and cytosine (C) appeared as
often as guanosine (G). These phenomena are meanwhile known as Chargaff’s rules:
#A #C
= =1 ⇔ #A = #T ; #C = #G.
#T #G
It was concluded that pairs of A:T, T:A, C:G and G:C make up the DNA. The
purine base adenine can form two hydrogen bonds with the pyrimidine base thymine
whereas three hydrogen bonds can be formed between the purine base guanine with
the pyrimidine base cytosine. One strand of DNA is thus paired with another opposing
strand. The sequences are referred to as complementary to each other, e. g.:
5’ AGCCTGTAC 3’
3’ TCGGACATG 5’
The sequence of nucleosides is linked by phosphodiesters creating a regular “back-

bone” of the DNA molecule. The phosphate bridges are formed between the 3’
hydroxyl group of a nucleotide and the 5’ phosphate group of a subsequent nucleotide.
If not indicated otherwise, sequences are usually given in the 5’→3’ direction. As the
phosphodiester bonds at the backbone of the DNA molecule are negatively charged at
physiological pH, they form a polar outer shell that protects the hydrophobic organic
bases at the inner core of the molecule in aqueous solution of the nucleus. The 3D
structure of the DNA molecule is a double helix, in which the two opposing DNA
strands are held together firmly by all the hydrogen bonds between the base pairs.
There are three different arrangements of DNA double helices: A-DNA, B-DNA
and Z-DNA. These forms vary in their number of base pairs per circle, the size of
the grooves and whether the spiral is left-handed or right-handed depending on the
sequence modifications and the chemical properties of the solvent. The most com-
mon structure under physiological conditions inside cells is B-DNA. RNA exhibits
much more diverse structures ranging from helices to loops and hairpins.
2.2 Organization of Genetic Information
2.2.1 Gene Elements, Histones and Chromatin
Given the size of the human genome with ≈ 3 × 109 base pairs (summing up to a
length of more than a metre [88]), our DNA cannot just be loosely packed within
the nucleus but needs to be tightly wrapped to fit in. DNA is coiled around a core
element of 4 × 2 = 8 histones. A total of 147 base pairs of DNA fit 1.7 × around
such an octamer of histone proteins. This constitutes a structure similar to “beads
on a string” (Fig. 2.2). Together, DNA and histones constitute a nucleosome, which
is a dynamically regulated, modular structure [71], and represents the basic unit
of chromatin. The regulation of the nucleosomes involves histone modifications,
e.g. acetylation or methylation of histone tails (Fig. 2.2). As these modifications
are not directly readable from the genetic code, they are summarized as epigenetic
information (see Sect. 4.4). The histone modifications therefore represent a regulatory
layer on top of the DNA sequence. They determine the structural arrangement of DNA
and proteins, which is summarized as chromatin.
Depending on the cellular state, chromatin can exist in different conformations.
If the chromatin is accessible, it is referred to as “open chromatin”. This means that
stretches of DNA are not (tightly) bound to histones and can thus be read, replicated
and transcribed. On the contrary, in regions of “closed chromatin”, nucleosomes
are tightly packed and cannot be accessed unless they are modified and rearranged.
The efficient wrapping of DNA around histones within closed chromatin reduces
the volume that the macromolecules overall need. This arrangement gives rise to
a particular structure called “heterochromatin” as opposed to “euchromatin” with
mostly open chromatin.
heterochromatin euchromatin
closed open
DNA
H3 H4 H3 H4
Ac H2A H2B H2A H2B Me
histone
tail
Fig. 2.2 Histones are proteins with each two of the four subunits: H2A, H2B, H3 and H4. DNA
is wrapped around. Histone tails can be chemically modified, e.g. acetylation (Ac) or methylation
(Me)
2.2 Organization of Genetic Information 15
The heterochromatin revealed a higher level of a structural organization unit of the

genetic information: the chromosome.
2.2.2 Chromosomes, Genes and Heredity
The word chromosome is derived from Greek and means “coloured body” because
chromosomes were studied during cell division (see Sect. 7.3.2) by staining with
certain dyes. The division is a part of the cell’s process of duplication (i.e. replication),
which is an essential step in life as such. In this process, two states of the chromosome
can be discriminated by their 3D conformation. The metaphase chromosome is the
state during the cell division that was discovered first as it is very condensed and thus
well structured (Fig. 2.3). The geometry is not well understood, but subunits can be
described. The metaphase chromosome is composed of two bar-shaped structures,
called chromatids. Each of the two chromatids of a chromosome contains a copy
of the same DNA. The pair of chromatids of a chromosome is referred to as sister
chromatids. They are held together and linked by the centromere located in the centre
of the metaphase chromosome, dividing each chromatid into a short arm and a long
arm. The terminal ends of each chromatid are called telomeres, which are important
to prevent shortening of chromosomes during replication (see Sect. 2.3), which takes
place prior to the metaphase of the cell division process.
Most of the time, the chromosome exists in the interphase state. The interphase
is the time between two cell divisions, as derived from the Latin term inter meaning
“between”. The interphase chromosome is characterized by a loose arrangement
of DNA, less condensed than metaphase chromosomes. Despite being more loosely
arranged, the DNA of interphase chromosomes is not randomly distributed in the
nucleus. Instead, there are chromosome territories in which regions of the DNA are
predominantly located during the interphase [28]. These spatially restricted areas
can be visualized by means of fluorescence in situ hybridization.
Fig. 2.3 Schematic

conformation of a metaphase short arms
chromosome. Two
chromatids with identical
DNA constitute the centromere
condensed structure that is
held together by the long arms
centromere that divides the
chromatids into a short arm
and a long arm. Terminal telomere
ends are called telomeres chromatid chromatid
Fluorescence in situ Hybridization aka. FISH

DNA or RNA is labelled in its original location (i.e. “in situ”) by binding to a
fluorescent probe that can be detected under a fluorescence microscope. Cells
are fixed and short nucleotide sequences are injected as labels to bind through
hybridization (i.e. the formation of hydrogen bonds) to complementary regions
of the cellular nucleotides in their original regions. The labels are not mere
nucleotide stretches, but they are most commonly coupled to fluorescent dyes,
which emit light when excited by a laser at a certain wavelength, which can
be detected under a fluorescence microscope to visualize the localization of
DNA or RNA inside a cell as for the diagnosis of breast cancer [88].
Another method to investigate distinct sites of a chromosome is called chromo-

some conformation capture (“3C”). The idea behind this technology is that distant
regions of a chromosome are brought into spatial proximity and interact if they are
active as open (eu)chromatin at the same time. Then, these regions can be stuck
together, in a process called ligation (derived from Latin ligare, which means to
tie). These stretches of DNA being regulated together can be between 200 kilobases
(kb) and 1.5 megabases (Mb) long. They are referred to as topological domains.
Two topological domains are separated by so-called boundary elements [29]. The
precise regulation of chromosomes’ structure is a precondition for the preservation
of a cell’s hereditary information. Every human cell contains two copies of each
chromosome. Ploidy refers to the number of complete sets of chromosomes, and
since human cells have two copies, they are referred to as diploid. In total, there are
46 chromosomes, consisting of a pair of sex chromosomes, which determines the
human sex (X X = female, X Y = male; even though there are several intermediate
states), and additional 22 pairs of body chromosomes called autosomes.
The most important functional subunit of hereditary material is a gene. A gene is
a stretch of DNA, that encodes the information to build a molecule with a particular
function. Molecules encoded by genes can be ensembles of either nucleotides of an
RNA or amino acids of a protein. Every gene is located at a certain position on a
particular chromosome. The gene’s position (i.e. location) is called its locus. The
gene exists on both duplicates of the pair of chromosome in that locus. The gene can
be present in different versions, which are called alleles. For instance, the ABO gene
is located on the human chromosome number 9. The ABO gene has three different
alleles that encode our blood types: A, B and 0. If one copy of the pair of chromosome
9 harbours the A allele, the other copy of the pair of chromosome 9 harbours the B
allele, the blood type is AB. In this case, the two alleles of the ABO gene are different
from one another, so they are called heterozygous. If the blood type is AA, BB or
00, the ABO gene is homozygous because the two copies of the gene exist in the
same version.
The entire genetic composition of a cell or an organism is called their genotype.
The physical outcome of this genetic composition (e.g. the blood type) is called the
phenotype, which is a characteristic that can be passed on from one generation to
2.3 The Genetic Code 17
another, and as such represents a heritable trait. Sometimes, one allele of a gene is
dominant over the other one, so a single copy of this allele is sufficient to confer a
certain phenotype. Otherwise, recessive alleles, which are inferior to the dominant
forms, are required as two copies to show the phenotype. In our blood type example,
the A and B alleles are dominant over the 0 allele. Consequently, A0 and 0A result
in blood type A, B0 and 0B result in blood type B and only 00 results in blood
type 0.
2.3 The Genetic Code
A set of rules is required for the conversion of genetic information into phenotypic
output. The link between the nucleotide sequence of the gene and the amino acid
sequence of the protein is encrypted by what is called the genetic code. Genetic
information is storable within cells and can be inherited from one generation to
another. To transfer the information stored in the DNA sequence to functional proteins
an intermediate layer is involved: RNA. The conversion from the DNA to the RNA
sequence is called transcription. It is straightforward as both macromolecules are
closely related and structurally very similar. Sequences are complementary to each
other and use the same letters A, G and C, while the DNA nucleotide thymidine (T)
is replaced by uridine (U) in RNA.
The conversion from a nucleotide sequence to an amino acid sequence is rather
complex because there are only four different nucleotides ∈ {A; U; G; C}, which need
to be mapped to 20 different amino acids to form proteins. Information encoded by
RNA nucleotides are processed as triplets. Given the four different nucleotides, there
are 4 × 4 × 4 = 64 possible triplets. Since all of them are being used, the information
is redundant, because 64 triplets encode only 20 different amino acids. The genetic
code is thus degenerated. The reason for this extent of complexity and redundancy
is three-fold [102]:
• Diversity of amino acids required.

• Robustness against errors during information processing.
• Minimization of metabolic costs.
The triplets are called codons and, of course, 20 different codons are required at least
to encode for the 20 amino acids, which serve as building blocks for proteins (see
Sect. 1.1). Chemically similar amino acids are encoded by triplets that are confused
by chance. The readout of the codons is an erroneous process and if a nucleotide is
likely to be mistaken, the corresponding correct and incorrect amino acids are likely
to be the same or at least comparable in their polarity, side-chain characteristics
or other biophysical properties. Too many redundant codons do not exist because
their maintenance is metabolically costly [98]. E.g. methionine and tryptophan are
encoded by only a single codon. These three aspects explained above steered the
evolution of the genetic code and rendered it modular (Table 2.2). With only a few
exceptions, for instance, in Mycoplasma, the genetic code is universal.
Table 2.2 Amino acids are encoded by triplets of nucleotides (in bold). Amino acids and codons are
given by the following one-letter code: F = phenylalanine, L=leucine, I = isoleucine, M = methionine
and start codon, V = valine, S = serine, P = proline, T = threonine, A = alanine, Y = tyrosine, Z = stop
codon, H = histidine, G = glutamine, A = asparagine, K = lysine, D = aspartic acid, E = glutamic acid,
C = cyteine, W = tryptophan, R = arginine, G = glycine
1st U C A G
2nd
U F F L L L L L L I I I M V V V V
C S S S S P P P P T T T T A A A A
A Y Y Z Z H H Q Q N N K K D D E E
G C C Z W R R R R S S R R G G G G
3r d U C A G U C A G U C A G U C A G
2.4 DNA Replication
For maintenance of DNA integrity and genomic stability throughout generations,

DNA has to be replicated robustly. The production of daughter cells requires DNA
synthesis in the so-called S(ynthesis) phase of the cell cycle (see Chap. 7) prior to
cell division. DNA replication is precisely regulated in space and time [35]. Starting
points for these processes within the genome are the origins of replication. The
human genome contains roughly 10,000 of these origins, and thousands can be
activated simultaneously within a single cell but only once within a single round
of DNA replication. Otherwise certain regions of the genome would be amplified
causing genomic instability.
An origin of replication (ORI) is defined as a site within the genome where
DNA replication starts. Every origin consists of two distinct elements. One region is
recognized by a molecular machinery to form the pre-replication complex. Another
region recruits the enzyme for DNA synthesis, namely DNA polymerase I in eukary-
otes. Due to these two distinct sites at the origin, the DNA replication process can
be discretized into two subsequent events:
1. Licensing: The origin of replication is recognized by an origin recognition com-

plex that recruits additional proteins for the formation of the pre-replication com-
plex.
2. Firing: The enzymes required for DNA replication are directed to the sites for
DNA synthesis in a cell-cycle-dependent manner.
The DNA double helix has to be unwound for replication. ORIs are AT-rich because
only two hydrogen bonds exist between adenosines and thymidines and therefore
these nucleotides can be easily prised apart. The double strand needs to be opened
like a zipper thereby forming a replication fork so that the DNA sequence can be
read and copied. To unzip DNA, hydrogen bonds between the two DNA strands
of the double helix have to be removed (Fig. 2.4). As single-stranded DNA is less
stable, these unzipped strands have to be stabilized to prevent their degradation or
2.4 DNA Replication 19
Fig. 2.4 DNA double helix

needs to be unzipped for
replication unzipped
end
DNA double helix
reformation of the double strand. During the copying of the DNA template, molecules
precede the replication machinery, which clear the way and adhere the complex to
the strand. Thus, many specific factors are involved in DNA replication.
2.4.1 Molecular Components Involved in DNA Replication
• Helicase unwinds double-stranded DNA under ATP hydrolysis.

• Topoisomerase relieves tension of twisted DNA.
• Single-strand binding protein (SSB) stabilizes single strands of unwound DNA.
• Primase provides RNA primer as a starting point for DNA synthesis
• DNA polymerase synthesizes DNA as a complementary copy of DNA template
strand.
DNA replication begins with a helicase that prises the two strands of the DNA double
helix apart. Energy from ATP hydrolysis is required for this unzipping enzyme to be
propelled forward. While the helicase opens the replication fork, the twisting of the
yet unwound DNA double helix in front of the helicase creates a tension that needs to
be relieved for the replication machinery to proceed. DNA is cut partially to prevent
it from being wound up more tightly. The enzymes responsible for creating nicks
and resealing upon unwinding are topoisomerases. Once the DNA replication fork
is opened up, single-strand binding proteins ensure that the fork is not prematurely
collapsing. For the actual DNA synthesis to begin, it needs some starting material,
which is an RNA primer provided by a primase. This enzyme belongs to the family of
RNA polymerases because it synthesizes RNA complementary to the DNA template.
The DNA polymerase can elongate the RNA primer in the 3’ direction with DNA
complementary to the template strand. The DNA polymerase adds nucleoside 5’-
triphosphates upon hydrolysis of pyrophosphate thereby creating a phosphodiester
bond between the 5’ phosphate group of the nucleotide and the free 3’ hydroxyl
group of the strand that is to be elongated.
The human DNA polymerase I processes ≈ 100 nucleotides per second. The
nucleotides complementary to the template are energetically favoured and therefore
bind faster. If there is an error, such as A not matched to T or C not matched to G,
the DNA polymerase slows down and uses its proof-reading activity. The enzyme
tries to immediately correct the wrong nucleotide by removing it and waiting for the
binding of the nucleotide that is complementary to the template strand. If there is a
correct match, the phosphodiester bond between the adjacent nucleotides is formed
and the DNA polymerase proceeds in the 5’→3’ direction of the nascent DNA
strand. Despite its proof-reading activity, the human DNA polymerase I creates an
error every 107 nucleotides. This error rate adds up to ≈ 300 mismatches within
Fig. 2.5 For DNA replication, the double strand is unwound by the helicase creating a replication
fork. On the leading strand, DNA polymerase I continuously synthesises DNA in 5’→3’, whereas,
on the lagging strand, Okazaki fragments are discontinuously added
a single round of replication of the cellular genome. To prevent these mismatches

from being propagated throughout subsequent generations, additional DNA repair
mechanisms exist (see Sect. 7.2.2). A so-called repair polymerase is also involved
in filling the gaps that were created upon removal of the RNA primer sequence.
When a nuclease cuts the RNA nucleotides out of the newly synthesized strand, the
repair polymerase fuses DNA nucleotides in. A ligase links the 3’ hydroxyl group
of the synthesized DNA strand with the 5’ phosphate of the inserted nucleotides via
a phosphodiester bond, which seals the loose ends.
To replicate the entire genome of a cell, multiple DNA polymerase I molecules
have to be present at every single replication fork. The sequences of the two strands
of the DNA double helix are complementary to each other and their direction is anti-
parallel. DNA synthesis by DNA polymerase I can only occur in 5’→3’ direction of
the nascent DNA strand. This implies that the replication machinery moves in oppo-
site directions along the two loose ends of the replication fork. On one strand, DNA
polymerase I follows the helicase towards the DNA region that is being unwound
and DNA synthesis is continuous. On the other strand, DNA polymerase always
approaches the end of the fork and its DNA synthesis can only occur discontin-
uously (Fig. 2.5). The fragments that are synthesized between the provided RNA
primer and the end of the strand are named after their discoverer: Okazaki frag-
ments. As the continuous DNA synthesis is faster than the discontinuous addition
of Okazaki fragments, the strand of continuous DNA synthesis is considered the
leading strand, while the other one is the lagging strand.
The problem at the very end of the chromosomes is that the RNA primer on
the lagging strand cannot be replaced. To prevent chromosome shortening due to
loss of this single-strand overhang, telomerases create a repetitive DNA sequence
at the terminal ends of the chromosomes called telomeres. The repetitive DNA
sequences rely on an RNA template that is provided by the telomerase itself. Attrition
of telomerases declines telomere maintenance and is linked to ageing and disease
[14]. Telomeres prevent the terminal sites of chromosomes from being recognized
as double-strand breaks and unwanted recombination.
2.4.2 DNA Recombination to Secure Integrity and Diversity
Errors are made while the DNA is replicated. Ideally, an exact copy of the cellular
DNA template is generated, but changes in the normal DNA sequence can occur.
A deviation of the DNA sequence from the original sequence is called a mutation.
An error in this case means, for instance, that an G(uanosine) is introduced oppo-
site of an A(denosine), whereas T(hymidine) would be the correct complementary
base. The observed frequency of such errors during DNA replication is around 10−4 .
The observed frequency of observed mutations, however, is only 10−9 per copied
nucleotide. This high accuracy stems from the “proof-reading” activity of the DNA
polymerase, the enzyme that synthesizes new DNA. The proof-reading mechanism
was studied not only in human cells but also in bacteria, mostly in the rod-shaped
bacterium Escherichia coli (abbreviated E. coli). E. coli is called a “model organ-
ism” because it served traditionally as the main subject of experiments in molecular
biology, and many experimental techniques were established with this bacterium. E.
coli is a prokaryotic cell and has three different types of DNA polymerases, all of
which with proof-reading activity. Two of the DNA polymerases in eukaryotic cells
(e.g. human) are able to perform proof-reading.
The synthesis of DNA through both, prokaryotic and eukaryotic DNA poly-
merases, happens in 5’ → 3’ direction. Proof-reading is ensured by a 3’ → 5’
exonuclease activity, which means that the DNA polymerase can go back in the oppo-
site direction of the newly synthesized strand and cut out the previously inserted,
wrong nucleotides. Upon pause and removal of the incorrectly inserted nucleotide,
the template strand of the DNA is copied again. If base pairing is happening correctly,
the synthesis proceeds in 5’ → 3’ direction.
Besides copying errors, DNA is exposed to other damage-causing agents such
as ultra-violet (UV) light that may ultimately lead to the generation of cancer (see
Sect. 9.1.2). Mutations that affect only a single nucleotide, which is changed com-
pared to the original (i.e. normal) DNA sequence, are referred to as point mutations.
These point mutations will be propagated if they are not corrected. In addition to
proof-reading, there are several DNA excision-repair systems to prevent mutations
from being established in the genetic code. These systems all work in a similar
way. The damaged segment of a single DNA strand is cut out, and the created gap
is refilled by the DNA polymerase complementary to the original template. Three
types of excision-repair systems are distinguished.
• Base excision-repair: Most common in humans is a point mutation changing the

original C(ytosine) to T(hymidine) thereby creating an erroneous GT pair instead
of the correct GC nucleotide pair. The incorrect pair is detected, because G and T
do not fit as well together as G and C. It is hard for the DNA repair machinery to
recognize whether the mutated nucleotide is the G or the T. But since the mutation
from C to T is much more likely, the system evolved to replacing the T of the
incorrect GT pair with a C by default.
• Mismatch excision repair: A general case of the base excision-repair that elim-
inates other point mutations, or insertions or deletions of several nucleotides.
Insertions mean pasting of more nucleotides in synthesized strand than the tem-
plate. All these errors occur during DNA replication by DNA polymerase. It is
not fully understood how the system discriminates the correct template from the
erroneously synthesized strand. Some proteins detect and bind to the ill-shaped
mismatch of incorrect nucleotide pairs, and subsequently guide the cutting and
replacement.
• Nucleotide excision repair: This applies to single nucleotides that are chemically
modified and then interact with neighbouring nucleotides of the same DNA strand.
The prime example is a dimer (i.e. a pair) of two adjacent T(hymine)s in the same
DNA strand. Since one T is chemically added to the subsequent T, it is also
referred to as a “chemical adduct”. The T + T structure results in a local distortion
of the DNA double helix. A helicase is recruited to open and unwind this part of
the helix, which facilitates the excision (i.e. cutting out) and replacement of the
wrong T + T dimer.
Two other systems use DNA recombination instead of excision and repair to keep
DNA integrity. DNA recombination is the exchange of DNA snippets between very
similar regions, e.g. the same locus of the two copies of the same chromosome in a
diploid cell, or following replication, (see above, Sect. 2.2.2). Recombination-based
DNA repair happens when both strands of the DNA are ruptured as if one cuts a
telephone wire (i.e. the DNA double helix) with a pair of scissors (usually caused by
irradiation or drugs). The resulting disjoint structure is called a “DNA double-strand
break”. The loose ends of the DNA can cause gross structural rearrangements of the
chromosome, which has to be prevented to keep the genetic information intact. DNA
double-strand breaks can be repaired by two different mechanisms.
1. Homologous recombination: Overhangs are created by cutting out opposing

strands from the two broken ends of the DNA double helix through the exonu-
clease activity of enzymes. The thus-created overhangs are then hybridizing (i.e.
binding) to the homologous (i.e. similar) region of the other copy of the same chro-
mosome. The broken ends are extended through DNA replication of the template
strand from the other copy of the (homologous) chromosome. Once the former
loose ends are connected, the junctions between the homologous chromosomes
are cut (Fig. 2.6). This process leads to the repair of DNA double-strand breaks
and involves exchange (i.e. recombination) of genetic information between the
homologous chromosomes.
2. Non-homologous end joining: The loose ends of the broken DNA double strand
are merged together with each other or with similar regions in the proximity of
the same chromosome. This involves cropping of the terminal overhangs to create
blunt ends, which leads to the loss of several nucleotide base pairs.
Once DNA integrity is ensured for replication of genetic information, the hereditary
material can be decoded for the propagation of functional properties of the cells.
Fig. 2.6 A DNA

1) break
double-strand break (1) is cut
to generate overhangs (2),
which bind to the
complementary regions of 2) overhangs
the other copy, and DNA
replication (dashed line)
completes the second strand 3) recombination
(3). Subsequent cutting of
X-like “holiday” structure
(4), and connecting the black cutting
4)
strands (5). 5’ → 3’
direction indicated by
arrowheads
5) completion
Summary
• Nucleotides are building blocks of DNA and RNA.
• Genetic information is encoded in DNA, which is wrapped around histone
proteins to form chromatin structure in chromosomes.
• The genetic code degenerated because 64 possible combinations of three
nucleotides encode for only 20 amino acids.
• DNA replication ensures integrity and maintenance of hereditary material
through different proof-reading and repair mechanisms.
Transcription and Translation
3
Contents
3.1 The Genome is Read and Transcribed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2 Processing of Transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.3 Transcripts are Translated into Polypeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4 Measuring Cellular Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Introduction
The mere maintenance of genetic information and its structure is insufficient to
carry out cellular functions. Genetic information has to be expressed. Therefore, the
information stored in the DNA sequence needs to be decoded, i.e. transcribed, into
a messenger molecule consisting of RNA. Mostly, this message in turn needs to
be translated into a molecule, i.e. a polypeptide, which, upon folding into a protein,
exerts the function of the gene with a certain property (Fig. 3.1). Reading, writing and
translating genetic information are fundamental cellular processes, and their main
principles are evolutionary conserved. The molecular processes of gene expression
can be investigated in bacteria as well as in human cells. Importantly, it is not sufficient
to just replicate DNA, transcribe it to messenger RNA ( mRNA) and translate this
to protein. In biology, structure and function are intimately related. The protein at
first is a steady chain, which has to fold into a three-dimensional (3D) structure.
The protein cannot function properly unless its correct conformation is reached.
Erroneously folded proteins can aggregate and plug the cell or do harm otherwise.
That is, the reason why unfolded (i.e. incorrectly folded) proteins will be erased (i.e.
degraded) from the cell.
With modern technologies it is possible to measure all cellular DNA, or RNA, or
all proteins of a bulk of cells. If these entities are measured at this global scale, the
processes are referred to as “-omics”. For instance, the measurement of all genetic
information in the form of DNA is referred to as genomics. The measurement of

https://doi.org/10.1007/978-3-662-65357-9_3
26 3 Transcription and Translation
replication
transcription translation folding
DNA mRNA protein property
genotype gene expression phenotype
Fig. 3.1 The genetic information stored in DNA is replicated and transcribed into a messenger
molecule of RNA (i.e. mRNA), which in turn is translated to a protein that needs to fold into a
particular 3D conformation to obtain a structural and thus functional property. The process of gene
expression links a genotype to a phenotype
all transcripts (i. e. mRNA) is referred to as transcriptomics. Measuring all proteins

of cell populations is called proteomics. Analogously, the whole DNA of a cell is
its genome, all transcripts are called the transcriptome, and all proteins make up
the proteome. The sequence of DNA and RNA can be read by machines, thus the
measurements of genome and transcriptome are referred to as “sequencing”, whereas
individual proteins are analysed based on their mass and electrical charge as ions.
The proper interpretation of such datasets is still challenging, but it allows a global
perspective on fundamental cellular processes of life.
3.1 The Genome is Read and Transcribed
To read and transcribe the genetic information, a reading device is needed. A molec-
ular machine capable of reading and copying DNA is the DNA polymerase (see
Sect. 2.4.1). A polymerase is an enzyme capable of synthesizing and outputting a
long molecule of many units (i.e. a polymer, poly from Greek meaning “many”).
In case of the DNA polymerase, the output is a DNA polymer because the DNA is
copied. To exert the function of a gene, the genetic information in the form of DNA
needs to be transcribed into messenger RNA transcripts first. The enzyme in this case
has to output RNA, and is thus an RNA polymerase. Bacteria have only a single
type of RNA polymerase, which, however, consists of several subunits with specific
functions such as providing the primer (i.e. the first building block) for the synthesis
of the RNA polymer.
The genome of eukaryotes is more complex, and so are the structures of their
RNA polymerases. Humans, for instance, have at least three diverse types of RNA
polymerases: I, II and III. The messenger RNA (mRNA) transcripts are generated
among other RNA molecules (see Sect. 4.2) by RNA polymerase II. The entire pro-
cess of transcribing DNA into mRNA and their translation into a protein, which folds
into a functional structure, is referred to as gene expression, because the information
encoded in a gene is thereby decoded and finally expressed.
3.1 The Genome is Read and Transcribed 27
3.1.1 Start the Transcription
The first step of gene expression is the initiation of the transcription of the DNA. A
starting point has to be found to mount the process. The DNA sequence is indexed
relative to the transcription start site (TSS). The TSS is the origin (0) and the DNA
template strand to be transcribed is displayed in 3’ → 5’ direction. The left side
of the TSS is referred to as “upstream” with a negative index number of nucleotide
bases, and right to the TSS is the downstream sequence with positive index numbers
of nucleotide bases. The region where transcription and thus gene expression is
initiated is called promoter region (Fig. 3.2).
In bacteria, the module that detects the TSS is the σ factor, a protein tightly asso-
ciated to the bacterial RNA polymerase. It binds to a consensus sequence upstream
of the TSS to find the position to initiate transcription. A consensus is a sequence
region that is very similar among different species and thus evolutionarily conserved.
The consensus sequence for the σ factor to bind the DNA to be transcribed is around
the positions −35 and −10 (Fig. 3.2). The spacing between these regions varies
around 17 base pairs. The consensus sequence at −35 is TTGACA, and the consen-
sus sequence at −10 is TATAAT. This does not mean that upstream of every TSS
in bacteria this exact sequence can be found, but it is the most frequently observed
sequence among bacterial species. For eukaryotes such as humans, the DNA-binding
region for the initiation of transcription is around the position −25 upstream of the
TSS. This sequence is less conserved than in bacteria, and it is referred to as the
TATA box rather than giving a consensus sequence.
While in bacteria, the σ factor together with RNA polymerase is sufficient to
initiate transcription, eukaryotes require additional proteins capable of DNA binding
to recruit the RNA polymerase II; so-called transcription factors. The events happen
in the following order:
1. The TATA box-binding protein (TBP) as subunit of the transcription factor TFIID
(standing for T ranscription Factor, RNA polymerase II, D) recognizes the TATA
box in the promoter region of the gene to be transcribed.
2. Other general transcription factors (TFIIB, TFIIE and TFIIH) are recruited to the
DNA of the TATA box as well as the RNA polymerase II. Altogether, they form
the so-called transcription initiation complex.
3. The transcription factor TFIIH serves as a helicase that unwinds the DNA powered
by ATP hydrolysis. In addition, TFIIH chemically modifies the C-terminal domain
(CTD), a side arm of RNA polymerase II.
-35 -25 -10 0 +10

upstream TSS downstream
Fig. 3.2 Promoter region usually symbolized by a bent arrow around the transcription start site
(TSS). DNA nucleotide positions in the direction of transcription are downstream and positively
indexed, whereas the opposite, upstream, is negatively indexed
4. The RNA polymerase II synthesizes an RNA primer and changes its conformation
to cause the release of the general transcription factors. Thereupon, transcription
starts from the nucleotide at the position +1 of the DNA template strand.
3.1.2 Quality Control During Transcription
Besides its role in transcription initiation, the transcription factor TFIIH is also
implied in transcription-coupled nucleotide excision repair (see Sect. 7.2.2). When
RNA polymerase II becomes stalled at T + T dimers, TFIIH is recruited again to
unwind the DNA for repair. Once the transcription is successfully initiated, the RNA
polymerase runs along the RNA polymerase runs along the DNA template strand
in 3’ → 5’ direction and synthesizes a nascent polymer from complementary RNA
nucleotides in 5’ → 3’ direction. This process is called elongation. In contrast to
DNA polymerases, RNA polymerases lack proof-reading ability, because they do
not have any 3’ → 5’ exonuclease activity. Instead, there is a kinetic proof-reading
phenomenon, which applies during the elongation of mRNA synthesis. If an RNA
nucleotide is not the correct one (i.e. not-complementary to the DNA template strand,)
then the RNA polymerase slows down because hydrogen bonds cannot form prop-
erly between the nucleotides and no energy is released that fuels the process. The
longer waiting time at this position increases the probability that the RNA nucleotide
falls off again since the binding and the unbinding are in equilibrium. By chance, the
wrong RNA nucleotide is released and replaced by the correct one, which causes the
formation of hydrogen bonds, the release of energy and the subsequent acceleration
of the elongation of the nascent RNA polymer. Due to the kinetic “proof-reading”,
the error rate of RNA polymerases is as low as ≈10−4 per nucleotide. This is much
higher than the error rate of DNA polymerases, but in contrast to copied DNA, mRNA
transcripts will not be inherited. Wrong mRNA molecules can cause only little dam-
age to a cell. Being single-stranded, mRNA molecules will be rapidly hydrolysed (i.e.
degraded). If the mRNA is translated to a wrong protein, there are additional steps
of quality control for it to be removed (see Sect. 3.3.3) Despite eventual deceleration
by the kinetic proof-reading, elongation continues until it is signalled to terminate.
3.2 Processing of Transcripts
3.2.1 Stop the Transcription
In prokaryotes, there are two different ways to terminate the transcription from DNA
into mRNA by the RNA polymerase.
1. Rho-independent termination: The nascent RNA polymer forms secondary struc-

tures, such as a hairpin, through binding of repetitive regions that are comple-
mentary to each other within the same mRNA molecule. The secondary structure
pulls the transcript away from the RNA polymerase (Fig. 3.3).
3.2 Processing of Transcripts 29
Fig. 3.3 An mRNA hairpin

is formed between repetitive,
complementary regions, that
physically pull off the mRNA
nascent mRNA molecule hairpin
from the RNA polymerase RNA polymerase
and the DNA template
strand, which is transcribed
3’ 5’
along its 3’ → 5’ direction
2. Rho-dependent termination: The RNA-binding protein Rho is recruited to the

nascent RNA polymer and slides towards the RNA polymerase to unzip the hybrid
between DNA template and mRNA transcript. Since hydrogen bonds between
the complementary nucleotides have to be broken, energy is required, which is
obtained through ATP hydrolysis by Rho. As such, Rho is a helicase that unwinds
the DNA-mRNA helix, and an ATPase.
In eukaryotes, the nascent mRNA transcript is processed during its synthesis. Ter-
mination of transcription is regulated within this process.
3.2.2 Splice the Transcript
The processing of the eukaryotic mRNA involves three distinct steps, all of which
start, while the DNA is still being transcribed in the nucleus (Fig. 3.4). The structure
of a gene is important to understand the necessity of the processing. A gene sequence
consists of coding and non-coding regions. Coding regions contain the blueprint for
the protein that will be translated from the mRNA transcript. Non-coding regions
have regulatory functions (see Sect. 4.2), but they do not contain information for the
protein to be synthesized. Coding regions are called “exons”, while the non-coding
regions of a gene are so-called “introns”. Introns are still part of the nascent mRNA
strand synthesized from the DNA template by the RNA polymerase II, and have
to be cut out during mRNA processing. Cutting out introns from the pre-(mature)
mRNA, leaving a mature mRNA containing only exons is referred to as splicing.
The prediction of introns and splicing sites from the mRNA sequence is a non-trivial
task. There is a consensus sequence, which, however, can occur just by chance on
the transcript without determining the splicing sites of the intron:
GU A YYYYYYYYY AG
5’ splicing site branch point poly-pyrimidine tract 3’ splicing site
To cut out the intron, the adenosine at the branch point loops forward and binds to
the 5’ splicing site. The 3’ end of the adjacent exon (to the left of the consensus
sequence above) is subsequently free and can connect to the exon at the opposing end
of the intron. The joining of the two exons releases the intermediate intron as a lariat
structure with a loop between the 5’ splicing site and the branch point, and the poly-
Fig. 3.4 RNA processing exons

involves the addition of the
5’ cap, splicing of introns so
that only exons remain in the
mRNA transcript, and the introns
addition of the poly-A tail
5’ AAAAAA 3’
pyrimidine tract as a tail. The exons are finally connected in a continuous sequence,
and the excised intron is recycled into single nucleotides for another mRNA polymer.
In addition to splicing, the nascent mRNA polymer receives a 5’ cap and a poly-
A(denosine) tail at its 3’ end. Once the synthesis of the 3’ end of the mRNA is
completed, the nascent polymer is cleaved from the RNA polymerase II. Subse-
quently, a poly-A polymerase is recruited to the mRNA to catalyse the synthesis
of a poly-A tail at the 3’ end of the mRNA. Once the poly-A tail is generated, it
is bound by poly-A binding proteins that stabilize the mRNA. The 5’ cap is a 7-
methyl G(uanosine), which is linked through a 5’–5’ triphosphate bridge to the first
nucleotide at the 5’ end of the mRNA. The 5’ cap stabilizes the single-stranded
RNA molecule, because it protects it from 5’ → 3’ exonucleases, which degrade
incorrectly processed mRNA molecules. In addition, the 5’ cap guides the mRNA
orientation, facilitates its export from the nucleus to the cytoplasm and initiates the
translation of the mRNA molecule (see Sect. 3.3.1).
3.3 Transcripts are Translated into Polypeptides
The structure of proteins is important to understand the translation of mRNA tran-

scripts to proteins. The basic, modular unit of a protein is an amino acid. An amino
acid has a conserved core structure with an amino (NH2 ) group, a carboxyl(ic acid,
i.e. COOH) group, and a hydrogen (H) group around the central carbon atom (C)
as well as a variable residue (“R”) that determines the type of the amino acid. The
amino group of one amino acid can bind to the carboxyl group of another amino
acid, thereby forming a peptide bond. A molecule consisting of two amino acids
that are linked by a peptide bond, is called a dipeptide. Depending on the number
of amino acids in such a molecular chain, the molecule is called a di- (2), tri- (3)
or poly- (>10) peptide (Fig. 3.5). Every peptide has an N-terminus (i.e. the end with
the unbound NH2 group), and a C-terminus (i.e. the end with the unbound COOH
group). Polypeptide chains that are ultimately folded into a 3D structure are mature
proteins. To translate the mRNA transcript into the encoded amino acid sequence,
another molecular assembly line (similar to the transcription machinery) has to be
initiated.
3.3 Transcripts are Translated into Polypeptides 31
Fig. 3.5 A tripeptide H H H

consisting of three amino H2N C CO NH C CO NH C COOH
acids characterized by their
variable residues R R R R
3.3.1 Initiation of Translation
To initiate the translation of the mature mRNA molecule into a nascent polypep-
tide chain, the mRNA (as a substrate of this reaction) has to be recognized first.
Detection happens through the poly-A binding proteins at the 3’ end of the mRNA.
The 5’ cap of the mRNA is bound by the eukaryotic initiation factor 4E (eIF-4E),
which cooperatively interacts with another one of these factors called eIF4G. Next,
a carrier is required to link the mRNA sequence to the encoded amino acids. Every
three nucleotides form a triplet, which determines the corresponding amino acid (see
Sect. 2.3). Every triplet on the mRNA molecule encoding an amino acid is referred to
as a codon. This carrier, that transfers the amino acid to the correct codon, is called
a transfer RNA (tRNA). The tRNA contains an RNA stretch complementary to the
codon, called the anti-codon. Depending on the anti-codon, the tRNA is loaded with
the amino acid encoded by the corresponding complementary codon (Fig. 3.6). The
first codon of an mRNA molecule is called the start codon and always stands for
methionine. To initiate translation, a ternary complex (i.e. with three components)
needs to form. This complex must contain the eukaryotic initiation factor eIF2, the
initiator tRNA and the methionine loaded on that tRNA. The place where the trans-
lation is carried out is the ribosome.
Fig. 3.6 The tRNA contains

id
ac
an anti-codon, which is
o
complementary to the codon

in
am
on the mRNA. Depending on

these triplets, the tRNA is
loaded with the
corresponding amino acid
tRNA
anti-codon
3’ 5’
5’ 3’
codon mRNA
3.3.2 Ribosomal Structure and Function
Ribosomes are modular structures composed of a small and a large subunits, which
differ in size between bacteria and eukaryotes. The size of the ribosomes and
their subunits was indirectly measured by a centrifugation gradient with the unit
S(vedberg) corresponding to the sedimentation of the molecules that does not scale
linearly with their size. With respect to the measured sedimentation, ribosomes and
their subunits were named accordingly:
Ribosome Bacteria Eukaryotes

Small subunit 30S 40S
Large subunit 50S 60S
Total: 70S 80S
In eukaryotes, the ternary complex, harbouring the initiator tRNA loaded with
methionine for the start codon at the mRNA bind to the 40S (i.e. small) riboso-
mal subunit, thereby completing the assembling of the pre-initiation complex. The
interaction between eIF4E, which is bound to the 5’ cap of the mRNA, and eIF4G,
which is bound to the poly-A binding proteins at the 3’ end, causes the mRNA to
form a circle. The loop formation ensures that the mRNA was correctly processed,
otherwise, eIF4E and eIF4G could not interact. In addition, the circular structure
facilitates the recruitment of many ribosomes. An ensemble of many ribosomes is
referred to as polysomes.
The small subunits of the assembled ribosomes are already equipped with the
initiator tRNA and the methionine. Methionine is always the first amino acid of the
translated polypeptide chain, but it will be cleaved by a protease (an enzyme that
can degrade proteins). The assembled ribosome moves along the mRNA in their
5’ → 3’ direction (i.e. from the cap towards the poly-A tail). The ribosome exhibits
three distinct sites where codons of the mRNA strand are placed and tRNAs loaded
with corresponding amino acids are recruited. These sites are called the A(cceptor),
the P(eptide bond formation) and the E(xit) site, respectively (Fig. 3.7).
Fig. 3.7 The assembled

ribosome contains three
distinct sites where large subunit
subsequent codons of the
mRNA are placed: The
E P A mRNA
A(cceptor), the P(eptide
bond formation) and the 5’ 3’
E(xit) site
small subunit
3.3 Transcripts are Translated into Polypeptides 33
The polypeptide chain is assembled in a four-step process:
1. The specific tRNA with the anti-codon complementary to the codon of the mRNA
is recruited to the vacant A site of the ribosome. The tRNA is loaded with the
corresponding amino acid.
2. A peptide bond is formed between the amino acid of the tRNA at the A site and
the amino acid (chain) of the tRNA at the P site of the ribosome.
3. The ribosome translocates so that the tRNA, which has previously been at the P
site, is now at the exit site while the tRNA at the former A site is now at the P site
and carries the polypeptide chain.
4. The tRNA at the E site of the ribosome is released and the cycle can continue to
assemble the polypeptide chain starting from the N-terminus to the C-terminus.
Similar to the hybridization between DNA and RNA nucleotides during transcription
catalysed by the RNA polymerase, also the ribosome exhibits kinetic proof-reading
activity. If the tRNA with the wrong anti-codon and amino acid binds to the codon
of the mRNA, the ribosome slows down. This deceleration increases the chances
that the wrong tRNA falls off the A site and is replaced by the correct tRNA with
the anti-codon complementary to the codon, which is energetically favoured. During
translation, the mRNA template is already de-capped and de-adenylated, in other
words: The 5’ cap and the poly-A tail are removed to accelerate the breakdown
of the mRNA into individual nucleotides and their recycling to the next round of
transcription.
Translation is terminated at the last and so-called stop codon. Once the A site of
the ribosome reaches a stop codon, termination factors are recruited, the ribosomal
subunits fall apart and the polypeptide chain is released. The region between the start
codon and the stop codon is called “open reading frame”.
3.3.3 Protein Folding
The synthesized polypeptide chain is at first a one-dimensional sequence of amino

acids. To exert its function as a protein, the molecule needs to fold into a 3D structure.
The problem is that the angle of the bond between the amino group and the central
carbon atom as well as the angle of the bond between the central carbon atom
and the carboxyl group can rotate almost freely. As a consequence in theory 1030
possibilities exist to fold a polypeptide sequence that consists of 100 amino acids
into a 3D structure. Folding took almost infinitely long if all possible conformation
would be randomly tested until the correct one was found. In fact, polypeptide chains
fold into their 3D structure quite rapidly, once they are released from the ribosome.
This discrepancy between the theoretically long folding time and the factual quick
folding is known as Levinthal’s paradox. The cell contains other proteins, which assist
polypeptide folding. These so-called chaperones can also trigger stress responses to
clear incorrectly folded proteins thereby preventing their aggregation and further
harm (see Sect. 5.3.1).
3.4 Measuring Cellular Content
For the first time, modern biology allows to experimentally assess gene expression
on a global scale. This means that all genes can be measured at once, either at the
genome level of DNA, or the transcriptome level of mRNA, or the proteome level
of proteins and their modifications. Previous studies focused on the investigation
of individual genes, their DNA sequence, mRNA abundance or protein level. Now,
the whole entity of DNA, RNA or protein can be measured even quantitatively, with
absolute numbers of DNA sequence variants, mRNA molecules or proteins per gene.
Thus, every instance of gene expression (c.f. Fig. 3.1) can be assessed globally. These
measurements of the genome, the transcriptome or the proteome are referred to as
the omics. Despite the steady development of new methods and improvement of
existing technologies, limitations have to be considered and data should be analysed
with great care.
3.4.1 DNA Sequence Variants
“Reading” (i.e. measuring) of the DNA sequence is referred to as DNA sequencing.

Due to improvements in technology, costs of DNA sequencing dropped significantly
in the last years: https://www.genome.gov/sequencingcosts. Since 2008, the reduc-
tions in DNA sequencing costs even outpace Moore’s Law, a well-established and
widely applied model for the development of technology based on the assumption
(among others) that computer power doubles every 2 years. Improvements in sensi-
tivity and cost-efficiency of DNA sequencing methods accelerated research findings
in the field of genomics. Increasing complexity and diversity of sequenced genomes
has been witnessed thanks to the next-generation sequencing technologies [40].
DNA is sequenced to access the genotype of an organism (c.f. Fig. 3.1). The geno-
type can vary between individuals as they acquire mutations. The word mutation is
derived from the Latin verb mutare, which means “to change”. The DNA sequence
of a mutant has changed compared to the “wild-type”, which serves as a null ref-
erence. At a larger scale on the level of chromosomes, changes to the DNA can be
either insertions or deletions. Insertions can be subdivided into inversions (i.e. flip
of two chromosomal regions), duplications (i.e. copy and paste of a chromosomal
region) or additions of genetic material (Fig. 3.8). An example of the latter one is the
addition of a DNA region of the human chromosome number 9 to the chromosome
number 22. This “translocation” of a chromosomal region creates a fusion gene:
BCR-ABL1, a mutation strongly associated with cancer formation [91]. It is appar-
ent that changes in the chromosome level can have drastic consequences for a cell
or the entire organism.
At a smaller scale on the level of individual nucleotides, the consequences of muta-
tions are constrained by the fact that the genetic code is degenerated (see Sect. 2.3).
It can well be that a change of a single nucleotide does not have any consequences
because the mutated triplet also encodes for the same amino acid as the wild type
does. If this is the case, the mutation is referred to as “silent”. If the mutations of
3.4 Measuring Cellular Content 35
AB A A AB
BA AA AC A
inversion duplication addition deletion
Fig. 3.8 Mutations on the level of chromosomal regions A, B and C are either insertions (inversion,
duplication or addition) or deletions
a single nucleotide generate a triplet that encodes for a different amino acid, it is
referred to as “missense”, because the sense of the sequence might change. The
mutant amino acid will have a different spatial arrangement and maybe even other
properties (e.g. hydrophilic/-phobic) than the wild-type amino acid, which will influ-
ence the 3D structure and thus the function of the polypeptide chain. The most drastic
case is the creation of a stop codon through the mutation of a single nucleotide. This
mutation will cause the termination of translation at this point and create a frag-
mented polypeptide chain. The creation of a stop codon is therefore referred to as a
“nonsense” mutation. If there is only a single nucleotide (ex)change, the mutation
is referred to as “point mutation” (Table 3.1). A change in a single nucleotide can
cause many (Greek: poly) differently shaped (Greek: morphos) organisms. Another
name for these mutations is therefore single-nucleotide polymorphism (SNP). It is
hard to prove that a mutation in a single nucleotide is solely responsible for changes
in the phenotype. That is why genome-wide associations are studied only to find
loose connections between SNPs and a disease. The discovered associations need to
be validated mechanistically. An example are the mutations of a single nucleotide in
the genes MLH1, PMS2 and MSH5. Point mutations in these genes seem to be risk
factors for male infertility [50]. In this case, the polymorphism means that the wild
type exhibits normal sperm, whereas the mutant exhibits crippled sperm.
Mutations at the nucleotide level can involve insertions or deletions that affect
more than a single nucleotide. Collectively, insertions and deletions are abbreviated
“indels”. If the number of inserted or deleted nucleotides is not a multiple of three,
the reading frame will be shifted, because the genetic code is processed in triplets
(see Sect. 2.3). A frame shift will alter all encoded amino acids downstream of the
Table 3.1 Point mutations (in grey) of single nucleotides at the DNA level and their consequences
on the RNA level of the codon and the amino acid level of the protein. Missense mutation to lysine
is very similar to wild-type arginine but different from threonine
Wild-type Mutation
Silent Missense Nonsense
DNA TCT TCC TTT TGT ACT
RNA AGA AGG AAA ACA UGA
Protein Arginine Arginine Lysine Threonine Stop codon
mutation. In the following example, the deletion of a single nucleotide altered already
the start codon, inserted a stop codon and changed all other encoded amino acids:
wild-type codons AUG CAU UUG AGA AGG ACA UGA

wild-type amino acids M H L R R T Z
mutant codons AGC AUU UGA GAA GGA CAU GA
mutant amino acids S I Z E G H
All such variants can be measured by DNA sequencing. If the sequencing is not
“deep” enough, some sequence stretches will not be detected. Count matrices are
obtained (Table 3.2), which can be analysed for statistical significance of the presence
or absence of certain mutations under different conditions (e.g. healthy versus disease
condition).
3.4.2 Omics Data
Independent of the platform and its specifications during the experiment and the
measurement, processed genome, transcriptome and proteome data have the same
format. Per genetic region and condition, obtained levels of sequence variants (i.e.
DNA), transcripts (i.e. mRNA) or protein are given in absolute or relative units
(Table 3.2).
Recent advances in transcriptomics allow the rapid and standardized acquisition
of mRNA numbers per gene (i.e. protein-coding genetic regions), with every con-
dition being a single cell. Why is it important to measure the mRNA globally at
the resolution of single cells? Because the cellular composition of a tissue can be a
heterogeneous mix of different cell (sub)populations, which cannot be discriminated
when being averaged out in a bulk measurement. Single-cell RNA sequencing can
discover rare cell types, which have to be characterized based on their transcriptome.
The identification of a unique set of marker genes that distinguish one subset of cells
from another one is a non-trivial task. Recent technological advances allow to resolve
heterogeneity in a spatial context including physical interactions for the improved
assessment of cellular identity and biological function [38]. Computational pipelines
help to understand that the acquired data represents snapshots of dynamic temporal
processes [2], e.g. the progression of a disease such as liver fibrosis.
Table 3.2 For genomics, transcriptomics and proteomics processed output is displayed as a counts
table for genetic regions under different experimental conditions
Condition 1 Condition 2 … Condition n
Genetic region 1 32 NA … 1
Genetic region 2 NA 15 … NA
… … … … …
Genetic region n 6 NA … 7
3.4 Measuring Cellular Content 37
800
15000
# Genes
600
# Cells
10000
400
200 5000
0 0
0 5 10 01 5 10 15 20
log2(transcripts+1 per cell) log2(transcripts+1 per gene)
Fig. 3.9 Distributions of the numbers of measured transcripts per cell (left) and per gene (right).
Dashed vertical line indicates cut-off (i.e. 400 transcripts) for quality control. Data from [49]
The obtained data matrices (c.f. Table 3.2) are sparse with missing entries for many
gene(tic region)s/conditions. Therefore these omics data require proper processing.
Raw data has to be inspected carefully to learn something about their distribution
(Fig. 3.9) for quality control. A normalization procedure with proper controls needs
to be established to compare datasets from different experiments. Because both, the
transcriptome and the proteome, are intricately regulated at several layers, global
trends in gene expression are non-intuitive. Ideally, published findings are freely
available as raw data as well as processed data together with curated open-source
code to reproduce results sustainably.
An example is the data in [49], which can be inspected and (re)analysed with all
the information provided on a repository under: http://bit.ly/2JvmlD7.
Summary
• For transcription of DNA into mRNA, conserved sequences have to be bound
by transcription factor molecules.
• Transcription is terminated and transcripts are processed by splicing: Introns
are cut out, while exons are being pasted together.
• mRNA is translated by ribosomes into a polypeptide chain, which needs to be
folded and shuttled to the correct location.
• Total DNA, mRNA or protein content of cells can be measured with current-
gen/transcript/prote“-omics” technologies.
Regulation of Gene Expression
4
Contents
4.1 Transcriptional Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.2 Regulatory RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.3 Protein Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.4 Epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Introduction
The mechanisms of gene expression (i.e. the decoding of genetic information as a
blueprint to synthesize proteins with a certain structure and function) are conserved
between bacteria and eukaryotes. With additional complexity scales the possibility
for regulatory layers to fine-tune processes during gene expression. In biology, struc-
ture and function are tightly coupled. The composition of DNA, RNA and protein
allow interactions among themselves and each other. Interactions lead to the creation
of regulatory networks rather than a linear chain from DNA to mRNA to protein
(Fig. 4.1). These networks evolved with time and allowed adaptation to changing
conditions. If more structural proteins are needed to allow the rapid development of
more complex cellular structures and multi-cellular tissues of an organism, all steps
from DNA transcription to RNA translation and protein folding need to be balanced
to prevent bottlenecks in the production. An increased pool of mRNA might facili-
tate rapid translation once the respective protein is needed. Therefore, micro-RNA
(miRNA) exist, which can for instance block translation without necessarily affect-
ing the production of mRNA. On the other side, miRNA can lead to the degradation
of mRNA, which needs to be precisely balanced.
Dynamic synthesis and degradation, as well as the possibility for acceleration or
deceleration at every step of gene expression, can save cellular resources. Because a
protein does not have to be produced from an incorrectly processed mRNA. Instead,

https://doi.org/10.1007/978-3-662-65357-9_4
40 4 Regulation of Gene Expression
chaperones
miRNA
replication
transcription translation folding
DNA mRNA protein property
binding proteins
genotype gene expression phenotype
Fig. 4.1 The concept of gene expression does not follow a linear process but contains several
feedback and feedforward mechanisms resulting in a complex regulatory network
the process can be abrogated as soon as an error occurs. Maintenance of such reg-
ulatory mechanisms also requires energy and metabolites but allows the cell more
valuable flexibility when facing a change in conditions.
4.1 Transcriptional Regulation
While many paradigms were disproved in the light of modern technologies, the
key concepts for regulation of gene expression dating back to the work of Jacob
and Monod in the 1960s do still hold [48]. From their pioneering work in bacte-
ria, they proposed the existence of proteins, which they called trans-factors, that
are capable of binding DNA at specific sites, which they called cis-elements. When
trans-factors bind to cis-elements, they recruit the molecular machinery to initi-
ate transcription. The trans-factors are meanwhile called transcription factors. In
eukaryotic cells, these transcriptional control mechanisms were extensively studied,
too. Today’s common notion is that transcription factors bind to DNA, recruit cofac-
tors and the transcription apparatus as well as chromatin remodelling enzymes to
regulate gene expression on the transcriptional level.
In eukaryotes, transcription factors cannot only bind to the promoter region of
a gene but also to the distal (i.e. far away) sites upstream of the transcription start
site. The distal DNA regions, bound by transcription factors, are called enhancers,
because they enhance the recruitment of RNA polymerase II. The presence of the
general transcription factors TFIIB, TFIID, TFIIE and TFIIH in addition to RNA
polymerase II is sufficient to initiate transcription (see Sect. 3.1.1). To bring distal
regulatory sites and their transcription factors in close proximity to the transcription
start site, the so-called “Mediator” complex is required, too. Binding of the mediator
to transcription factors and RNA polymerase II leads to a bending of the DNA
and helps to efficiently launch transcription (Fig. 4.2). As the organization of the
eukaryotic genome is complex, a chromatin-remodelling complex is also part of the
transcription machinery to ensure open chromatin and accessible DNA. Remodelling
of chromatin is achieved by histone-modifying enzymes (see Sect. 4.4).
There are also regulatory mechanisms that inhibit the initiation of transcription.
GC(-rich) boxes at the DNA sequence tend to be chemically modified by the addition
4.2 Regulatory RNAs 41
enhancer site
Mediator
RNA
transcription polymerase II
DNA factor
TFIIB,D,E,H
promoter site
Fig. 4.2 The mediator complex bridges the RNA polymerase II and the transcription initiation
factors TFIIB, D, E and TFIIH at the promoter site with a transcription factor at the enhancer site
of the DNA
of a methyl group. These methylated nucleotides impede the binding of transcription

factors and, instead, recruit histone-modifying enzymes that create heterochromatin,
which cannot be transcribed and is thus considered “transcriptionally silenced”.
4.2 Regulatory RNAs
As transcription is a very essential cellular process, its misregulation is closely linked

to diseases ranging from cancer (see Chap. 9) to autoimmune disease, diabetes and
developmental disorders [68]. Mutations that cause such deleterious consequences
can either affect the genes encoding for a transcription factor or their DNA-binding
sites. The latter ones are the cis-elements, which can be either located at the core
promoter or distal sites.
The complexity of an organism does neither correlate with DNA Content nor with
the number of protein-coding Genes, which is known as the C-value paradox and
the G-value paradox, respectively. For instance, the genome size of common wheat
Triticum aestivum exceeds with 16 × 109 base pairs the size of the human genome
with 3 × 109 base pairs by far. Despite its reduced complexity compared to humans,
the roundworm Caenorhabditis elegans contains with ≈20,000 the same number of
protein-coding genes as Homo sapiens. These discrepancies can be resolved when
looking at the number of regulatory elements in the genome, because the number
of non-protein-coding sequences per genome scales with the organismal complexity
[100].
Among the RNA molecules transcribed from DNA, which do not encode for
proteins, the two most prominent are:
• long non-coding RNA and

• micro-RNA (miRNA).
Both types of these non-coding RNA are synthesized mainly by RNA polymerase II.
While the function of the majority of long non-coding RNAs is not known, the role
of miRNAs as an important regulatory player in gene expression is well understood.
After being transcribed, miRNAs exist as a pri-miRNA usually in the shape of a
hairpin (c.f. Fig. 3.3). This pri-miRNA is then shortened by an enzyme called Drosha,
which releases a pre-miRNA. The pre-miRNA is subsequently exported from the
nucleus to the cytoplasm. The cytoplasmic pre-miRNA is cleaved by another enzyme

called DICER. The resulting double-stranded miRNA (called “miRNA duplex”) is
separated into single strands of mature miRNA, each ≈21 nucleotides long, by a
helicase. The mature miRNA is loaded onto a protein called Ago to form the RNA-
induced silencing complex (RISC). Silencing (i.e. shutting down gene expression)
can happen in two ways:
1. Perfect match of the miRNA on the RISC with a recruited mRNA causing the
degradation of the latter.
2. Imperfect binding of the miRNA on the RISC with an mRNA causes a block of
ribosome binding and thus inhibits translation initiation.
The first scenario almost entirely abrogates the expression of the gene transcribed
into mRNA that matches the miRNA perfectly: ⇒ 0% protein.
The second scenario slows down translation but keeps the mRNA intact and thus
only reduces the expression of the respective gene: ⇒ [20%; 80%] protein.
Either way, regulatory RNAs interfere with the initiation of translation.
4.3 Protein Control
Once translation is successfully initiated, protein synthesis (i.e. the translation of

mRNA into polypeptide chains) is mainly controlled through the translation rate, in
other words, the speed of the ribosome sliding along the mRNA. Mostly, the ribosome
is physically blocked and therefore halts in its progression. An example is the control
of iron metabolism. If a cell is being starved from iron (i.e. Fe2+/3+ ions), it needs
less ferritin, a molecule that can bind free iron, but more of the transferrin receptor,
which facilitates the import of iron into the cell. In case of low cellular iron levels,
aconitase is bound to an mRNA hairpin structure in front of the ferritin mRNA and
behind the transferrin receptor mRNA. Thus, only the transferrin receptor mRNA is
translated, and more transferrin receptor is produced, which in turn accelerates the
iron uptake into the cell. In case of iron access, the Fe2+/3+ ions bind to aconitase
and cause a conformational change in the protein and its release from the mRNA.
As a consequence, the ferritin mRNA can be translated to produce more ferritin and
bind free iron, while the mRNA of the transferrin receptor is degraded (Table 4.1).
This fine balance is kept through selective blockade of the ribosome.
4.3.1 Post-Translational Modifications
Once the polypeptide chain has been synthesized and the mature protein folded
correctly, its activity can be controlled by chemically modifying it. Since these mod-
ifications of the protein occur after the translation of the mRNA, they are referred to
as “post-translational modifications”. Modifications are enzymatic reactions, which
facilitate the addition or the subtraction of groups of molecules.
4.3 Protein Control 43
Table 4.1 Cellular iron metabolism is regulated by the control of translation. The more protein
of the transferrin receptor (CD71) is synthesized, the more of the missing Fe2+/3+ ions can be
imported into the cell. The more of the ferritin protein is present, the more of the excessive iron can
be bound
Ferritin CD71 Ferritin CD71
Fe2+/3+ Aconitase mRNA mRNA protein protein
Starvation Bound to Blocked Protected ⇓ ⇑
hairpin
Excess Bound to iron Translated Degraded ⇑ ⇓
An enzyme (E) is a protein converting a substrate molecule (S) thereby creating

reaction product (P), with an intermediate state of an enzyme-substrate complex
(ES). The enzyme as a molecular machine is not needed for the reaction itself (→)
to occur, but it speeds up (“catalyses”) the reaction:
E + S ES → E + P.
The velocity v, which represents the reaction rate, can be expressed by the following
ordinary differential equation:
d [P] Vmax · [S]

v= ⇒ , (4.1)
dt K M + [S]
with d [P] /dt describing the change d of the concentration of the product [P] with
the change d of time t. [S] denotes the concentration of the substrate S. Vmax denotes
the maximum velocity of a reaction assuming saturation with substrate. The param-
eter K M is known as the Michaelis-Menten constant, which specifies the substrate
concentration at which the reaction rate is half-maximal. This becomes evident if
K M in Eq. 4.1 is substituted with [S]:
Vmax · [S] Vmax · 1 1

v| K M =[S] = ⇒ ⇒ · Vmax . (4.2)
[S] + [S] 1+1 2
The concentration of the enzyme [E] is not even part of the equation because the
substrate concentration [S] is the rate-limiting step. The molecular machinery of the
enzyme E works so efficient that very little amounts are saturating. A dose-response
curve of an enzymatic reaction showing the velocity of the reaction v depending on
the concentration of the substrate [S] resembles a hyperbolic function (Fig. 4.3).
An example of an enzymatic reaction is the hydrolysis of guanosine triphosphate
(GTP), which is the substrate S of the enzyme, to guanosine diphosphate (GDP)
and phosphate, which are the products P of this reaction (Fig. 4.4). The enzyme
catalysing the hydrolysis of GTP is a hydrolase; a GTPase to be precise. GTPases are
fundamental enzymes for the regulation of cellular processes as it will be described
in Sect. 5.2.1.
d[P]/dt
Vmax
½Vmax
KM
[S]
Fig. 4.3 Dose-response curve showing velocity of the reaction v = d [P] /dt depending on the
concentration of the substrate [S], with K M being the substrate concentration of the half-maximal
velocity of the reaction
enzyme enzyme-substrate enzyme
GTP
GTP GDP P
substrate complex products
Fig. 4.4 Enzymatic reaction of a GTPase enzyme, forming a complex with its substrate GTP to
hydrolyse it and release GDP and a phosphate group (P) as a product
Modifying residues of proteins through enzymatic reaction is a way to control their

function without changing their absolute amounts, even though post-translational
modifications can lead to a direct change in absolute amounts as is the case for
targeted protein decay.
4.3.2 Protein Decay
Protein levels cannot only be controlled by the production rate through the regulation
of transcription and translation, but absolute protein abundance can also be controlled
through targeted degradation. While RNA molecules are susceptible to hydrolysis
and thus decay by themselves with a certain half-life, proteins need to be broken down
actively. Proteins are labelled with small molecules, which flag them for degradation.
The most prominent mark for protein degradation is the small molecule ubiquitin.
Its name is derived from the fact, that the molecule is “ubiquitously” found in almost
every cell and every tissue. Protein degradation through ubiquitination is tightly
regulated. First, proteins subjected to degradation are labelled with a linear chain of
≥4 ubiquitin molecules. This degradation flag is recognized by a cellular machinery
called the 26S proteasome - the cellular protein dumpster. Similar to the ribosomes
(see Sect. 3.3.2), the size of the proteasome is indicated with the unit S(vedberg).
The 26S proteasome consists of a central core unit and two regulatory caps at the
top and the bottom of the core (Fig. 4.5). The ubiquitin-tagged proteins are unfolded,
4.4 Epigenetics 45
cap core cap
Fig. 4.5 Proteins labelled with a linear chain of at least four ubiquitin (Ub) molecules are being
degraded into small fragments inside the 26S proteasome
which requires energy from ATP hydrolysis. The polypeptide chain is subsequently
pulled into the core of the 26S proteasome and chopped into small fragments of
seven to nine amino acids in length. These fragments and the ubiquitin molecules
are recycled.
Degrading proteins prevents their accumulation and helps to keep a protein bal-
ance (i.e. “protein homoeostasis” or “proteostasis”) in the cell. However, the logic
behind this regulation is more complex, as there are proteins, which are labelled
with ubiquitin, but they will not be degraded, and there are proteins, which are not
labelled with ubiquitin, but they will be degraded [26]. The dynamic production and
degradation of proteins leads to the fact that protein content is not inherited from one
cell to another throughout generations. If a cell divides to form two daughter cells,
proteins of the mother cell are distributed among the daughter cells (see Sect. 7.3.2),
but the half-life of a protein is usually much shorter than the generation time (i.e. the
duration between two cell divisions).
4.4 Epigenetics
On top of genetic information encoded in DNA that will be maintained and inherited
from one generation to another, there are chromatin states that determine whether
genes are accessible and can be expressed, or not. This additional layer above the
genetic code is referred to as epigenetics (from Greek epi as prefix meaning “over”
or “on top of”). This means the epigenetic information is an additional layer on top
of the inheritable DNA sequence, which describe the chromatin state within a cell.
Despite adding a regulatory layer on top of the DNA, the chromatin state is not
independent of the DNA sequence.
4.4.1 Mechanisms of Epigenetic Regulation
Different factors are involved in the regulation of the chromatin state. The molecular
complexes introducing modifications can be classified as:
• writers—creating modification marks,

• readers—interpreting written modification marks or
• erasers—removing written modification marks.
The local change of the chromatin state between hetero- and euchromatin (see
Sect. 2.2.1) is a process called remodelling and means the structural rearrangement
between closed and open chromatin.
Whether chromatin is tightly or loosely packed depends mainly on the chem-
ical modifications of the DNA itself, and of histone proteins, around which the
DNA is wrapped. Common modifications at the DNA level include the addition
of methyl groups in particular to cytosine bases. Modifications of histone proteins
mainly involve the addition of methyl or acetyl groups or a combination of those.
At the DNA level, methyl groups are transferred to cytosine bases by DNA
methyltransferase (DNMT) enzymes. DNMT enzymes are thus the writers of DNA
methylation. The cytosine base of a CG nucleotide pair is often referred to as “CpG”.
Methylated cytosine bases can be bound by methyl-CpG-binding domain (MBD)
proteins. Those MBD proteins are readers of DNA methylation. Once bound to
methylated DNA, MBD proteins recruit other proteins that can modify the chro-
matin structure. These “chromatin remodelling factors” induce locally the formation
of heterochromatin, which is closed and does not allow for gene expression. Thus,
methylated cytosine bases, particularly in the proximity of promoter sequences, com-
monly lead to the silencing of gene expression.
Removal of methyl groups from cytosine bases mostly occurs passively, when
DNMT enzymes are not active. Methylated cytosine bases can be converted to
thymine bases by spontaneous deamination (i.e. the removal of an amino group:
–NH2 ). This conversion creates a TG nucleotide pair from the original, correct CG
nucleotide pair. This erroneous conversion of methylated cytosine to thymine in
CpG nucleotide pairs can be corrected by “base excision-repair” (see Sect. 2.4.2).
Thus, there are no directly active erasers of DNA methylation (i.e. DNA demethy-
lases). Among the chromatin remodelling factors that are recruited to the DNA in the
presence of DNA methylation and MBD proteins are histone-modifying enzymes
(Fig. 4.6).
A prime example of how histone modifications regulate gene expression is the
silencing of the inactive X chromosome [45], through the addition of three methyl
groups at the Lysine residue number 27 at the histone 3 (“H3K27me3”). Histone-
Fig. 4.6 Methylated (Me) cytosine residues in the DNA are recognized by methyl-binding domain
(MBD) proteins, which in turn recruit histone deacetylase (HDAC) enzymes that remove acetyl
(Ac) groups from histone tails and repress gene expression
4.4 Epigenetics 47
Table 4.2 Histone modifications by respective enzymes

Histone modification Enzyme Class
Acetylation Histone acetyltransferase Writer
Deacetylation Histone deacetylase Eraser
Methylation Histone methyltransferase Writer
Demethylation Histone demethylase Eraser
modifying enzymes can be recruited upon the change of the methylation pattern of
DNA nucleotides. Addition of methyl groups directly to nucleotides in the DNA
sequence thus represents another epigenetic layer highlighting the complexity of
regulatory mechanisms of gene expression.
Modifications of histone proteins are introduced by writers of the different chem-
ical residues, which are added to the histone tails, and removed in the reverse reaction
by erasers of histone modifications (Table 4.2).
Depending on the chemical properties of the added modifications (e.g. electrical
charge), the histone-DNA assembly can either be disrupted (by repulsive forces) or
tightened (by attractive forces). For example, the addition of a negatively charged
acetyl group to a histone tail through a histone acetyltransferase (HAT) enzyme
leads to the repulsion of the negatively charged backbone of the DNA (see Sect. 2.1),
because like charges repel each other. Consequently, histone acetylation induces
unwrapping of the DNA from the acetylated histone protein, which in turn allows for
gene expression from the locally formed euchromatin. The reverse reaction is catal-
ysed by HDAC enzymes removing acetyl groups from histone tails. The remaining
unmodified lysine residues of the histone tail are positively charged and thus attract
the negatively charged backbone of the DNA. The tight wrapping of histone and
DNA in this locally induced heterochromatin conformation does not allow for gene
expression.
Due to the combinatorial complexity of modified histone residues, it is hard to
predict the effect of a single histone modification on the recruitment of chromatin
remodelling factors and gene expression. The functional consequences of the modifi-
cation of a single histone residue depend on the presence or absence of other histone
modifications. The overall pattern of all present histone modifications is referred to
as the histone code, which influences chromatin structure and function by creating
or removing binding sites for other chromatin remodelling factors.
These chromatin remodelling factors are the readers of histone modifications.
E.g. acetylated Lysine residues of histone tails are recognized by the bromodomain
of transcription factors (see Sect. 4.1), which activate gene expression. This example
shows that the effect of the chromatin remodelling factors (i.e. recruitment of tran-
scription factors → activation of gene expression) can oppose the physicochemical
properties of the histone modification itself (i.e. negative charge of acetylation →
deactivation of gene expression), which introduces an additional layer of epigenetic
regulation.
4.4.2 Causes and Consequences of Epigenetic Regulation
The link from the DNA sequence to the property of an encoded protein (see Fig. 4.1)
can be modulated through mechanisms of epigenetic regulation. Therefore, the con-
nection between a genotype and a phenotype (see Sect. 2.2.1) is ambiguous.
Organisms with the same genetic information (i.e. the same DNA sequence) can
respond differently, e.g. when being exposed to different environmental conditions.
In some turtles, the sex of the progeny is determined by the temperature surround-
ing the egg. In this case, an environmental factor (i.e. temperature) influences the
demethylation of, again, the three methyl groups at Lysine residue number 27 on his-
tone 3 (“H3K27me3”), which in turn regulates the expression of the sex-determining
gene Dmrt1. Two genetically identical eggs can give rise to male progeny at 26 °C
or female progeny at 32 °C, due to epigenetic regulation induced by the environment
[37].
This example shows that epigenetic regulations play a prominent role in the devel-
opment of higher organisms. Unless mutations are introduced, all our body cells
contain the same DNA sequence. The differences in cellular identity originate from
the fact which genetic information is expressed, and which genetic information is
repressed—either through chromatin states or regulatory RNAs [31].
Evidently, the regulation of gene expression at different levels adds plasticity to
the possible programs exerted by the genetic code, which gave rise to complexity
and diversity during both, development and evolution.
Summary
• Regulatory RNA molecules can directly interfere with gene expression, e.g.
by binding of miRNA to mRNA, inhibiting their translation or inducing their
degradation.
• Proteins are extensively modified upon translation, which influences their
activity and stability.
• Epigenetic modifications occur on the level of DNA (e.g. methylation) and
histones (e.g. acetylation).
Membranes and Intracellular
Transport 5
Contents
5.1 Composition and Assembly of Cellular Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
5.2 Targeted Protein Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5.3 Vesicular Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Introduction
All reactions that occur within a cell can proceed more efficiently if they are spatially
separated from each other. Emergence of life from a self-replicating compartment
required, besides the hereditary material, a reaction chamber. The walls of this reac-
tion chamber are membranes made out of lipids. But these membranes do not only
separate the intracellular reactions from the exterior but also wrap cellular cargo in
vesicles for shipping within the cell or secretion to the outside (Fig. 5.1). As both,
plasma membranes and cellular vesicles, are composed of lipids, they can be regu-
lated together. To secrete the material they carry to the extracellular space, vesicles
can be fused with the plasma membrane. The reverse reaction takes place, too, if
vesicles are budding from cellular membranes to take up cargo for transport and
processing.
There is a dynamic exchange between the outside and the inside of a cell in
response to the environment. Intracellular transport demands frequent re-organization
of membranes. The adaptation of cellular compartment structures to fuel reactions
and production chains is essential for the cell to survive. The creation, merging
and budding of membranes are made possible by their biochemical composition.
Lipids are amphiphilic molecules, which means that, in part, they prefer both, aque-
ous and non-aqueous solutions. Therefore, they assemble into bilayers (Fig. 5.1) to
protect their hydrophobic part from water and expose the hydrophilic part to the
surrounding aqueous solution. Regulatory components are additionally required to
orchestrate membrane exchange and intracellular trafficking. These processes are an
intriguing subject because they highlight once more that complex biological systems

https://doi.org/10.1007/978-3-662-65357-9_5
50 5 Membranes and Intracellular Transport
Fig. 5.1 Lipid bilayers form

and separate cellular exterior
from the interior thereby
creating reaction
compartments
can be assembled from rather simple building blocks yielding sophisticated molec-
ular apparatuses such as the endoplasmatic reticulum (ER) made out of lipids for
specific modifications of proteins.
5.1 Composition and Assembly of Cellular Membranes
The cell is a biochemical container full of specific molecules reacting together. There
are separating borders to prevent reactions of these components with molecules of
separate compartments or the outside medium. These reaction barriers are composed
of lipids in a so-called bilayer. Two ply sheets of lipids are penetrated by proteins
to allow the targeted import and export of molecules for information exchange. The
composition and dynamic assembly of cellular membranes is a vital task that is
carried out by all living cells. The plasma membrane needs to be continuous and
elastic to enable integrity despite deformations and growth, which is achieved by the
biochemical properties of the membrane components.
Lipids consist of a polar head, which is therefore soluble in water. Phospholipids
such as phosphatidylcholine are a common membrane component. The phosphate
group at the head of phospholipid is negatively charged and forms electrostatic attrac-
tions with water molecules and is thus considered “hydrophilic”. This water-loving
side of the lipids is opposed by a pair of long water-fearing tails. These carbon chains
are neither charged nor polar and therefore not loving water but “hydrophobic”. If
lipids are put into an aqueous solution they will arrange in the energetically most
favourable way. This means the polar head will be exposed to water, whereas the
uncharged tail will shun water. The tails will form an inner core of two opposed
sheets of lipids with an outer shell of polar heads. To prevent exposure of water to
the tails at the edge of this lipid bilayer, the sheets have to bend and enclose in a
three-dimensional (3D) vesicle (Fig. 5.2).
The bended membrane has to seal its interior. The lipid bilayer has to be flexible
without rupturing. Whether a membrane is rather rigid or rather fluid results from
the tails of the individual lipids. These chains contain between 14 and 24 carbon
atoms. The tails of the most common lipids contain 18–20 lipids. These dimensions
build up membranes with an average thickness of 5 nm. If the tails of lipids are
shorter, interactions with one another are less likely, which makes the membrane-
less rigid. The fluidity is also influenced by the saturation of the carbon atoms in
the tails with hydrogen. If all carbon atoms are saturated, the chain is rather linear
and straight. If two sequential carbon atoms within the tail are unsaturated, double
bonds are formed between them, which introduces a kink in the chain. Phospholipids
5.2 Targeted Protein Transport 51
Fig. 5.2 A droplet-like vesicle with a bilayer shell is the energetically ideal 3D arrangement of
lipids in aqueous solution. Polar heads of lipids are exposed to H2 O, while the uncharged tails are
separated from water within the bilayer
Table 5.1 Summary of membrane properties. A densely packed membrane is rather rigid, which
could be due to long or saturated lipid tails or insertion of many cholesterol molecules. Loosely
packed, fluid membranes can occur because of many short or unsaturated lipid tails or only few
cholesterol molecules within the bilayer
Packaging Viscosity Lipid tails Bonds Cholesterol
insertions
Densely Rigid Long Saturated Many
Loosely Fluid Short Unsaturated Few
with unsaturated and therefore kinky chains cannot be packed as densely as lipids
with saturated and thus straight chains. Loosely packed lipids make a membrane
more fluid. The same holds true for cholesterol, one of the most common membrane
proteins. If cholesterol is inserted into the bilayer, it fills lateral gaps between the
phospholipids, which makes the membrane stiffer and less flexible. These properties
are summarized in Table 5.1.
5.2 Targeted Protein Transport
Lipid membranes form borders to separate compartments for specific reactions within
a cell. The nucleus is thought to be evolved from the plasma membrane that turned
inside into the cell to surround and separate the DNA. The process of a membrane
folding inwards is called invagination. This hypothesis for the creation of cellular
organelles is supported by the fact that the nuclear envelope and the ER are composed
of an outer and an inner membranes, which assemble through the invagination of
the plasma membrane. The space between the two membranes is called the lumen
and forms a continuum between nucleus and ER. Since mitochondria and chloro-
plasts are derived from procaryotes (see Sect. 1.3), parts of their protein synthesis
are independent. But in general, most of the proteins are synthesized at ribosomes
in the cytoplasm of the cell and need to be dispatched to their correct destination for
the maintenance of cellular functions and growth.
The address label is contained in the amino acid sequence of the proteins. It is
between 15 and 60 amino acids long and often but not always cleaved upon delivery.
The sequence, which directs the protein to the correct compartment, is called the
sorting signal. The physical properties such as charge and hydrophobicity are more
important than the exact sequence of the sorting signal. Sites of protein delivery can
be nucleus, mitochondria, chloroplasts, peroxisomes or ER. The protein remains in
the cytoplasm in absence of a sorting signal. Proteins destined for the nucleus are
delivered through nuclear pores, which are holes in the nuclear envelope. Proteins
that have to be transported across membranes to mitochondria, chloroplasts or the
ER need to unfold. Specific protein translocators facilitate this process. Proteins
shipped from the inside of the ER to the Golgi apparatus, lysosomes or endosomes
are ferried within vesicles.
5.2.1 Protein Transport through Nuclear Pores
The nucleus contains the DNA. For gene expression and its regulation (see Chap. 4),
molecules are shuttled in and out of the compartment with high frequency. The
nucleus is perforated with nuclear pore complexes that span the inner and the outer
membranes of the nucleus. More than 1000 translocations occur every second at
every single pore. Despite the fast rates, trafficking between cytoplasm and nucleus
is specifically regulated. The inner diameter of the pore is ≈ 40 nm, which allows
small, water-soluble molecules to pass freely. But larger proteins cannot simply
diffuse through the nuclear pore complex [53], which is composed of up to 1000
building blocks, so-called nucleoporins, from ≈ 30 different protein families [47].
Some polypeptide chains of the nucleoporins hang loosely into the inner space of
the pore thereby creating a diffusion barrier. Active transport through this meshwork
requires energy that is provided by the hydrolysis of GTP. This reaction is catalysed
by a small GTPase (see Sect. 4.3.1), named Ran. There is a concentration gradient
between Ran bound to GTP in the nucleus and Ran bound to guanosine diphosphate
(GDP) in the cytoplasm [79].
For a protein to be imported into the nucleus, the required sequence is the nuclear
localization signal. It is recognized by nuclear import receptors that mediate the way
through the fibrils of the nuclear pore complex. The nuclear import receptors bind
the protein with the nuclear localization signal and direct it through the pore to the
nucleus. Upon delivery of the cargo to the nucleus, the nuclear import receptor binds
to a Ran-GTP complex and is thereby shuttled back to the cytoplasm where the GTP
bound to Ran is hydrolysed to GDP and phosphate in presence of a GTPase-activating
protein (GAP). The resulting high concentration of Ran-GDP in the cytoplasm leads
to rapid recycling of the complex to the nucleus, where the GDP bound to Ran
is exchanged by another GTP. This reaction is catalysed by a guanine nucleotide
exchange factor (GEF) and maintains high amounts of Ran-GTP in the nucleus
thereby driving rapid export along the concentration gradient. Ran-GTP is shuttled
back from the nucleus into the cytoplasm (Fig. 5.3). Ran-GTP directed from the
nucleus to the cytoplasm can also carry proteins along that contain a sequence called
5.2 Targeted Protein Transport 53
cytoplasm
Ran-GDP + Pi
GAP
Ran-GTP
Ran-GDP
GEF
Ran-GDP Ran-GTP
Ran-GTP Pi
Ran-GTP
nucleus
Fig. 5.3 Nuclear import from the cytoplasm. Protein with nuclear localization signal is bound by
nuclear import receptor in the cytoplasm and dragged through a nuclear pore complex into the
nucleus. After the release of the cargo, the nuclear import receptor binds Ran-GTP. Export of this
complex is facilitated by the Ran-GTP concentration gradient
the nuclear export signal. Both targeted transport processes, the shuttling from the
cytoplasm to the nucleus and the export from the nucleus to the cytoplasm, were
recently shown to be light-inducible by biotechnological engineering [81,82].
5.2.2 Unfolding of Proteins for Mitochondria and Chloroplasts
Mitochondria and chloroplasts (see Sect. 1.3) contain their own DNA and thus pro-
duce some of their proteins by themselves. However, the majority of mitochondrial
and chloroplast genes are transcribed in the nucleus and translated in the cytoplasm
of the eukaryotic cells. These proteins need to be imported into the mitochondria
or chloroplasts for exertion of their specific tasks and maintenance of the organelle.
Unlike the folded proteins entering the nucleus through pores, transport through the
outer and inner membrane of mitochondria or chloroplasts requires proteins to be
unfolded. The polypeptide chain is directed through translocator proteins in the
outer and inner membrane like the thread through the eye of a needle. Chaperones
pull the imported proteins and help them to fold correctly once they reached the
inside.
For proteins to be translocated into mitochondria or chloroplasts, they need a
signal sequence, which is recognized by an import receptor. This sequence is cleaved
as the destination is reached and the mature protein is correctly folded. Transport
to a specific site within the organelle requires an additional sorting signal, which is
exposed upon the removal of the first sorting signal. Thereby a polypeptide chain can
e.g. be inserted into the inner membrane of mitochondria. Membranes are of vital
importance for the functionality of mitochondria and chloroplast. Phospholipids are
transported upon synthesis in the ER to proximal membranes of mitochondria and
chloroplasts by lipid-carrying proteins that enable distinctive compositions of these

membranes.
5.2.3 Co-Translational Import into the Endoplasmatic Reticulum
Proteins destined for the Golgi apparatus, endosomes, lysosomes, the plasma mem-
brane or the cell’s exterior are all shuttled through the ER. All proteins that are to be
transported there will enter the ER lumen, which is the interior space of the organelle.
Transmembrane proteins will be incorporated into the ER membrane. Respective
proteins are imported into the ER from the cytoplasm and will never be returning to
the cytoplasm along their journey to their target compartment. To be transferred to
the ER, proteins need a sorting signal sequence at their N-terminus. The mRNA is
translated in 5’-3’ direction by ribosomes into nascent polypeptide chains from the
N-terminus to the C-terminus so that the sorting signal is synthesized at the begin-
ning. Once exposed, the sorting signal is recognized by a signal recognition particle
(SRP). Binding of the SRP to the nascent polypeptide chain slows down translation
and drags the complex to the ER where the SRP engages with an SRP receptor.
Upon this interaction, the polypeptide chain is threaded across the ER membrane
through a channel of a translocator protein in the vicinity of the SRP receptor. The
translocation occurs while the protein synthesis is resumed by the ribosome at the ER
membrane. Co-translational import of nascent polypeptide chains from ribosomes
into the ER led to the observation of the rough ER, which is membranes covered by
ribosomes that produce proteins destined for intracellular trafficking or secretion.
Transmembrane proteins are not fully pulled through the translocator protein in the
ER membrane. Instead, they contain a stop-transfer sequence, which is hydropho-
bic and therefore stalls the translocation process. The stop-transfer sequence becomes
the membrane-spanning domain that is released from the translocator protein channel
laterally into the lipid bilayer of the ER membrane. The N-terminus of this protein
is located in the ER lumen, while the C-terminal part remains in the cytoplasm. If a
transmembrane protein contains multiple membrane-spanning domains, hydropho-
bic pairs of ER sorting signal and stop-transfer sequence can be found within the
polypeptide chain. Post-translational incorporation of these proteins into the ER
membrane occurs similarly to the mechanism described above but without cleavage
of the sequences. In a stitching-like mechanism, the hydrophobic regions are dragged
into the translocator protein channel within the ER membrane and remain there while
the rest of the polypeptide chain is pulled through.
Both, water-soluble molecules ending up in the ER lumen and transmembrane
proteins embedded in the lipid bilayer of the ER, can be further transported. As
the ER, the Golgi apparatus, lysosomes, peroxisomes and endosomes are forming
an interconnected network of membrane-enclosed organelles, they are summarized
as the endomembrane system. No matter if proteins are secreted from the cell
targeted to the lumen of another organelle of the endomembrane system or the plasma
membrane, onward-trafficking from the ER involves cellular vesicles.
5.3 Vesicular Transport 55
5.3 Vesicular Transport
Vesicles constitute the intracellular shipping and delivery system. Senders and recip-
ients are always different membranes of the endomembrane system. Cargo is protein
within the lumen of the vesicles. Transportation has to be specific in three ways:
1. Only proteins that need to be delivered should be wrapped by a lipid bilayer inside
a vesicle.
2. The composition of vesicles should preserve the integrity of donor and target
compartment.
3. Fusion of vesicles with target membranes should be directed to ensure delivery
only at the correct destination.
The composition of vesicles and target membranes allows for high specificity. First,
vesicles exhibit a distinctive protein coat on their outside. The protein clathrin is
the main component of so called clathrin-coated vesicles. This type of vesicle is
best-studied and therefore, budding is well understood. The outer clathrin coat is
connected via an adaptor protein called adaptin to specific receptors that span the
lipid bilayer of the vesicle facing the inside to bind the cargo molecules, which
display a transport signal. Once the cargo is bound by the receptor and the receptor
is trapped by the adaptin that connects it with the clathrin coat, the vesicle starts to
shape. When budding from the membrane of the donor compartment, a pit is formed,
and finally, the vesicle is cut loose by a spiral-shaped protein called dynein. Pinching
off the vesicle from the donor compartment requires energy as the lipid bilayer needs
to be torn apart. The energy is provided by GTP hydrolysis, a process catalysed by
dynein, which is therefore a GTPase. Once the vesicle is released to the cytoplasm,
the clathrin coat is removed to enable fusion with target compartments.
Docking is a precondition for delivery. Matching the vesicles to the correct target
membrane is ensured by Rab proteins and tethering factors. Rab proteins are
GTPases that are specific to the cytoplasmic surface of the vesicle. Unique sets of
Rab proteins are recognized by corresponding tethering factors. If there is a correct
match between vesicle and target membrane, the Rab proteins are captured by the
tethering factor. Additional strength in this interaction is provided by transmembrane
proteins called SNAREs. A v-SNARE of the vesicle winds up with a t-SNARE from
the target membrane thereby acting like a winch, pulling the vesicle close to the
target membrane, enabling fusion of the lipid bilayers. Upon membrane fusion, cargo
proteins inside the vesicles are released into the lumen of the target compartment.
5.3.1 Glycosylation of Proteins and Quality Control
Prior to onward transport, most of the proteins are chemically modified in the ER.
Contrary to the cytoplasm, the environment in the ER is reducing, which results in
the formation of disulfide bonds between reduced thiol groups from cysteine side
chains. These bonds stabilize the protein before it is incorporated into the plasma
membrane or secreted into the extracellular space. Rearrangements in S-S bonds

are catalysed protein disulfide isomerases (PDIs). Another chemical modification
of proteins in the ER is the attachment of sugar molecules. The process of adding
sugars to proteins is called glycosylation and serves several purposes:
• Increase of protein stability by protection from proteolysis.

• Change of solubility and affinity as oligosaccharides are hydrophilic.
• Quality control and labelling because chaperones can hold back proteins.
To fulfil these diverse functions, glycosylation patterns must exhibit a remarkable

diversity, which in fact they do despite maturing from a single precursor. Sugars are
not added as monosaccharides one by one but rather a preformed oligosaccharide
consisting of 14 sugar molecules that is covalently linked to the protein at once.
Dolichol is a membrane lipid that faces the lumen of the ER with two phosphate
groups and provides the sugar precursor. The oligosaccharide is transferred to the
amino group of an asparagine within the polypeptide chain as it enters the ER through
a translocator protein. The sugar transfer from dolichol to asparagine is catalysed
by an oligosaccharyltransferase (OST), which is only active in the lumen of the ER.
In subsequent reactions, the sugar precursor of the glycosylated protein is further
processed in the ER and the Golgi apparatus.
Proteins are not shipped onward from the ER unless folded correctly. There are
different chaperone molecules that steer correct folding of proteins in the ER. If
folding however fails, proteins are exported back to cytoplasm in a process known as
ER-associated protein degradation (ERAD). In case incorrectly folded proteins still
accumulate in the ER, the unfolded protein response (UPR) is triggered. Accumu-
lating protein is recognized in the ER by transmembrane protein kinases that facilitate
the expression of additional chaperone genes while decelerating translation of other
proteins by trapping ribosomes for the co-translational import of chaperones into the
ER. As soon as proteins are folded correctly, and as long as they do not carry an ER
retention signal, they are packaged into vesicles and exit the ER towards the Golgi
apparatus.
The Golgi apparatus is a multi-layered structure with a polar morphology con-
sisting of several flattened sacks called “cisternae”. The side of the Golgi apparatus
facing the ER is called cis-Golgi apparatus, opposed to the part facing the plasma
membrane, which is called trans-Golgi network. Vesicles fusing to the cis-Golgi
apparatus are originating from the ER. Vesicular transport from the ER to the cis-
Golgi apparatus is referred to as anterograde trafficking. Cargo is immediately sent
back if proteins contain the ER retention signal. This reverse route is referred to as
retrograde trafficking. Within the Golgi apparatus, proteins are sorted according to
their glycosylation patterns that are further processed, while proteins are migrated
through the cisternae from the cis-Golgi apparatus to the trans-Golgi network. The
sugar molecules linked to the proteins serve as a barcode that directs them to their des-
tination. Vesicles budding from the trans-Golgi network are either directed through
the early and late endosome to the lysosome for digestion, or they are sent within
secretory vesicles to the plasma membrane. Vesicles with transmembrane proteins
Fig. 5.4 Vesicles are

transported from the ER to
the Golgi apparatus
(anterograde trafficking) and
back (retrograde trafficking).
At the Golgi apparatus,
secretory vesicles are
budding that are delivered to
the plasma membrane and
cell exterior. Vesicles are
also transported from the
Golgi apparatus to the early
endosome. The late
endosome develops from the
early endosome, which is
exchanging material with the
plasma membrane. From the
late endosome, cargo is
eventually transported to the
lysosome for degradation
can be incorporated into the plasma membrane. Cargo within secretory vesicles or
from vesicles of the early endosome can be released into the extracellular space. With-
drawal of receptors from the plasma membrane or uptake of extracellular material
occurs by inward budding of vesicles from the plasma membrane. Early endosomes
are created that mature into late endosomes, which transport cargo to lysosomes for
digestion. Transportation routes of vesicles form a directed network with several
intermediate stops (Fig. 5.4).
5.3.2 Exo- and Endocytosis
Along the major routes of vesicular transportation, two main directions can be dis-
criminated:
1. Exocytosis: cytoplasm → Golgi apparatus → plasma membrane and

2. Endocytosis: plasma membrane → endosome → lysosome
For transmembrane proteins to be incorporated into the plasma membrane or proteins

to be released into the extracellular space, the secretory pathway is followed in the
process of exocytosis. The address label that directs proteins to the plasma membrane
is a diacidic motif: DXD or DXE, where D stands for aspartic acid, E stands for
glutamic acid and X can be any amino acid but proline. This motif is recognized
in the Golgi apparatus and wrapped in secretory vesicles that fuse with the plasma
membrane. There is a side-path from the trans-Golgi network to the cell periphery.
Proteins that are glycosylated with mannose 6-phosphate are shipped through early
and late endosomes to the lysosome. This compartment is thus provided with many
different hydrolysing enzymes for the digestion of proteins, nucleic acids, sugars and
lipids. Thereby building blocks for cellular components (see Sect. 1.1) are recycled
into the cytoplasm.
The lysosome is loaded with material for digestion and recycling from vesicles that
are budding from the plasma membrane in the process of endocytosis. During vesicle
formation, all membrane components are non-specifically taken up in the lipid bilayer
of developing early endosomes. Maturation of late endosomes involves activity of
ATP-driven H+ pumps to maintain low pH within the endosome compartment. The
acidic environment leads to the release of cargo from membrane-bound receptors,
which can therefore be recycled directly back to the plasma membrane or degraded
in the lysosome. Eventual uptake of membrane components by endocytosis needs
to be balanced with membrane fusion of secretory vesicles to maintain the overall
surface area of the plasma membrane constant.
A prominent example of receptor-mediated endocytosis is the uptake of choles-
terol, an important component of cellular membranes. Cholesterol is secreted from
the liver into the blood stream. Cholesterol is surrounded by particles of low-density
lipoprotein (LDL) because it is water-insoluble. LDL is recognized by specific recep-
tors on the cell surface and thereupon taken up into clathrin-coated vesicles. Due to
the increased acidity in the endosomes, LDL particles are released from the recep-
tors. In the lysosome, LDL particles are broken down by hydrolysing enzymes and
the cholesterol molecules are freed into the cytoplasm for new membrane synthesis.
The cell’s ability to regulate vesicular fluxes between the compartments is reflected
by the three vesicle types that can be discriminated by their coat proteins. Besides
clathrin-coated vesicles, there are two other types of vesicles: COP-I vesicles and
COP-II vesicles. COP stands for coat protein. These three types of vesicles differ in
their donor compartments, from which they are budding, and their target compart-
ments, to which they are fusing (Table 5.2).
Table 5.2 Vesicle types, their donor and target compartments

Vesicle type Donor compartment Target compartment
Clathrin trans-Golgi network Late endosome
Clathrin Plasma membrane Early endosome
Clathrin Early endosome Late endosome
Clathrin Secretory vesicle trans-Golgi network
COP-I cis-Golgi apparatus trans-Golgi network
COP-I trans-Golgi network Plasma membrane
COP-I cis-Golgi apparatus Endoplasmatic reticulum
COP-II Endoplasmatic reticulum cis-Golgi apparatus
Procedure
Several technologies exist to identify and follow transport processes of proteins.
• There are conditional mutant strains of yeast, in which some proteins become
inactive upon a temperature shift. As these mutant proteins are involved in
transportation, respective processes are stalled once the temperature changes.
For instance, proteins would accumulate in the ER, if the mutant were playing
a role in COP-II mediated shuttling between ER and cis-Golgi apparatus.
• Proteins in certain organelles can be detected after radioactive labelling.
Therefore, the polypeptides are translated in a cell-free system in vitro using
radioactive amino acids. The labelled proteins can subsequently be incubated
with isolated organelles. Co-sedimentation with these organelles in a centrifu-
gation gradient implies that labelled proteins are indeed imported into the
respective organelle.
• In contrast to the artificial cell-free system with protein synthesis from radioac-
tive amino acids, the green fluorescent protein (GFP) allows to trace trans-
portation processes in living cells by live-cell microscopy. GFP can be tagged
by means of genetic engineering to potential cargo. As GFP is rather small, it
hardly interferes with the function of the tagged protein [104]. GFP “glows”
in green upon excitation with UV light, thus tagged proteins can be followed
along the transportation routes within the cell.

Summary
• Cellular membranes consist of a lipid bilayer, and the more long and saturated
lipid tails they contain, and the more cholesterol is inserted, the more rigid are
these membranes.
• Proteins are shuttled between nucleus and cytoplasm through nuclear pore
complexes.
• Vesicular transport is the major way of intracellular trafficking, exocytosis and
endocytosis.
Signalling
6
Contents
6.1 Molecular Switches between On- and Off-State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
6.2 Molecules and Physicochemical Properties as Input from the Sender . . . . . . . . . . . . . . . . . 64
6.3 Receiving Cues via Cellular Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6.4 Signal Conversion in Protein Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6.5 Characteristic Motifs and Behaviours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
6.6 Emergent Properties as Output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
6.7 Signal Input, Processing and Output Happens on Various Levels . . . . . . . . . . . . . . . . . . . . . 73
Introduction
Signalling is a non-biological paradigm for information processing. Biologists bor-
rowed concepts from foreign disciplines, e.g. mechanical engineering, to understand
the signal transduction of living systems. Conceptually, all cells communicate, in
other words: they exchange information. Such processes exist on many different
levels in space and time [61] and exhibit stunning parallels to information process-
ing in technical devices (Table 6.1). A good source for spatial and temporal scales
in biology is http://bionumbers.org. On the intercellular level, information is sent,
received and processed between cells (Fig. 6.1). This concept applies, for instance,
when the hormone insulin is released to inform other cells to increase sugar uptake.
Likewise, all cells in our body serve as information processing units, which could
be described as living computers (http://stanford.io/1qGnvrn).
The conversion from the input signal (S) to the output response (R) in biol-
ogy is most commonly classified as either linear, hyperbolic or s-shaped functions
(Fig. 6.2). The functional relation between S and R depends on the type of biological
data and the measurement technique. Linear correlation is expected for protein abun-
dance vs. messenger ribonucleic acid (mRNA) abundance in the physiological range
because more mRNA should result in more protein. However, there is a tight and non-
linear interconnection between mRNA and protein regulation (see Chap. 4). Hyper-
bolic curves resemble enzymatic processes (see Sect. 4.3.1). A biological processor
with a steep input-output conversion acts as a switch. If a switch-like mechanism is

https://doi.org/10.1007/978-3-662-65357-9_6
62 6 Signalling
Table 6.1 Networks deal with information. Parallels from technology to biology can be found at
various levels
Technology Biology
Internet Population
Computer Organism
Hardware Tissues
Microprocessors Cells
Logic section Compartments
Transistors Molecules
inter-cellular signalling inter-cellular signalling
input output
intra-
infor- infor-
decoding cellular encoding
mation mation
signalling
signal S response R
sender cell receiver/sender cell receiver cell
Fig. 6.1 Inter- and intracellular signalling. A sender cell sends information that are decoded as
an input signal S by receiver cells. Intracellular signalling events process the input and encode an
output response R that is sent as information to another receiver cell
R biological
data
linear
fit
hyperbolic
fit
s-shaped
fit
Fig. 6.2 Functional relations between the input signal S and output response R can be linear,
hyperbolic or s-shaped, given experimental data (circles). Best fit depends on the biological context
investigated (see Sect. 6.5), or the detection of the output is insensitive to low S and
saturates for high S, s-shaped curves will be recorded.
6.1 Molecular Switches between On- and Off-State
Apart from the information processing behaviour of entire cells, biological switches
can be found on the molecular level. Inside cells, switches are molecules that can be
toggled between the on-state (i.e. active) and the off-state (i.e. inactive) by binding or
unbinding other molecules. Binding and unbinding can both, activate or inactivate,
depending on the binding partners. The phosphate molecule (PO3− 4 ), for example,
6.1 Molecular Switches between On- and Off-State 63
Fig. 6.3 Molecular switch phosphate- phosphate-

unbound state phosphatase: bound state
depicted as a lever between a de-phosphorylation
molecular phosphate
(PO3−
4 )-unbound state (left) kinase:
and phosphate-bound state phosphorylation
(right). Converting enzymes molecule
for de-/phosphorylation are molecule PO43-
called phosphatases/kinases PO43-
triggers the switching process. Enzymes that tack a phosphate onto another molecule
are called kinases, whereas enzymes that pluck off the phosphate are called phos-
phatases (Fig. 6.3). An example for activation through phosphorylation (i.e. addition
of PO3−4 ) is the transcription factor signal transducer and activator of transcription 5
(STAT5) (see Sect. 6.4) with one of its kinases Janus kinase 2 (JAK2) (i.e. activator)
and phosphatases (i.e. de-activators) in the nucleus.
Guanosine triphosphate (GTP) and GDP work similarly to phosphate. Molecules
can become active or inactive by being associated with GTP or GDP. G-protein
coupled receptors serve as a paradigm (see Sect. 6.3.2). The conversion from GTP to
GDP by GTPases (see Sect. 4.3.1; e.g. Ras as link to the Raf-MEK-ERK cascade, see
Sect. 6.4.2) is called hydrolysis (as a water molecule is incorporated and a phosphate
group is released). The reverse reaction is realized by an exchange of GDP with a
new molecule of GTP. The catalysing enzyme for the latter substitution is classified
as a guanine nucleotide exchange factor (GEF) like for the shuttling of molecules
between cytoplasm and nucleus (see Sect. 5.2.1).
These regulatory principles are common throughout the entire signal transduction
network, ranging from the perception of the input signal to processing events and
the final output response (Fig. 6.4).
signal signal transduction response

input information processing output
gene expression
receptor
metabolism
second
messenger cell architecture
extra- intra-
cellular cellular
conversion/
amplification integration alteration
decoding
Fig. 6.4 Overview of signal transduction. Extracellular molecules can be sensed by receptors that
convert the information input into an intracellular cue. Second messengers amplify the relayed
signal and multiple signalling branches can be integrated to finally alter cellular structures and
processes in response. Adapted from [6]
64 6 Signalling
6.2 Molecules and Physicochemical Properties as Input

from the Sender
The first layer of signal processing in biology is the information input. Diverse stimuli
can be classified.
• Signalling molecules can serve as mediators of communication between cells

(i.e. intercellular), such as hormones, growth factors and cytokines. Prominent
examples are insulin and erythropoietin (Epo).
• Metabolic compounds play a role, too. Glucose, amino acids or adenosine triphos-
phate (ATP) are a valid readout for conditions within cells (i.e. intracellular).
• Chemicals (acids [H+ ], bases [OH− ], metal ions [Fe2+ /Fe3+ ] and radicals [· O− 2 ])
characterize the cellular environment. Spatial surroundings could be acidic (cells
in sour medium), or hypoxic (low-oxygen stress mediated through O2 radicals).
• Physical properties such as light, temperature or pressure can also serve as sig-
nalling cues (e.g. heat shock in bacteria).
An example of mechanical confinements as input that influences emergent proper-

ties (see Sect. 6.6) is the physical pressure that seems to be the reason for altered
(a)symmetry of cell divisions in mouse cells [103], though the precise mechanism
remains to be resolved.
6.3 Receiving Cues via Cellular Receptors
The way incoming information is processed by a cell crucially depends on the layer
of perception. Input is sensed by molecules called receptors that usually convert the
signal from the outside of the cell (i.e. extracellular) to the inner, cytoplasmic part (i.e.
intracellular). Some receptors however sense their ligands and trigger intracellular
signalling from the cytoplasm only. Input molecules recognized by the receptor are
called ligands.
There are three main types of receptors that can be discriminated by their structure
and thus their functions. In the following, the three types will be introduced separately.
6.3.1 Ion Channels Maintain the Flow of Ions and Thus Information
Ion channels pass ions through the membrane with a simple pore-like structure. The
driving force is the concentration gradient of the ions across the membrane. Ions are
charged and thus create an electric potential. There is no need for an active transport.
Channels can always be open, with ions diffusing passively through, or the pore
opening and closing can be regulated.
An example is the sensation of salty (i.e. sodium chloride NaCl) taste. Our tongue
is full of taste buds that contain taste receptor cells with so-called epithelial sodium
ion (Na+ ) channels. Those channels are always open and sodium ions from salty
6.3 Receiving Cues via Cellular Receptors 65
food diffuse through the pores into the cell to level out the concentration gradient
(extracellular: high Na+ ↔ intracellular: low Na+ ). The sodium influx induces the
opening of calcium ion (Ca2+ ) channels (taste signal transduction reviewed in [19,
69]). Subsequent diffusion of Ca2+ into the cells leads to membrane depolarization
(i. e. inversion of the electric potential across the cell membrane). This electric signal
(i.e. action potential) can then be transmitted to the brain, which decodes the taste
from the activation pattern of the taste buds’ cells.
6.3.2 G-Protein Coupled Receptors Enable Switching Mechanisms
G-protein coupled receptors are capable of activating effector domains upon stim-
ulation. Besides the extracellular part, which recognizes the ligand, they always
contain seven transmembrane domains that penetrate the cellular envelope. On the
cytoplasmic side of the membrane, they are associated with a GTP-binding protein
(i.e. the “G-protein”). The activation process of G-protein coupled receptors can be
summarized as follows:
ligand binding to the receptor

↓
conformational change in the structure of the receptor
↓
G-protein binding to the receptor
↓
GDP-GTP exchange (i.e. G-protein activation)
↓
subsequent target activation by the G-protein
The exchange between GDP and GTP provides a molecular trigger for intracellular
signalling (see Sect. 6.1).
6.3.3 Enzyme-Coupled Receptors Regulate Complex Processes
Enzyme-coupled receptors either exhibit their own enzymatic activity or they are
situated adjacent to enzymes. The purpose is to integrate different signalling inputs,
which is strengthened by the fact that
#(receptor types) #(ligands). (6.1)
The combinatorial complexity and structural diversity of receptor complexes allows

enzyme-coupled receptors to fulfil a myriad of functions. They are very sensitive and
can be activated even by concentrations of growth factors in the pico-molar range
(i.e. a single growth-factor molecule within more than a thousand trillion water
molecules). The receptors are most commonly coupled to kinases, e.g. JAK2 for the
66 6 Signalling
erythropoietin (see Sect. 6.2) receptor (EpoR), which exhibits a K D (i.e. dissociation
constant) for Epo of 1.64 · 10−10 M [12]. This means, in equilibrium, the molar
ratio of the concentration of unbound ligand [Epo] and free receptor [EpoR] to the
concentration of ligand-receptor complex [Epo-EpoR] is K D :

Epo · EpoR
KD = . (6.2)
Epo-EpoR
If an enzyme-coupled receptor possesses intrinsic enzymatic activity, it is most

commonly a tyrosine kinase. This means they are capable of transferring a phosphate
group to tyrosine residues of other proteins (see Fig. 6.3). Tyrosine kinase receptors
form dimers of single membrane-spanning domains, which phosphorylate each other.
Phosphorylated tyrosine residues on the intracellular tails of the receptors serve as
anchor points for adaptor molecules that relay the signal.
6.4 Signal Conversion in Protein Networks
The topological feature that diverse extracellular cues are integrated by a rather small
set of core components is referred to as bow-tie structure [58] (Fig. 6.5).
Intracellular molecules are capable of amplifying, integrating or distributing the
signal (Fig. 6.4). Three different transduction features can be classified, a combina-
tion of which enables regulatory complexity.
6.4.1 Second Messengers to Spread Information Rapidly
As the name implies, second messengers are a class of molecules that forms the
next instance of signal transduction beyond the receptor. Second messengers are
mainly induced via G-protein coupled receptors (see Sect. 6.3.2). Membrane-bound
components are recruited and small molecules are subsequently released and diffuse
quickly through the cytoplasm to spread the information. Some components serve
as paradigms that should be known, e.g.:
• phosphoinositide 3-kinase (PI3K), producing

– phosphatidylinositol 3,4 bisphoshpate (PIP2 ) and
Fig. 6.5 Bow-tie structure of

signal transduction
components. A
diversity
heterogeneous ensemble of PI3K/AKT

MAPK/ERK
receptors feeds into a set of JAK/STAT
four core branches that divert NFκB
into a multitude of various
effectors and transcription
factors signalling
receptors molecules effectors
6.4 Signal Conversion in Protein Networks 67
– phosphatidylinositol 3,4,5 trisphoshpate (PIP3 ),

serves as a model system to understand regulatory features of tumour suppression
(reviewed in [23]). Or
• phospholipase C, producing
– diacylglycerol and
– inositol 3,4,5 trisphoshpate (InsP3),
which is in turn responsible for Ca2+ waves [64] that are visualized particularly
for fertilization http://bit.ly/1s22ycg (see Sect. 7.1).
A paradigm for a soluble second messenger that is not bound to the membrane
but diffuses within the cytoplasm is the adenylate cyclase, which produces cyclic
adenosine monophosphate (cAMP) that has been implicated, for instance, in nutrient
sensing of yeast [101].
6.4.2 Kinase Cascades for Signal Relay
Connecting multiple kinases in a linear chain offers additional layers of interference

for fine-tuning. Consecutive phosphorylation is sometimes facilitated by a scaffold
protein, which keeps the kinases in spatial proximity. Each kinase can be regulated by
incoming (e.g. negative feedbacks, see Sect. 6.5) and outgoing (i.e. additional target
substrates) connections. In a prominent case, the hierarchy of kinases within the
cascade can be inferred by the name: The extracellular signal-regulated kinase (ERK)
is a mitogen-activated protein kinase (MAPK), while its kinase, mitogen/extracellular
signal-regulated kinase (MEK), is a MAPK kinase (MAPKK), and the kinase of MEK
is Raf, a MAPKK kinase (MAPKKK).
This kinase cascade (less Ks mean substrate) is involved in proliferation of blood
progenitor cells in mice [94].
6.4.3 Transcription Factors Control Gene Expression
Downstream to receptors and possible second messengers in the signal transduction

hierarchy, there are proteins modulating the initiation of transcription (see Chap. 4)
that are referred to as transcription factors. They can represent a direct link from the
receptor to target-gene expression. Upon signalling, transcription factors are shuttled
to the nucleus and bind to the DNA. The order of signalling events is as immediate
as follows:
receptor activation
↓
transcription factor activation
↓
shuttling from the cytoplasm to the nucleus
↓
target-gene control
68 6 Signalling
Such a route is direct and thus highly efficient and specific without intermediate
branching steps. The EpoR-JAK2-STAT5 axis serves as a paradigm in this respect.
Despite the immediate link from the receptor EpoR with its adjacent kinase JAK2
to the transcription factor STAT5, there is the possibility for regulatory mecha-
nisms. STAT5 is activated through phosphorylation (see Fig. 6.3), and two activated
molecules form a complex. These dimer structures exhibit distinctive transport and
DNA-binding kinetics as a base for mechanistic diversity [16].
6.5 Characteristic Motifs and Behaviours
The elements of signal transduction that were introduced above are highly inter-
connected in molecular networks. The wiring (i.e. the network structure) encodes
distinctive functions in motifs and sub-modules. Pathways must not be thought of as
linear chains. There are many interconnections. Feedback loops are either positive
or negative with respect to the conversion of the input signal S to the response R
(Fig. 6.6). Positive feedback loops further increase R with increasing S, while R is
reduced in negative feedback loops by rising S.
These loops render molecular pathways non-linear. The shape of signal-response
curves depends on the topology of the underlying network motif. The structure of
the sub-modules and their respective function are intimately related and gave name
to exemplary “sniffers”, “buzzers” and “toggles” [106].
A sniffer for example is a motif that responds transiently to signal input, but the
steady state of the response R is independent of the signal S. The sniffer is named after
the molecular pathway responsible for the perception of odour within the sensory
neuronal cells of our nose. Its structure (Fig. 6.7A) entails a negative feedback loop
because the component X that is induced by the signal input S diminishes the response
R. More S results in less R, which indicates a negative feedback. In simple terms:
For high input, the systems show a transiently high response. But high S results
in high X and a correspondingly strong reduction of R, which thus always returns
to the same steady state (Fig. 6.7B). This return is called “adaptation”. The system
adapts to the input signal and its response returns to basal activity. This feature can
be further exploited as the model files are provided as online material to be simulated
with the freely available D2D Software.
nothing reaction catalysis inhibition
a b
S S S
R R R
X X X
Fig. 6.6 Process diagrams of feedback loops. (A) Positive feedback loop further increases response
R with more signal S. Production of R is additionally catalysed by intermediate species X . (B)
Negative feedback either via inhibition of synthesis of R through X (left motif) or degradation of
R catalysed by X (right motif). Visualisation adopted from [59]
6.5 Characteristic Motifs and Behaviours 69
a b Signal S Variable X Response R
2 2 1.8
concentration [nM]
concentration [nM]
concentration [nM]
1.5 1.5 1.6
1 1 1.4
S X
0.5 0.5 1.2
R
0 0 1
0 5 10 15 0 5 10 15 0 5 10 15
time [min] time [min] time [min]
Fig. 6.7 Sniffer network motif and dynamic behaviour. A) Signal input S induces production of
response R and production of intermediate species X , which catalyses the degradation of R. B)
Temporal evolution of the three components S, X and R. With more S, R is transiently increased
but reset by X to its basal steady-state level. Adopted from [106]
Thus, our sensory system is always reset to its basal minimum state and we adapt
even to strong smells. However, excitation (i.e. a response) occurs for every new
input smell. Similarly, bacteria sense nutrients in their surrounding. Adaptation is
an evolutionarily conserved property. Bacteria adapt to chemical gradients but can
respond to every new input via molecular feedback loops [1]. Thereby they robustly
sense nutrients and swim towards the attractant. Robustness in this context is defined
as a small change in the variable X for small changes in the parameter p:
δX
. (6.3)
δp
If the system’s parameter p underlies minor fluctuations, be it through some input

perturbation, the readout variable X remains unchanged. In contrast to robustness,
sensitivity means that the system responds to subtle changes in p with a large change
in X (Eq. 6.3). In summary:
• a system is robust if its output is not altered despite perturbation and

• a system is sensitive if small changes can be sensed and alter the output.
6.5.1 Signal-Response Curves
The term sensitivity is also used in the context of drug research. If the effect of a
newly developed drug is investigated, the response (of the cellular system, the animal
or the patient) to different doses of the substance is evaluated. That is why the signal-
response curve is referred to as a dose-response curve and sensitivity can be depicted
in such a curve.
A measure for sensitivity is the dose at which a half-maximal response is observed
(Fig. 6.8). This parameter is denoted as EC50 (i.e. half-maximal effective concen-
tration) [27] if the response increases with dose. In case an inhibitor is tested and
the response is thus diminished with increasing doses of the drug, the parameter is
called I C50 (i.e. half-maximal inhibitory concentration). Anyway, it signifies the
70 6 Signalling
R
max
H
half
amplitude
min
EC50
S
Fig. 6.8 Dose-response curve. Response R changes with input S between minimum min and
maximum max with slope H and a half-maximal effective concentration at S = EC50
dose at which half the response amplitude between minimum min and maximum
max is reached. A measure for the steepness of the curve is the Hill coefficient H
that indicates the level of cooperative effects of the drug. Cooperative binding would
mean that binding of a single ligand to the receptor facilitates binding of another
one. A formula for a four-parameter Hill equation with those parameters is
min − max
R = max · S −H . (6.4)
1 + EC50
The parameters can be estimated given experimental data and the dose-response
behaviour can be quantified. The sensitivity towards the drug indicates susceptibility
of the system towards the substance as well as the efficiency of the drug.
6.5.2 Time-Course Curves
Dose-response behaviour is no static property, it underlies a dynamic. The drug

needs to be taken up by the body (pharmacokinetics) and cells respond with time
(pharmacodynamics).1 Sensitivity (see Fig. 6.8) can be reflected in temporal profiles
of signal transduction, too. If one parameter fluctuates, the systemic variables might
also change. Furthermore, all biochemical processes are stochastic (Brownian motion
of particles that diffuse through a cell) and exhibit a certain degree of noise. Sources
of noise can be:
• intrinsic noise: fluctuations in cellular protein concentrations,

• extrinsic noise: alterations in kinetic rates of cellular processes and
• measurement noise: errors and imprecision during experimental proceedings and
data acquisition.
1 Pharmacokinetics is what your body does to the drug. Pharmacodynamics is what the drug does
to your body.
6.5 Characteristic Motifs and Behaviours 71
Signalling events are commonly investigated in time-course experiments via a tech-

nique called immunoblot or western blot.
Immunoblot aka. Western Blot

Cells are lysed—the membrane (see Chap. 5) is chemically disrupted—so that
their proteins become accessible. The cellular proteins are then separated by
size and transferred to a membrane where they can be detected via antibodies
(see Sect. 8.3.2). As readout serves the signal intensity for the detected protein
that is scanned from the membrane. The bigger the spot (called a “band”) of the
respective protein on the membrane, the stronger the corresponding biological
response.
In this exemplary case, mouse erythroid progenitor cells are stimulated with Epo and
the corresponding phosphorylation (i.e. activation) of STAT5 with time is measured.
Figure 6.9 shows the detected amounts of phosphorylated STAT5 (pSTAT5) at the
given time points. The bigger the spot (called a “band”), the more pSTAT5 is present
(Fig. 6.9). As the transcription factor is shuttled to the nucleus during its course of
activation (see Sect. 6.4.3), STAT5 was analysed in the cytoplasm and the whole-cell
lysate (i.e. cytoplasm + nucleus).
Once the measurement is quantified, the time-course curve can be plotted and
characterized (Fig. 6.10). The amount of the protein of interest at the beginning of
the experiment at t = 0 = t0 is referred to as its initial concentration or basal level.
Local maxima that are reached in the course of the experiment are called “peaks”
and the time until they are reached is the respective peak time. Afterwards, the
system usually adapts to a steady-state level. Dynamics can be either transient, if
the steady state is low (Fig. 6.10), or sustained, if the steady state remains high after
peak activation.
* * time
30 480 60 30 240 5 120 0 360 15 0 [min]
pSTAT5
P P
STAT5 STAT5 STAT5 STAT5
STAT5 STAT5
P P P P
STAT5 STAT5 STAT5 STAT5 STAT5
STAT5
P P STAT5
STAT5 STAT5 STAT5 STAT5 STAT5
Fig.6.9 Western blot data of phosphorylated STAT5 (pSTAT5) in whole-cell lysate (*) or cytoplasm
(otherwise). Erythroid progenitor cells from mice were stimulated with 5 U/ml Epo for indicated
times. Order of samples was randomized to reduce correlated errors [93]. The detected amount of
pSTAT5 was quantified and plotted in Fig. 6.10. Data from [4]
72 6 Signalling
8 whole-cell
log10(phosphorylated STAT5) [BLU]

peak lysate
cytoplasmic
lysate
7
steady-state
level
6
initial, basal
5 level
0 100 200 300 400 500

tp time [min]
Fig. 6.10 Time-course experiment. Erythroid progenitor cells from mice were stimulated with
5 U/ml Epo. The stimulation was terminated either via whole-cell or cytoplasmic lysis. The phos-
phorylation of STAT5 was analysed via western blot (Fig. 6.9). After the peak time t p of 15 min,
signalling returns to a low steady state on a logarithmic scale. BLU = Boehringer Light Units
STAT5 activation is transient because it is reset by negative feedback regula-

tors (see Sect. 6.5). Both cytokine-inducible SH2 domain-containing protein (CIS)
and suppressor of cytokine signalling 3 (SOCS3) inhibit STAT5 activation and thus
reduce the amount of pSTAT5 with time.
6.5.3 Complex Interactions
After the first peak, dynamics of biological systems are not always monotonous.
Temporal responses ddtR could resemble periodic behaviour. Oscillations occur if
certain preconditions are met [83]:
• feedback regulation (see Fig. 6.6),

• sufficient level of non-linearity and
• separated time scales.
Separated time scales mean that the reactions exhibit different velocities. Kinetics are
balanced and occur with a sufficient delay to allow changes that are about to be lev-
elled out. Due to dependence on the mentioned preconditions, oscillations occur only
in a subregion of the parameter space. An example of molecular oscillations is the
core signalling component (see Fig. 6.5) nuclear factor kappa-light-chain enhancer
of activated B cells (NFκB) that is known to cycle periodically between cytoplasm
and nucleus for the control of gene expression [77].
The interplay and interdependencies of molecular components become more com-
plex as the network size grows. Pathways exhibit crosstalk and sophisticated response
patterns are created [95]. Complexity is required because the outcome of signalling
events are decisions to alter cellular properties in response to the multi-parametric
environment, so the cell has to process and integrate several inputs (see Sect. 6.2) at
a time.
6.7 Signal Input, Processing and Output Happens on Various Levels 73
6.6 Emergent Properties as Output
Cells integrate multiple signals to choose their fate in terms of:
• survival or programmed cell death,

• proliferation or quiescence,
• migration or residence,
• metabolic alterations and
• differentiation.
The term “emergent property” means literally the outcome of signalling events.
Multiple properties have to be measured to assess the output upon information pro-
cessing. To decide, whether a cell undergoes programmed cell death, morphology
and molecular components need to be evaluated (see Sect. 9.2.1). In the exemplary
case of the EpoR-JAK2-STAT5 axis, the signalling input Epo is connected to the
output response of cell survival. Among the target genes of STAT5 are some factors
that inhibit cell death. But how is the amount of Epo that is sensed by the cells con-
verted to the alteration of gene expression? Fine regulation via the negative feedback
regulators, CIS and SOCS3, allows erythroid progenitor cells in mouse to respond
specifically to a wide range of Epo concentrations. By mathematical modelling, it
was shown that the amount of activated STAT5 (i. e. pSTAT5) in the nucleus of
the cells is a good predictor for the fraction of surviving cells in a population [10].
Cell morphology and different molecular components implicated in cell death were
measured to assess the emergent property of survival as an output response of the
erythroid progenitor cells to the input signal Epo.
6.7 Signal Input, Processing and Output Happens on Various

Levels
Cellular signalling can be diverse as many different components are involved and
interconnected. As introduced in Fig. 6.5, there are a few central components that
should be known as well as some core features of molecular networks (Fig. 6.6).
The overview given in Fig. 6.4 is completed by names for molecules that serve as
paradigms in Table 6.2, most of which were introduced in this chapter.
Table 6.2 Paradigms for different signalling pathway components. IN = input, REC = receptor,
KIN = kinase, 2nd = second messenger, INT = integrator, TF = transcription factor, TG = target,
OUT = output, REF = reference
IN REC KIN 2nd INT TF TG OUT REF
Epo EpoR JAK2 – – STAT5 CIS, SOCS3 Survival [10]
PI3K AKT – Cyclin D2, Cell cycle [3]
Cyclin G2
– ERK – – Proliferation [94]
74 6 Signalling
Summary
• Receptors sense information input into the cell by binding cognate ligand
molecules and triggering intracellular reactions.
• Information are being processed inside the cell through signal transduction
pathways.
• Inhibition and activation can be intertwined in feedback loops, which render
cellular output highly non-linear in space, time and dose-response.
Cell Cycle
7
Contents
7.1 Cell-Cycle Model Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
7.2 Checkpoints Regulating the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
7.3 Conceptual Views on Cell-Cycle Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Introduction
The basis for reproduction of all organisms is a process called the cell cycle [75].
As the name implies, the cell cycle is an iterative, periodic chain of events, in which
the cell undergoes repetitive cycles of growth and division. Proper timing and execu-
tion of the cell-cycle programme is vital for the development and maintenance (i.e.
homeostasis) of our tissues and organs. Malfunctioning of the cell cycle is connected
to uncontrolled growth and tumour formation. The cell cycle is therefore tightly reg-
ulated by cellular signal transduction networks (see Chap. 6). Several checkpoints
exist to ensure that the cell cycle only progresses if all processes are performed cor-
rectly. Unless the preconditions are fulfilled (e.g. doubling of the DNA before cell
division), the process is halted.
In principle, the cell cycle can be depicted as a circle (Fig. 7.1) with two clearly
defined events, namely DNA synthesis (S-phase) and mitosis (M-phase). In between
those two discrete events, the cell prepares for DNA doubling and division in so-
called gap (G)-phases. Throughout the cell cycle, the cell replicates its contents
e.g. membranes, cytoplasmic organelles, structural proteins, rRNA and mRNA. This
continuous process is difficult to observe compared to doubling of the chromosome
set during S-phase and the distribution of cellular mass to an upcoming pair of
daughter cells during M-phase. Thus the name G-phase is a bit misleading. In fact,
cells actively grow in the G1 phase if they are not interrupted at a quality control
checkpoint.

https://doi.org/10.1007/978-3-662-65357-9_7
76 7 Cell Cycle
Fig. 7.1 Overview of cell cycle. Two defined phases of mitosis (M) and DNA synthesis (S) are
interrupted by two gap phases (G1 and G2 ), in which the cell grows and prepares (during G1 ) for
replication of DNA, and (during G2 ) for cell division ( ). Length of cell-cycle phases adapted
for a 20 h cell cycle of HeLa cells from [42]
Besides accuracy, the correct timing of the cell cycle is essential. The length of the
individual phases needs to be well coordinated. Cells dividing too rapidly can develop
malignancies while slowly dividing cells can impair tissue regeneration following
lesions. It is very important to maintain the balance for fast and precise completion
of the cell cycle. What are the components that constitute the underlying molecular
machinery? The following section will shed light on the discovery of core cell cycle
components and their mode of action.
Conventions
Non-dividing cells are sometimes supposed to be arrested (i.e. stopped) in a
phase called G0 prior to entering cell cycle.
For convenience all phases except the M-phase are often summarized as
Interphase = G1 + S + G2 .
7.1 Cell-Cycle Model Systems
Many important observations regarding the cell cycle have been made in model
organisms such as yeast, fruit fly, nematode worm and frog. Table 7.1 gives an
overview of some species predominantly used in cell-cycle research.
Yeast Their genomes are simple, their doubling times are short and their tradition
as research subjects is long-standing, even though the two unicellular fungi, budding
yeast and fission yeast, exhibit slightly different mechanisms of division. Haploid
cells can be maintained in culture and genetically manipulated. Conditional mutants
were used to identify essential genes for cell-cycle progression. Genes were non-
functional under certain conditions, e.g. low temperature. These conditional mutants
were arrested in cell cycle when shifted to low temperature. Otherwise, the wild-type
cell-cycle progression was observed. Thus, factors were identified that are respon-
7.1 Cell-Cycle Model Systems 77
Table 7.1 Exemplifying species used in cell-cycle research and main advantages for their use
Organism Species Advantage
Budding yeast Saccharomyces cerevisiae Simple Eukaryote and straightforward
genetic analysis
Fission yeast Schizosaccharomyces pombe Simple Eukaryote and straightforward
genetic analysis
Fruit fly Drosophila melanogaster Cell cycle control mechanisms with
intermediate complexity
Nematode worm Caenorhabditis elegans Well studied for mitosis and division
Frog Xenopus laevis Giant unfertilized eggs, maternal
proteins in embryo
sible for chromosome duplication and segregation as yeast cells undergo repetitive
cycles of growth and division.
Fly The genome of the fruit fly contains ≈ 14,000 genes, twice as many as yeast but
only half the number of the human genome. Its chromosome set is diploid, which
makes genetic manipulation a bit more intricate than for haploid yeast. Changes in
cell cycles during the development of this multicellular organism make it nonetheless
a suitable subject to study control mechanisms. During the first rounds of cell cycle
after fertilization, only DNA and nuclei are doubled but neither cytoplasmic growth
nor cleavage is observed. Such a cell with multiple nuclei is called syncytium. Nuclei
locate at the outer rim of the embryo and are then encapsulated by membranes. This
process of cellularization depicts an important step during early divisions in the fruit
fly.
Frog The unfertilized egg spans a diameter of about 1 mm. It is possible to inject
various substances into this single cell or extract small parts and study them in a
test tube. This is how the maturation- or later mitosis-promoting factor (MPF) was
found. Mock fertilization of these eggs with an electrical pulse in a Petri dish can
trigger the release of calcium ions inside the cell (see Sect. 6.4.1).
Upon fusion of the haploid egg nucleus with the haploid sperm nucleus, the cells
of the early embryo grow rapidly because they are provided with maternal RNA and
protein as starting material.
7.1.1 Cellular Contexts to Study the Cell Cycle
Those model organisms are all suitable for the discovery of key, conserved mecha-
nisms due to their low complexity and the available analysis tools. Nonetheless, the
ultimate goal is the understanding of the mammalian cell cycle and finally interven-
tion in the context of deregulation and human diseases.
Sources of human material are limited. Biopsies provide healthy or diseased tissue
for patients and are thus very precious. As mice are genetically similar to humans
78 7 Cell Cycle
(≈ 85% sequence identity of protein-coding genes)1 , they are used as model organ-
isms for human disease such as type 1 diabetes [9]. Regulatory mechanisms of the
genomes were diverging during the evolution of the two lineages (only ≈ 50% of
non-coding DNA regions are shared between mouse and human) [21]. However,
newly established tools allow to study the function of certain genes in the living
animal (i.e. in vivo).
A tissue sample (so-called primary material) taken out (i.e. ex vivo) of the living
animal is only able to survive for a short time. The number of cell cycles and divisions
is limited. The environment during cultivation of the primary cells in a plastic dish (i.e.
in vitro) is too artificial. It does not resemble physiological conditions. Tumour cells
survive and divide almost infinitely even in vitro. They possibly acquired growth-
promoting mutations to become immortalized. Also, some non-tumour cells are
transformed so that they can sustain with serum and sufficient amounts of growth
factors in vitro. These cell lines can be expanded to generate enough biological
material for cell-cycle studies. Notably, such cell lines may exhibit up-regulated cell-
cycle-associated functions as they evolved towards rapid growth during cultivation
[85].
7.1.2 Discovery of Core Cell-Cycle Components
The Nobel Prize in Physiology or Medicine 2001 was awarded jointly to Leland
Hartwell, Tim Hunt and Paul Nurse for their discoveries of key regulatory compo-
nents of the cell cycle. They described the mode of action of cyclins and cyclin-
dependent kinases (CDKs) in regulation of DNA synthesis, chromosome separation
and cell division.
Hartwell identified cell division cycle (cdc) genes in budding yeast (Table 7.1). He
defined checkpoints for control of correct execution of preceding cell-cycle phases.
Nurse worked with fission yeast and found the gene cdc2 as a regulator for both,
transition from G1 to S-phase and transition from G2 to M-phase. The homologous
gene in budding yeast is CDC28 and the corresponding human gene is cdk1, a
CDK. Hunt discovered cyclins, the counterpart required for functioning CDKs. He
radioactively labelled those proteins and found them to be degraded periodically at
every M-phase in eggs of sea urchins.
While the concentrations of CDKs remain constant throughout the cell cycle,
cyclins, as their name implies, are repeatedly synthesized and degraded (Fig. 7.2).
CDKs depend in their activity on the presence of cyclins. Only if cyclins are bound to
CDKs, they can phosphorylate their substrates. This central mechanism is conserved
across species from yeast to insects, plants, animals and humans.
1 genome.gov/10001343, last opened March 10, 2022.

7.2 Checkpoints Regulating the Cell Cycle 79
Fig. 7.2 Concentrations of

cyclins and CDKs during
inter- and M-phase. While
cyclins are constantly
synthesized and degraded,
concentrations of CDKs do
not change, but their activity
depends on the presence of
cyclins
7.2 Checkpoints Regulating the Cell Cycle
In an ideal environment, unrestrained cell-cycle progression occurs. Cell cleavage

(i.e. division) without delay happens for cells of the early embryo, which do not
grow but rapidly progress from the 1-cell stage to the 26 = 64-cell stage. Such
accelerated divisions are also observed for cells (and unicellular organisms, e.g.
yeast) in culture because they are usually bathed in growth factors, and growth is
therefore not the rate-limiting step anymore [85]. Cell-cycle progression in tissues
of higher organisms is otherwise restricted to ensure maintenance of size and to
exert specific functions. The timing of the cell cycle is regulated by extracellular
and intracellular signalling cues. Not only nutrients and growth factors but also
physicochemical properties of the cellular environment compose regulatory factors
from the outside of the cell, as we have learned before (see Sect. 6.2). Intracellular
information consist of the signalling programme and gene expression status. For both,
extracellular and intracellular regulation of cell-cycle progression, exist a multitude
of involved components that differs in name and associated functions between species
and organisms.
Conceptually, the wheel of the progressing cell cycle (Fig. 7.1) is spinning unless
diverse braking mechanisms prevent the cells from cycling too fast. There is a restric-
tion point in the G1 -phase of the cell cycle that is often displayed as a point of no
return. It has been speculated that once all extracellular and intracellular requirements
for passing the restriction point are fulfilled, the cell completes the current cycle even
if growth factors are withdrawn. However, if the cell does not sense enough mito-
gens before passing the restriction point, it remains quiescent. Growth factors and
mitogens are occasionally distinguished by their function. Growth factors promote
growth and mitogens stimulate division. However, as we will see in Sect. 7.3, it is
sometimes hard to discriminate those two processes.
7.2.1 The Restriction Point Mediated by Rb and E2F
The all-or-none decision between cell-cycle progression or cell-cycle stop is achieved

by a switching mechanism (see Sect. 6.5) of the E2F protein [114]. E2F is a transcrip-
80 7 Cell Cycle
tion factor for genes involved in DNA replication and thus cell cycle progression to
S-phase. An important feature is the autocatalytic activity of E2F, which activates
transcription of its own gene. Such a positive feedback loop characterizes the bistable
switch between active and Rb-inactive E2F. Repression of E2F is mainly coordinated
by the retinoblastoma (Rb) protein. Rb contains a binding pocket for E2F and is there-
fore a pocket protein. If E2F is bound by Rb, it cannot bind to DNA and is repressed
in its function as a transcription factor. Phosphorylated Rb (pRb) releases E2F that
can exert its function.
Rb as such is the substrate of CDK2 or CDK4/6. These kinases are only
active when bound to their respective cyclins E or D. This redundancy of Rb-
phosphorylating enzymes ensures robustness of the pathway against perturbations
of individual components and enables integration of several signalling branches.
Extracellular growth factors activate signalling pathways through MAPK and AKT
(Table 6.2), which directly or indirectly lead to expression of the cyclin E and the
stable assembly of a complex between cyclin E and CDK2. This process is inhibited
the co-factors p21 and p27. The latter one represents also the internal state of the
cell because it is degraded as the cell cycle starts. This degradation is initialized
by mitogen-induced phosphorylation of p27, which is labelled for degradation by
ubiquitination (see Sect. 4.3.1). The ubiquitination is catalysed by a generic Skp,
Cullin, F-box containing (SCF) complex that specifically binds phosphorylated p27
by an adaptor protein called Skp2. The gene encoding for Skp2 is among the targets
of E2F [116] creating another feedback loop in this regulation scheme of cell-cycle
progression (Fig. 7.3).
Fig. 7.3 External stimuli and intracellular signalling determine cell-cycle progression to S-phase.
Growth factors and mitogens trigger complex formation between cyclin D and CDK4 or CDK6 that
phosphorylate Rb. Phosphorylated Rb releases E2F that activates transcription of its own gene as
well as S-phase promoting genes. Induction of Skp2 degrades p27, which otherwise would, together
with p21 inhibit complex formation between cyclin E and CDK2, which also phosphorylates Rb.
Inhibition: . Catalysis:
7.2 Checkpoints Regulating the Cell Cycle 81
7.2.2 The DNA-Damage Response
A safeguard mechanism exists to ensure correct duplication of the cellular DNA

content, once the cell passed the restriction point and entered S-phase. Eventual
DNA damage is sensed that triggers DNA repair and cell-cycle arrest. This response
can be considered a DNA damage checkpoint.
Damaged DNA is initially bound by one of the two factors adenine-thymine
mutated (ATM) or ATM- and Rad3-related (ATR), which are evolutionary conserved
and named after the context of their discovery. ATM and ATR are both kinases that
recruit other adaptors or mediators to the local site of DNA damage. Phosphorylation
of those regulatory proteins results in the activation of effector kinases, namely
checkpoint kinase (Chk)1 and Chk2. The mechanism of response and the involved
factors depend on the type of DNA damage. Three different scenarios exist. A DNA
damage response is triggered in severe cases of these three phenomena.
• Block of the progression of the DNA replication fork. A stalled replication fork
can be induced by abnormal DNA structures and vice versa. If DNA replication
does not proceed, M-phase entry is also blocked. Cell division prior to the com-
pletion of DNA duplication is referred to as mitotic catastrophe and should be
prevented to avoid genomic instability.
• Single-strand DNA damage. Errors on one strand of the DNA can be corrected
by base- or nucleotide-excision repair. The incorrect DNA stretch is cut out and
the complementary strand provides the template for re-synthesizing the original
sequence.
• Double-strand breaks. If both strands of the DNA do break, loose ends could
lead to chromosome rearrangements. Non-homologous ends are joined in G1 -
phase to prevent this. Disruption of protein-coding genes in humans is unlikely
given that more than 98% of the human genome are non-coding sequences [32].
Double-strand breaks can be repaired by homologous recombination with sister
chromatids after chromosome duplication upon S-phase completion.
ATR is recruited by the replication protein A (RPA) to the single DNA strands of
the stalled replication fork, or upon base or nucleotide excision. RPA is a single-
strand binding protein that stabilizes the DNA strand. Additionally, a sliding clamp
(9-1-1 complex) and a clamp loader (Rad17-RFC) are recruited similarly to con-
ventional DNA replication (see Sect. 2.3) for stabilization of the DNA structure. It
is important to stabilize the stalled replication fork that would otherwise collapse
and create more severe DNA damage. The inhibition of complexes between CDK2
and cyclin A blocks the initiation of DNA replication, while the replication fork
is stalled. Inhibition of the phosphatase Cdc25 leaves the complex between CDK1
and cyclin B inactive, which would be required for M-phase entry. Recruitment of a
DNA-unwinding helicase facilitates the resumption of DNA synthesis.
ATM is recruited by the Mre11, Rad50 and Nbs1 (MRN) complex to double-strand
breaks. The MRN complex holds together the otherwise loose ends of the broken
DNA helix. Histone modifications are mediated by ATM to alter local chromatin
82 7 Cell Cycle
structure. Phosphorylated histones lead to an increase in the local concentration of

cohesins that bring sister chromatids in closer proximity to allow for homologous
recombination. If double-strand breaks occur in G1 -phase and DNA was not yet repli-
cated, Chk phosphorylates the phosphatase Cdc25. Phosphorylated Cdc25 is targeted
for degradation by ubiquitination through the SCF complex. Inhibitory phosphoryla-
tion at CDK2 remains and cells therefore do not pass the restriction point and arrest
in G1 -phase.
Long-term DNA damage response involves p53, which induces expression of
target genes responsible for cell-cycle arrest and programmed cell death (see
Sect. 9.2.1). Regulation of p53 is threefold. First, p53 is phosphorylated by Chk
and therefore stabilized. The nuclear export signal of phosphorylated p53 is blocked
ensuring p53-mediated gene expression in the nucleus. Second, lysine residues of
p53 are acetylated by p300, which prevents p53 from ubiquitination and degrada-
tion. Last, p53 is regulated by the ubiquitin ligase Mouse double minute 2 homolog
(Mdm2) because it targets p53 for degradation and blocks the DNA-binding capac-
ity of p53. Activity of Mdm2 was reported to be reduced upon DNA damage [20].
Among the p53 target genes is also p21, which inhibits the complex between CDK2
and cyclin D (Fig. 7.3). B cell lymphoma 2 (Bcl2)-family members, which initiate
programmed cell death, are induced by p53 as last option to prevent detrimental
effects of propagated genomic instability in case DNA repair did not succeed.
7.2.3 Hyperproliferative Stress
The activation of p53 leads to cell-cycle arrest. Tumours are characterized by uncon-
trolled growth and division, which can be suppressed by p53. Therefore, p53 is
considered a tumour suppressor. On the other hand, genes encoding for factors
responsible for proliferation are called oncogenes as they could induce cancer if
they are overexpressed or hyperactive (see Sect. 9.2). Examples for oncogenes are
the genes encoding for Myc and Ras. Both are stimulated by growth factors and acti-
vate E2F for cell-cycle progression. A negative feedback exists to prevent cells from
uncontrolled proliferation in case of hyperactivation of these factors in a scenario
called hyperproliferative stress. It could be required if cells are exposed to an excess
of growth factors or oncogenes are mutated to become hyperactive or constitutively
expressed.
Mdm2 is inhibited upon upregulation of Myc or Ras, which in turn releases p53
to induce expression of p21. As we have learned, p21 represses the complex between
CDK2 and cyclin E, which is required for G1 /S transition (Fig. 7.3). This mechanism
renders cells more likely to arrest in cell cycle or induce p53-mediated programmed
cell death instead of growth and division without control.
7.3 Conceptual Views on Cell-Cycle Phenomena 83
7.3 Conceptual Views on Cell-Cycle Phenomena
It has been hypothesized that variability in cell cycle length mainly arises from
different growth times in the G1 -phase [89]. Once cells are stimulated by growth
factors, or committed to a developmental programme, they start to cycle. Cells that
are bigger grow for longer before they have acquired sufficient biomass to proceed in
the cell cycle, at least if growth and division in response to growth factor stimulation
occur subsequently in a cell. Both processes could also be regulated in parallel or
coordinated by diverging signalling pathways. These three scenarios (Fig. 7.4) are
not mutually exclusive and could be combined depending on the cell type, the internal
signalling state and the environmental condition.
A prerequisite for the understanding of the interdependence between cell growth
and cell division is to know the individual processes as such.
7.3.1 Cell Growth as Biomass Acquisition
The size of a cell scales with its DNA content. We have seen in a simple paradigm
(see Fig. 3.1): DNA → mRNA → protein; and protein contributes the most to dry
mass of a cell. If a cell needs to grow, it needs to acquire biomass, and thus it needs to
produce protein. For protein synthesis, translation turned out to be the rate-limiting
step because the transcription rate for induced genes exceeds usually the translation
of the respective mRNA molecules:
transcription translation
DNA −−−−−−→ mRNA −−−−−→ protein,
k1 k2
with k1 k2 . The cell needs more ribosomes to boost translation and therefore
increases the production of rRNA and ribosomal protein in response to nutrients and
growth factors.
Fig. 7.4 Growth and division triggered by growth factors and/or mitogens can be coordinated
in three different ways: hierarchical, in parallel or by branched signalling pathways. Mostly a
combination of these mechanisms exists that is hard to disentangle. GF: growth factor, MG: mitogen.
Adapted from [75]
84 7 Cell Cycle
The cell undertakes additional measures besides an increase in the number of ribo-
somes to grow and accumulate biomass. The entire metabolism is shifted. A central
regulatory component of cellular metabolism in mammals is the mammalian target
of rapamycin (mTOR). Its name is derived from the growth inhibitor rapamycin,
which blocks mTOR activity. Uptake of amino acids and expression of metabolic
enzymes is mediated by mTOR. Another downstream target activated by mTOR in
response to growth factor stimulation is the ribosomal subunit S6. Translation is fur-
ther enhanced through mTOR by phosphorylation of the eukaryotic initiation factor
4E (eIF-4E)-binding protein (4E-BP). Phosphorylated 4E-BP releases eIF-4E, which
then promotes initiation of translation (see Sect. 3.3.1).
The cellular biomass could be even more increased by further stabilizing proteins
thereby preventing their degradation. It has been observed that application of pro-
teasome inhibitors reduces activation of downstream components of mTOR such as
4E-BP [60]. But whether mTOR regulates cell growth also by directly interfering
with the proteasome is unclear.
7.3.2 Cell Division as Biomass Distribution
Accumulated biomass has to be distributed to daughter cells for division. This

dynamic process during M-phase includes not only re-organization of proteins but
also DNA. Mitosis represents the last phase of the cell cycle prior to cell division
and is divided into five stages that can be morphologically discriminated.
1. Prophase is initiated by condensing chromosomes in the nucleus. The chromatin

state is switched from the open euchromatin state, which still allows for DNA
replication and transcription, to the tightly packed heterochromatin state, in which
DNA is associated to histones and other proteins. The heterochromatin state in
the prophase of mitosis makes chromosomes visible in their classical X shape.
2. Prometaphase starts with the breakdown of the nuclear envelope. Microtubules
form a bipolar spindle apparatus and associate with kinetochores, which are
located at the centres of the replicated sister chromatids (see Sect. 2.1). Sister
chromatids are attached to opposite spindle poles. The spindle assembly check-
point prevents separation of sister chromatids before the entire protein machinery
is assembled [66].
3. Metaphase is characterized by correctly positioned sister chromatids, which are
aligned at the equator of the bipolar spindle apparatus (Fig. 7.5). The anaphase-
promoting complex (APC, see Sect. 7.3.4) facilitates the separation of the sister
chromatids so that they can be pulled to the opposing poles of the future daughter
cells.
4. Anaphase involves the split of sister chromatids away from each other. They are
forced by the spindle apparatus towards the microtubule-organizing centre at the
cell poles.
Fig. 7.5 Cell in metaphase of mitosis. Microtubules from mitotic centres attach with kinetochore
around centromeres of sister chromatids that are aligned in the equator region of the cell. Bipolar
spindle apparatus can then pull sister chromatids apart
5. Telophase exhibits loosening sister chromatids that start decondensation. Finally,

a new nuclear envelope is assembled around each set of sister chromatids so that
mitotic exit and cell division can follow.
Cell division separates the two nuclei by an encapsulating membrane. Synthesized

proteins in the cytoplasm are assumed to be well mixed and thus equally distributed
to both of the created daughter cells.
7.3.3 Find a Balance: Divide et cresce!
Division and growth of cells have to be well balanced to keep the right number of
cells with the correct size. Although cause and effect in the relation between cell
size and cell-cycle length are still debated, the following proportion is frequently
observed:
tˆG n
∝ ,
ˆtC V
with tˆG being the average length of G1 -phase and tˆC being the average duration time
of a complete cell cycle and n being the number of cells and V being the overall
volume of all cells.
However, the question remains whether the size of a cell is a mere consequence
of growth rate and division frequency or independently regulated to meet the cell’s
physiological requirements [39].
86 7 Cell Cycle
7.3.4 A Molecular Cell-Cycle Set-up for Oscillations
We have seen the stunning capability of the cell-cycle machinery to up- and down-
regulate individual components, such as cyclins (Fig. 7.2), repeatedly. The molecular
requirements for a signalling network to produce oscillatory behaviour have been
mentioned briefly (see Sect. 6.5.3) and apply for the regulation of the anaphase-
promoting complex (APC) in various model systems [34].
APC is activated by binding Cdc20. This process is rather slow but facilitated
through APC phosphorylation by M-phase complex of CDK1 with cyclin B (see
Table 7.2). This complex re-establishes itself via Cdc25.
APC is a ubiquitin ligase that, once activated, induces securin destruction.
Thereby, separase is unleashed that cleaves cohesin, which holds together sister
chromatids that can subsequently be pulled apart.
Another target of active APC in complex with Cdc20 is cyclin B that is degraded
and thus creates a negative feedback loop (see Sect. 6.5). Free CDK1 can then bind to
APC in the late M-phase or the early G1 -phase of the next cell cycle. This complex is
independent of APC phosphorylation and destructs cyclin A and cyclin B to prevent
premature entry into the S- or M-phase. But CDK1 is phosphorylated by the complex
between CDK2 and cyclin E in late G1 to release APC and make it available for the
next M-phase.
Timing plays an important role in cell-cycle regulation. Notably, the binding of
Cdc20 to APC is rather slow, creating a sufficient delay of this molecular network
motif (Fig. 7.6) required for oscillations [83].
7.3.5 The Cell-Cycle Programme
CDKs and cyclins mainly coordinate the cell cycle (Table 7.2). The process involves
many checkpoints and regulatory components to control precisely timed progression
Fig. 7.6 The complex of CDK1 with cyclin B promotes M-phase progression and is established via
Cdc25. Phosphorylation of APC by CDK1 promotes complex formation between APC and Cdc20.
This complex degrades cyclin B thereby creating a negative-feedback loop. Complex formation
between APC and Cdc20 is considerably slower than the degradation, creating a delay. APC forms
also a complex with CDK1 in late M-phase that degrades cyclin B. Inhibition: . Catalysis:
Table 7.2 CDKs and cyclins active in different cell-cycle phases in humans
G1 -phase S-phase G2 -phase M-phase
CDK 4,6 2 2,1 1
Cyclin D E A B
and prevent dysfunction. All these extracellular and intracellular conditions that are
monitored influence the cell-cycle programme. Cells cycle only if sufficient growth
factors or mitogens are available. They arrest in cell cycle, while DNA damage is
repaired.
In the following, a heuristics of the cell-cycle programme is given as pseudo-code
that the cell runs through during every division.
In G1 , the cell progresses if the external stimulus of growth factors or mitogens
exceeds a certain threshold. CDK4 or CDK6 forms a complex with cyclin D that
promotes gene expression via Rb phosphorylation and subsequent E2F release. This
promotes also complex formation between CDK2 and cyclin E to establish G1 /S
transition. Additionally, mTOR promotes growth by a metabolic shift towards protein
synthesis.
In S-phase, the cell replicates its genome unless DNA damage is detected that
needs to be repaired. Replication initiation is promoted by a complex of CDK2 or
CDK1 with cyclin A.
In G2 , the cell waits to prevent premature entry into mitosis thus avoiding mitotic
catastrophe.
In M-phase, the complex between CDK1 and cyclin B establishes binding between
APC and Cdc20, which leads to the separation of sister chromatids. Finally, cyclin B
is degraded to terminate the M-phase with cell division to start from anew.
% % G_1 p h a s e
i f s t i m u l u s > t h r e s h o l d % e x t e r n a l g r o w t h f a c t o r s or m i t o g e n s
C D K 4 /6 + c y c l i n D = { C D K 4 /6 c y c l i n D } % complex formation
gene . e x p r e s s i o n = TRUE % G_1 / S t r a n s i t i o n
CDK2 + cyclin E = { CDK2 cyclin E} % complex formation
mTOR . m e d i a t i o n = TRUE % growth
end
%% S phase
w h i l e DNA . d a m a g e = TRUE % cell - c y c l e a r r e s t
DNA . r e p a i r = TRUE
DNA . r e p l i c a t i o n = FALSE
end
CDK2 + cyclin A = { CDK2 cyclin A} % complex formation
DNA . r e p l i c a t i o n = TRUE
% % G_2 p h a s e
wait
%% M phase
CDK1 + cyclin B = { CDK1 cyclin B} % complex formation
APC + C d c 2 0 = { APC C d c 2 0 } % complex formation
cyclin B = 0 % degradation
return
88 7 Cell Cycle
Summary
• Different organisms serve as model systems to study the cell cycle because
they are easy to grow and/or manipulate.
• There are several checkpoints at which the cell cycle halts until proper growth
or maintenance of DNA integrity is ensured.
• The molecular programme for cell division is cycling as protein components of
the machinery (“cyclins”) are oscillating and other regulatory proteins (“cyclin-
dependent kinases”) depend on their activity on them.
Immunology
8
Contents
8.1 Components of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
8.2 Innate Immunity as First Line of Host Defence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
8.3 Adaptive Immunity as Ability to Strike Back . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Introduction
The immune system is vital to human health and therefore a major field of biomedical
research. Immunis in Latin originally means free from obligation or liability. The
immune system can be understood as an intricate network or machinery that protects
its host from foreign substances [24,57]. A key feature to exert this task is the
discrimination between own and foreign entities.
The main subject of immunology is the immune system of our human body. Many
phenomena in immunology carry human-related names such as recognition, memory
and defence. Substances are recognized as self or non-self. There is a memory of the
non-self substances and adaptation of the mounted immune response.
For all these phenomena to occur, a complex interplay is needed between the
variable components that constitute our immune system. To cope with the diver-
sity of immune responses, they are divided into the innate and the adaptive immune
response. While the innate immune response relies crucially on macrophages, neu-
trophils and mast cells, it is linked by dendritic cells to the adaptive response that
involves B lymphocytes and T lymphocytes. Adaptive immunity features a humoral
response in addition to the cellular response.
Not only the mechanisms underlying these processes are in the focus of interest
but also understanding of the immune system’s malfunctions is important for curing
disease. Errors in the immune system are referred to as autoimmunity, immunod-
eficiency or hypersensitivity and will mark the transition to cancer (see Chap. 9)
towards the end of this chapter.

https://doi.org/10.1007/978-3-662-65357-9_8
90 8 Immunology
8.1 Components of the Immune System
The immune system consists of a multitude of components, which are organized in

tissues as cells and molecules. The diversity of the immune systems’ components
reflects the plethora of causes of diseases, which have to be prevented. We as the host
have to be protected from pathogens, which by definition have the ability to make us
sick. Examples of pathogens are bacteria, fungi, parasites or viruses. The first line
of defence is our skin and mucosal surfaces that line organs with contact with the
outside world like lung and gut. Parts of our immune system recognize the intruding
enemy specifically to fight it. The soldiers of this army of our immune system are
the immune cells.
8.1.1 Sites and Non-Cellular Substances of Immune Reactions
A major home base of our immune cells is the bone marrow. Many immune cells
originate from there. Hematopoietic stem cells (HSCs) in the bone marrow have the
capacity to give rise to all immune and blood cells. White blood cells (leukocytes)
form the majority of the cellular immune response with a myeloid and a lymphoid
branch (see Sect. 8.1.2). Together with the thymus, the bone marrow is referred to as
primary lymphoid organs because lymphocytes are derived there. The lymphatic sys-
tem comprises vessels transporting fluids and cells through our tissues. This draining
substance with all its components is referred to as lymph. Specializing lymphoid
cells exert their tasks also in the periphery of our body. Lymph nodes, mucous, skin
and spleen are therefore referred to as the secondary lymphoid organs in the periph-
ery. Skin and mucous are the first physical barriers against pathogens. The mucous
is a fluid covering our respiratory and gastrointestinal tract with neutralizing sub-
stances (e.g. IgA), acidic milieu (i.e. low pH) and enzymes (e.g. lysozyme) or even
small peptides (e.g. defensin) that kill pathogens. Besides the cellular response, these
chemicals, enzymes and peptides, there are other molecules, mainly antibodies (see
Sect. 8.3.2) to fight pathogens. Antibodies are secreted to extracellular fluids, which
constitute humoral immunity (Fig. 8.1).
All components of the immune system need to be well orchestrated to guard our
body. Regulatory mechanisms and maturation dynamics were derived from the study
of hematopoiesis, a process that gives rise to all our immune and blood cells.
Fig. 8.1 There is an intersection in cellular components between innate and adaptive immune
responses. In addition to cells, the adaptive immune response is mediated by humoral components,
i.e. secreted antibodies
8.1 Components of the Immune System 91
8.1.2 The Hematopoiesis Tree and Its Branches
Immune cells are a subset of the hematopoietic system that is rooted in hematopoietic
stem cells. Because of their potency to give rise to several diverse cell types, they
are called pluripotent. Cells arising from HSCs subsequently lose this potential for
functional diversity. Progenitor cells become more and more committed to a certain
fate. Upon maturation, cells have acquired a very precise function and cannot produce
any other cell type. Every stage in this process is considered a compartment. There
are compartments of stem cells followed by compartments of committed progenitor
cells and finally compartments of mature cells.
The process of cellular reproduction within a compartment, involving iterated
rounds of cell cycle (see Chap. 7), is called proliferation. Differentiation refers to cells
acquiring specialized functions while changing their morphology or intracellular
structures thereby entering a new compartment. The balance between proliferation
and differentiation is important for homeostasis of the hematopoietic system. HSCs
are on top of a hierarchy that is referred to as the hematopoiesis tree with “stem” cells
and different branches of progenitor cells (Fig. 8.2). The arrows in the hematopoiesis
tree indicate that differentiation is unidirectional and cells cannot differentiate back
into a previous compartment. A more quantitative understanding could help to weigh
and lengthen these arrows according to the frequency and duration of the fluxes.
Many cells rapidly differentiating along a line would result in a thick but short
arrow, whereas few cells slowly leaving a compartment would be indicated with a
thin, long arrow.
In general, there is a differentiation influx parameter di into a compartment and a
differentiation efflux out of the compartment with the rate constant do . An average cell
Fig. 8.2 Schematic overview of the hematopoiesis tree. Arrows are arbitrarily scaled. HSC:
hematopoietic stem cell, ST-HSC: short-term hematopoietic stem cell, MPP: multi-potent progeni-
tor, CMP: common myeloid progenitor, CLP: common lymphoid progenitor, MK: megakaryocyte,
EB: erythroblast, MB: myeloblast, NK: natural killer cell, LC: large lymphocyte, Plate: platelet,
Trombo: trombocyte, RBC: red blood cell, Baso: basophil, Eosino: eosinophil, Neutro: neutrophil,
Mono: monocyte, Dendr: dendritic cell, T: t(hymus) lymphocyte, B: b(one marrow) lymphocyte,
Mast: mast cell, Macro: macrophage, Plasm: plasma cell
92 8 Immunology
Fig. 8.3 Cell x differentiates

into the shaded compartment
with parameter di and
progresses with do . The net
proliferation rate is
(λ − α) · x
within the compartment proliferates with λ and has a probability α to die (Fig. 8.3).
Please note that for HSCs there is no influx, and for mature cells, there is no efflux.
These rates for proliferation, differentiation and death characterize the hematopoietic
system and its flux control. Genetic labelling of HSCs and subsequent progeny in a
mouse model allowed researchers to establish a quantitative framework to determine
those rates [18].
It was found that an adult mouse contains ≈5000 HSCs contribute to normal
hematopoiesis. And both, the HSCs as well as their subsequent compartment, the
short-term (ST)-HSCs operate near self-renewal. This means that the outgoing dif-
ferentiation and death rate do not exceed proliferation and differentiation influx of
the ST-HSC compartment (Fig. 8.3):
di + (λ − α) ≤ do . (8.1)
Additionally, the residence time of an average ST-HSC was estimated to be 330

days [18]. This is an exceedingly long time spent in this compartment given that
the expected lifetime of a mouse is around 2 years. Without stem or progenitor cells
being exhausted, those rates allow the hematopoietic system to rapidly replenish
compartments after depletion. Amplification is achieved by massive proliferation of
multi-potent progenitor cells that are the branching point for the myeloid and the
lymphoid lineage.
The myeloid lineage contains important cells of the innate immune response
(see Sect. 8.2), namely basophils, eosinophils, neutrophils, mast cells (collectively
called granulocytes), monocytes and macrophages and dendritic cells. Macrophages
mature from monocytes, and together with granulocytes and dendritic cells, they
belong to the group of phagocytes. Phagocytes are cells that eat up their enemies.
Besides other functions, e.g. during inflammation (see Sect. 8.2.1), macrophages act
as scavenger cells indiscriminately clearing cell debris. Granulocytes were named
after the multitude of diverse granules in their cytoplasm. These vesicles contain
enzymes and toxins to destroy microorganisms and parasites. Unlike macrophages,
granulocytes are not always present but are called into action if needed. Mast cells
are less well defined. They are known to be mainly involved in allergic reactions.
Dendritic cells take up pathogens, but in contrast to macrophages and granulocytes,
their main task is not clearance but presentation of antigens. These pathogen-derived
fragments received their name from the fact that antibodies (see Sect. 8.3.2) are
generated by B lymphocytes in the adaptive immune response to antigen exposure.
Dendritic cells belong to the class of antigen-presenting cells (APCs). They exhibit
pathogen particles on their outer surface to expose them to naive T lymphocytes
that thereby become activated. Dendritic cells have arm-like structures to embrace
8.1 Components of the Immune System 93
as many T lymphocytes as possible. Dendritic cells were named after those arm-like
processes because they look akin to dendrites of brain cells.
During wound healing, platelets serve the purpose of clotting blood. Because they
make blood thick and sticky, they are also called thrombocytes. Although red blood
cells are not directly involved in immune reactions, they ubiquitously contribute to
the overall number of cells in our body and thus the hematopoietic system. Recently,
the number of red blood cells in an average male was estimated to be 2.5 × 1013 ,
which represents 84% of total cell counts [97]. Given the half-life of red blood cells
being 120 days [46], our body produces more than 2 million mature red blood cells
every second. These cells are needed because their vital task is to provide all our
organs and tissues with oxygen.
Procedure
Erythropoiesis means the maturation of red blood cells from erythroid progen-
itor cells. Optimal control of proliferation and differentiation is important for
erythroid progenitor cells to produce the right amounts of red blood cells in a
given time. To define the stages of erythroid differentiation is a non-trivial task.
Criteria for distinct subpopulations are morphology, expression of surface mark-
ers and functional assays. During maturation, erythroid progenitor cells undergo
morphological changes as they shrink and eject their nucleus. The expression of
the transferrin receptor CD71 and the glycophorin A-associated antigen GPA is
regulated because these surface markers are implicated in iron metabolism. Iron
ions are needed for haemoglobin synthesis, which is essential for oxygen-carrying
red blood cells. Functional assays characterize haemoglobin content in a blood
sample. Stages of erythroid progenitor cells are named burst- or colony-forming
unit-erythroid cells, respectively, depending on how they grow in a semi-solid
medium. To analyse thousands of single cells with respect to their morphology
and surface markers, a technique called flow cytometry can be applied (Fig. 8.4).

The lymphoid branch contains natural killer cells that belong to the innate immune
response. The common lymphoid progenitor cell gives rise to a minor subpopulation
of dendritic cells. As dendritic cells take up pathogenic particles and represent them
to naive T lymphocytes, they form an important link between innate and adaptive
immune responses. B lymphocytes and T lymphocytes exhibit an intricate mecha-
nism of regulation that will be further explained for adaptive immunity (see Sect. 8.3).
Whether our hematopoietic system is correctly represented by this tree-like struc-
ture (Fig. 8.2) is currently under debate. Especially the discrete branching between
the lymphoid and especially the myeloid lineage is challenged by most recent tech-
nological advancements. The progeny of individual barcoded cells can be traced
in vivo after transplantation into mice [87]. Single-cell RNA sequencing suggested
a robust maturation continuum based on gene expression profiles [86] rather than
discrete gating based on the presence of surface markers in two-dimensional scatter
plots (Fig. 8.4).
94 8 Immunology
Fig. 8.4 Scatter plot of erythroid progenitor cells upon flow cytometry measurement. Single cells
in a thin liquid stream pass laser beams. Recorded scatter light indicates the size and granularity of
the individual cells. Surface markers can be stained so that fluorescent signals correlate with their
abundance per cell. Gates are set to indicate cells positive for GPA or CD71, respectively. Every
circle encloses 10% of a population of 38,475 cells
8.2 Innate Immunity as First Line of Host Defence
If a pathogen tries to invade our body, it first encounters multiple layers of the
innate immune system. Mucous covers all wet surfaces of our body that are not
protected by skin, such as eyes, mouth, lungs and stomach. Preventing these tissue
layers (epithelia) from drying out stops cracking and thus keeps infectious agents
away. The lubricious substance acts as a conveyor belt because it houses trillions
of our bacterial tenants but flushes out pathogens. The importance of mucous is
underlined by the fact, that dysfunctional mucous production is linked to disease:
Mucoviscidosis aka cystic fibrosis. To further support tissue surface cleansing, white
blood cells (leukocytes) are sent to spots of infection. Leukocytes are also recruited
after wounding your skin because this first shield of your body is then permeable to
pathogens.
8.2.1 Inflammation and the Complement System
If you wound your skin, the site of damage will start to hurt, swell and feel warm.
This process is called inflammation. Microbes that breach the skin are recognized
by macrophages, which engulf the bacteria and destroy them. This process is called
8.2 Innate Immunity as First Line of Host Defence 95
phagocytosis. Besides eating up the enemy, macrophages release cytokines, which

are messenger molecules to coordinate the immune response among the cellular com-
ponents. A special class of cytokines, chemokines, attracts neutrophils and mono-
cytes to exit the bloodstream and enter the local site of infection. Cells penetrating
the blood vessels cause the characteristic pain of an inflammatory response. In addi-
tion, increased production of lymph flushes the invaders out of the tissue into nearby
lymph nodes to trigger more specialized adaptive immunity. The elevated production
of fluids and recruitment of immune cells cause swelling. Dilation of blood vessels
and increased local blood flow to transport all the cellular components to the site of
damage makes us feel heat, too.
Inflammation is enhanced by the complement system, which comprises a fam-
ily of more than 20 plasma proteins. The complement system triggers a cascade of
reactions to coat microbes with molecular patterns that are recognized by comple-
ment receptors on macrophages for phagocytosis. Proteins of the complement system
not only attract macrophages but also cluster microbes together in a process called
agglutination and lyse bacteria by punching holes through their cell wall and mem-
brane. When the inflammation sustains, macrophages are supported by neutrophils,
which are also capable of phagocytosis. The supply of macrophages is ensured by
recruited monocytes that differentiate into mature macrophages at the site of local
inflammation.
8.2.2 Pattern Recognition: Know Your Enemy
Precondition for any immune response is the recognition of pathogens. You have to
know your enemy in order to combat them. Despite their diversity, many pathogens
exhibit common motives on their surface that are essential and evolutionarily
conserved. These structures are known as pathogen-associated molecular patterns
(PAMPs). The presence of PAMPs is recognized by receptors that are called pattern-
recognition receptors (PRRs). The number of different PRRs to detect pathogens is
rather limited indicating the few core elements that discriminate pathogens from host
cells. Detected components range from sugars and lipids (see Sect. 1.1) to DNA and
RNA, in bacteria, fungi, parasites, viruses and its own host (reviewed in [5]). The
major class of PRRs is Toll-like receptors (TLRs), named after the founding member
Toll, which was initially identified in the fruit fly.
TLR4, for instance, a prominent member of the TLR family, recognizes
lipopolysaccharides on the outer membrane of bacteria. Cell wall components of
fungi can be detected by other TLRs, likewise plasma membrane anchors of para-
sites . Molecules of our own body should not but can trigger inflammation through
TLRs, too. Heat shock proteins are secreted during fever upon infection and act in a
pro-inflammatory way [105]. There are other molecules released by our own body
cells, which are recognized by PRRs. They are called damage-associated molecular
patterns (DAMPs), and as the name implies, they are released by stressed cells, which
are dying without an infection. Hereditary material of bacteria or viruses can also
be detected by TLRs. Unmethylated cytosine-guanine dinucleotides in a particular
96 8 Immunology
base context are characteristic of genomic DNA of bacteria and can therefore be
recognized by TLRs. The RNA, no matter single- or double-stranded, of viruses can
also elicit an innate immune response via TLRs because there is no “naked” single-
stranded RNA (mRNA is, for instance, capped, see Sect. 3.2.2) nor double-stranded
RNA under normal conditions in human cells. To study an anti-viral response, a
synthetic analogue of double-stranded viral RNA, polyinosine-deoxycytidylic acid
(poly I:C), is often used [8]. Activated TLRs trigger an inflammatory response by the
expression of inflammatory cytokines, such as interferon (IFN)-α. TLRs detecting
DNA or RNA are not located at the plasma membrane but at intracellular vesicles
of phagocytotic cells because the enemy has to be processed first before its inner
material can be exposed. Dendritic cells that process pathogens link the innate to the
adaptive immune response. They migrate from the local site of infection to lymph
nodes to activate naive T lymphocytes.
8.3 Adaptive Immunity as Ability to Strike Back
Processed pathogens have to be presented to the cellular components of the adap-

tive immune system to retaliate. Dendritic cells pick up antigens at the local site of
infection and migrate to regional lymph nodes to activate naive T lymphocytes. Dif-
ferentiation and proliferation give rise to functional forms of T lymphocytes, among
them T helper cells, which help B lymphocytes to mature and produce antibodies
in response to antigen exposure. The “T” in T lymphocytes stands for the thymus
where they mature even though they originate from the bone marrow. B lympho-
cytes originate from and mature in the bone marrow, but the “B” stands for bursa
of Fabricius, a lymphoid organ in birds from which they were first derived. Both, T
lymphocytes and B lymphocytes, carry antigen receptors.
Antigens exhibit immense diversity, which is encountered by a possible number
of 1015 different antigen receptors. It is impossible to maintain the presence of so
many different receptors all the time, especially because it exceeds the total number
of lymphocytes by a factor of ten thousand. Instead, only the cell that carries a
receptor that recognizes the exposed antigen should be amplified in number. It has to
be ensured that the receptor does specifically recognize the pathogen-derived antigen
but does not cross-react to cells or fragments of our own body. High numbers and high
specificity of so-called effector cells in the adaptive immune response are achieved
by the process of clonal selection.
8.3.1 Clonal Selection of Special Forces
Lymphoid progenitor cells (Fig. 8.2) give rise to a large number of lymphocytes,
each with an individual antigen receptor. All progeny of one of those cells will carry
the same receptor, which is therefore a clonal population. If the receptor reacts to
particles of the own body (i.e. a self antigen), it is discarded. Induced cell death of self-
reacting lymphocytes is referred to as clonal deletion. Only if the receptor recognizes
8.3 Adaptive Immunity as Ability to Strike Back 97
Fig. 8.5 Schematic overview of clonal selection for specific antigen-receptor cells. A lymphoid
progenitor cell gives rise to different cells, each carrying an individual type of antigen receptor.
Cells recognizing self antigens are deleted to prevent an auto-immune reaction. Cells specifically
recognizing the non-self antigen of the foreign pathogen expand in number to eradicate the non-self
antigen
specifically the antigen derived from the pathogen (i.e. a non-self antigen), the clone
proliferates in a process called clonal expansion. The progeny carrying all the same
receptor, which reacts specifically to the foreign antigen (Fig. 8.5). Thus, specialized
T lymphocytes form functionally distinct sub-populations and B lymphocytes give
rise to plasma cells that can produce the antigen receptor and secret it as an antibody.
8.3.2 Antibodies
Antibodies are an important weapon of the immune system. They detect pathogen-
derived antigens and neutralize them or activate further cells of the adaptive immune
response. These two functions of recognizing an antigen specifically and affecting
different cells in turn are represented in the Y-shaped structure of an antibody. Each
of the two upper arms of the Y binds to an antigen, while root at the bottom interacts
with cells. Antigens are very variable and so are the antibody regions detecting them.
The same two regions of an antibody that detect two antigens of the same type are
called variable regions because they differ from antibody to antibody, each being
specific for a single type of antigen. On the other end, there is a limited set of cell
types of the adaptive immune system interacting with the antibody. The antibody’s
effector function is thus exerted by a constant region.
The Y shape implies a vertical symmetry axis for antibodies. They are composed
of two identical heavy chains linked by disulfide bonds and two identical light chains
on the outer arms linked to each heavy chain by disulfide bonds, too. Heavy and light
refer to the approximate molecular weight of 50 kDa and 25 kDa, respectively. The
immense diversity between the variable regions of the individual antibodies is created
by shuffling of gene segments by means of DNA recombination (see Sect. 2.4.2) in
the different cell clones. The DNA stretches that are shuffled are the Variable gene
segment, the Diversity gene segment (only in the heavy chain) and the Joining gene
segment. The process is therefore referred to as VDJ recombination. In contrast to
the vast number of variable regions, there are only five different types of constant
98 8 Immunology
Table 8.1 The five different kinds of heavy chains determine the class of the antibody, called
immunoglobuline (Ig)
Heavy-chain type μ γ α δ
Immunoglobulin class IgM IgG IgA IgE IgD
regions for the heavy chains. They correspond to the different classes of antibodies
called immunoglobins (Table 8.1)
Antibodies are secreted by matured B lymphocytes in huge quantities, which
makes them the major component of the humoral (i.e. non-cellular) immune response
and suitable for being studied biochemically. However, antibodies are not the only
molecule capable of antigen binding. Both, the B cell receptor (BCR) and the T
cell receptor (TCR), were selected to specifically bind antigens. These receptors are
structurally and functionally related but not identical (Fig. 8.6). While BCRs exhibit
a Y shape, TCRs are rather I-shaped. Every BCR carries two antigen-binding sites
whereas each TCR can only bind a single antigen. The specific BCR can be bound
to the membrane of a B lymphocyte, and, the very same receptor, can be secreted as
an antibody. TCRs are always integrated into the membrane of T lymphocytes and
can only recognize antigens that are presented by antigen-presenting cells (APCs).
8.3.3 Regulation of T Cells for Killing, Help, Memory and Balance
T lymphocytes play a very central role in adaptive immunity. Naive T lymphocytes

can mature into cytotoxic T cells, which acquire the ability as effector cells to kill the
pathogen. T helper cells constitute another sub-population of T lymphocytes, which
helps B lymphocytes to mature into plasma cells for massive antibody production.
Besides, so-called regulatory T cells prevent the immune system from overshoot-
ing. How are these diverse functions orchestrated? Cytokines are important soluble
factors secreted by cells to coordinate the innate immune response (see Sect. 8.2).
Adaptive immunity relies on direct interaction between cells via receptors on the
surface of immune cells. These receptors are called clusters of differentiation (CD).
antigen-binding
site
light chain
variable region
disulfide bond
linker region α chain β chain
heavy chain
constant region disulfide bond
membrane
B lymphocyte T lymphocyte
Fig. 8.6 Structure of an antibody as secreted form of a B cell receptor and the membrane-bound T
cell receptor
They are traditionally used to classify sub-populations of the hematopoiesis tree.

More than 400 different CD molecules have been described in humans [33]. The
decision of whether a cell belongs to our body or not is nonetheless binary. To
discriminate between self and non-self, there is another important type of surface
marker: the major histocompatibility complex (MHC).
Type-I MHC molecules are expressed by all nucleated cells of our body. Except red
blood cells, all our blood cells identify themselves by carrying the MHC-I molecule
on their surface. The human MHC molecules are therefore referred to as human
leukocyte antigen (HLA). The name for MHC molecules was derived from the fact
that they define tolerance to tissues upon transplantation. If tissue is compatible,
the subtype of the major histocompatibility complex matches between donor and
recipient. As there are minor MHC antigens that could evoke an immune response,
suppression of the immune system is needed to avoid rejection of the tissue, even if
the MHC-I subtype matches. Cells lacking MHC I on their surface are eliminated by
natural killer cells as part of innate immunity. MHC molecules are no real receptors
but rather membrane-embedded carrier molecules, which expose certain antigens
to surrounding cells. Antigens displayed by MHC I are derived from cytoplasmic
particles of the cell upon viral infection. Antigens presented by MHC II are derived
from extracellular particles that were taken up and processed by antigen-presenting
cells (APCs) in intracellular vesicles.
While infected cells are carrying the viral particle loaded to MHC I have to be
eliminated to prevent the spread of infection, APCs need to be further supported and
orchestrated in fighting pathogens. These tasks are exerted by two distinct subsets
of T lymphocytes. The killing is done by cytotoxic T cells, and the mediation of
the adaptive immune response is performed by T helper cells. To prevent confusion
between the MHC molecules and the subsets of T lymphocytes, co-receptors are
expressed on their surface that corroborate correct detection. Besides the T cell
receptor on the surface of cytotoxic T cells detecting the viral antigen presented by
MHC I, a CD8 molecule binds MHC I to confirm the killing. In short, those cytotoxic
T lymphocytes expressing the CD8 molecules on their surface are referred to as
cytotoxic CD8 T cells. And apart from the T cell receptor detecting antigens presented
by the MHC II, there is a CD4 molecule on the surface of T helper cells binding
MHC II to confirm the release of cytokines, e.g. for activation of B lymphocytes and
antibody production.
Some of the matured T and B lymphocytes remain after the pathogen has been
defeated thereby forming the basis of immunological memory. These memory cells
can re-activate the adaptive immune response much quicker in case of another
encounter with the then “known” pathogen. The adaptive immune system thus
exhibits a memory because it remembers previous attackers. In theory, the prop-
erty of a system’s output depending on the history of input is known as hysteresis. In
the context of adaptive immunity, this means that the strength of the immune response
rises with the increasing strength of first pathogen exposure. Once exposed, the adap-
tive immune system remembers the pathogen and mounts a strong response even if
the pathogen exposure declines the second time (Fig. 8.7). Hysteresis was in fact
100 8 Immunology
Fig. 8.7 Adaptive immune response strength versus pathogen exposure strength. For the 1st expo-
sure, T lymphocyte activation rises with increasing antigen stimulation. For the 2nd exposure,
immune response remains strong, even if pathogen levels sink. The curve forms a hysteresis loop
that depicts memory. For the same pathogen exposure, the 2nd response adapted as compared to the
1st (dotted arrow)
shown with concentration of activated T lymphocytes versus antigenic stimulation

level [17].
If receptors are detecting self antigens, death of the cell carrying this receptor
can be induced (see Sect. 8.3.1). Apart from this self-induced cell death, there is a
population of functional T lymphocytes that suppresses immune responses. These
regulatory T cells (“Tregs”) exist as mature forms in the periphery of our body
and exert dominant-negative control over self- or hyper-reactive T lymphocytes to
balance the immune response (i.e. homeostasis). If regulatory T cells are functionally
deficient, chronic auto-immune diseases occur [92].
8.3.4 (Auto-)immune Disease
Our immune system involves the following features:
• diversity of the repertoire of antigen receptors,

• specificity for pathogen-derived antigens,
• discrimination between self and non-self antigens and
• memory of past antigen exposures.
Mechanisms have to be well balanced for a reaction with correct timing, specificity
and strength. Too weak immune response will let the pathogen spread and an over-
reaction exhausts resources, both harmful to our body. To sum up, diversity of the
antigen receptors is generated by genetic shuffling called VDJ recombination, while
specificity is achieved by the clonal selection of antigen-receptor cells (Fig. 8.5).
After the clonal selection, discrimination between self and non-self is additionally
ensured by MHC molecules identifying our body cells or displaying antigens of
invaders. Some T and B lymphocytes that orchestrate cellular and humoral immune
response persist after the initial exposure to strike back more efficiently at the second
attack. This is a strong call for vaccinations to prevent infections. Because if the body
is exposed to dead and therefore harmless particles derived from a pathogen, memory
cells specifically recognizing these antigens can form and provide a long-lasting
protection against the disease.
Problems occur if the process loses control. In cases of autoimmune diseases,
components of ourself are falsely detected and fought as the enemy. Our own cells
then make us sick, and the host itself becomes the pathogen. An example of an
autoimmune disease is type I diabetes. In this disease, over-reactive CD8 T lym-
phocytes and B lymphocytes lead to the destruction of insulin-producing cells in
our body. Over-reacting lymphocytes can also give rise to lymphoma, a cancer of
the lymph nodes. On the other hand, natural killer cells and cytotoxic T cells could
be used to specifically eradicate cancer cells. But immediate questions are: What
is cancer, and how to detect, characterize and combat it? These questions will be
addressed in the final chapter.
Summary
• Cellular and non-cellular (i.e. “humoral”) components constitute the immune
system to defend an organism from disease-causing entities.
• All immune cells are highly specialized in their function and arise from com-
mon and shared progenitors all originating back to hematopoietic stem cells.
• Whereas innate immunity is about pattern recognition of common pathogens,
adaptive immunity is triggered against novel invasions through selection of
highly reactive non-/cellular components.
Cancer
9
Contents
9.1 Different Types of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
9.2 Pro et Contra Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
9.3 Combat Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Introduction
Cancer is one of the most devastating diseases of our time. It is a global challenge
to find new ways and improve existing approaches to prevent, diagnose and combat
cancer. Oncology is the name of the subject that is dedicated to understanding the
cause and the progression (i.e. the “aetiology”) of the disease(s). The biggest hurdle
in fighting cancer is that there is no single enemy. Cancer represents a multitude
of diseases with a huge variety of modes of action. Therefore, we have to join
forces from different fields of medicine, physics, biology and mathematics: surgery,
pharmacology, radiology, molecular biology and dynamic modelling.
Cancer represents the final chapter of this book because we need to consider
all aspects of modern biology to understand the basics of cancer research, from the
change of cellular architecture and the deregulation of gene expression to signal trans-
duction, cell cycle control and the role of the immune system. Despite the complexity
of cancer, there is one characteristic that all different types of cancers share to a certain
extent: The deregulation of cell proliferation. Proliferation is phenomenologically
a straightforward process: One cell becomes two in a given time. This duplication
involves DNA synthesis, cell growth and division. In normal, healthy cells under
physiological conditions, proliferations are tightly controlled. If this regulation is
lost, for various reasons, healthy cells transform into cancer cells that proliferate in
an uncontrolled manner (Fig. 9.1). Erroneous DNA synthesis, excessive cell growth
and frequent cell division lead to the formation of tumours and metastases.

https://doi.org/10.1007/978-3-662-65357-9_9
104 9 Cancer
Fig. 9.1 Cancer is

characterized by healthy cell
uncontrolled cell
proliferation. While a
healthy cell doubles only
once in a given time, a cancer
cell grows and divides much cancer cell
faster in the same time to
form tumours and metastases
time
There are many physiological anti-cancer mechanisms that keep cell proliferation
under control. Once a former normal cell escapes and is transforming into a cancer
cell, drug interventions can help to constrain the tumour in its size or localization
inside the body. But cancer cells divide and therefore evolve rapidly. They develop
resistance against therapeutic approaches. Besides, interfering with a generic process
such as proliferation can also have severe side effects and harm healthy body cells.
But recent discoveries regain us hope that integrated and personalized approaches
may lead to a life without cancer in the future if we combine our expertise from the
diverse areas of modern biology.
9.1 Different Types of Cancer
Cancer is diverse. There is not only a single type of cancer. There is lung cancer and
breast cancer, colon cancer and blood cancer, tumours in the brain and the prostate,
just to name a few. But even within one and the same organ, there can be different
subtypes of cancer, at subsequent stages of severity of the disease. However, cancer
cells arise from normal cells, tumours form within healthy tissues. Given this origin,
there must be some shared characteristics. Cancer was named after the brown crab
Cancer pagurus because the shape of tumours in Ancient Greece reminded people of
this animal [76]. According to this comparison, the body of crab refers to the primary
tumour, which is its core, its site of origin. The distant legs and claws represent
ensembles of cancer cells spread to other places, called metastases. Tumours that
do not grow out, form metastases and invade other sites, but stay local, are called
benign, whereas tumours that invade other tissues are called malignant. If tumours
originate from epithelial cells, sheets that line inner and outer surfaces of tissues,
they are called carcinomas, which make up 80 % of all human cancers. Within the
carcinomas, one can discriminate between squamous carcinoma, originating from
protective epithelial cell layers, and adenocarcinoma, generated by epithelial cells
that secrete substances to inner cavities. As such there can be both, squamous and
adenocarcinoma, in the lung, depending on the originating cells.
A major group of cancers, which do not derive from epithelial cells, are the
sarcomas that stem from connective tissue, consisting largely of so-called “mes-
enchymal cells”. Sarcomas constitute only 1% of tumours. An example of sarcomas
is liposarcoma that affects fat tissue. Tumours of the hematopoietic system are called
9.1 Different Types of Cancer 105
leukaemia if they refer to myeloid cells or lymphoma in case the lymphoid lineage
is concerned. The last major group of cancers relates to neuroectoderm cells and thus
the brain. This group of cancers involves gliomas, glioblastomas, neuroblastomas,
schwannomas and medulloblastomas. Again, the name depends on the cell type of
origin or where are they localized.
There is a challenge to this dogma, because (cancer) cell types and their local-
izations can change during the generation of a tumour. This feature is called
“transdifferentiation” and means change in cell type. Most prominently, epithe-
lial cells can become mesenchymal cells during a process, which is referred to as
epithelial-mesenchymal transition (EMT). In simple terms, a surface cell is becom-
ing a cell of connective tissue properties, which is important for the cancer cell to
invade neighbouring tissues [11].
9.1.1 Phenotypic Tumour Development
Cancer is diverse. An example of some of the vocabulary introduced above can be

the following common path of colon cancer development. The type of cancer is
routinely defined based on the site of its first occurrence, i. e. the primary tumour in
case of colon cancer in the colon, which is the last part of the gut before the rectum.
Other tissues can be affected during the course of the disease, e.g. metastases in the
liver. It starts with hyperplasia of the epithelial cell layer in the colon. This means
that all cells increase in numbers while still looking healthy. Once cells stop looking
healthy, the phase of dysplastic growth is entered. How does an unhealthy cell look
like? An example is an increased ratio between the volume of the nucleus and the
volume of the cytoplasm. Therefore, a biopsy is taken, and from a thin slice of the
epithelial layer of the colon, DNA is stained for histology (“the study of tissue”).
This abnormal cell growth will soon be macroscopically visible by the naked
eye in the form of a polyp. Since the epithelial cell layer in the colon is capable
of secreting substances, the polyps can be referred to as adenomas. Yet they are no
adenocarcinomas, because the polyps do not penetrate through the basal membrane
on the tissue and thus do not invade underlying tissue. Adenomatous polyps are
benign.
Once the abnormal cells break through the basal membrane and invade other
tissues, they are referred to as (malignant) carcinoma cells. The process of abnormal
cell growth in disparate places such as epithelial cells in the underlying stroma of
the colon is called neoplasia, because new tissue types are being formed (Fig. 9.2).
Some people use the term “neoplasm”, which is the product of neoplasia, collectively
for malignant tumours. A malignant tumour in the colon is certainly colon cancer,
potent of undergoing EMT and forming metastases in other organs.
In general, a neoplasm can also be benign. There are even cases, in which almost
normally looking cells are found in disparate places, a phenomenon called “meta-
plasia”. This example highlights the plasticity in the context of cancer development.
There is not a single way of tumour formation. For the sake of the example above,
the most common way of cancer generation can be depicted as follows [109]:
106 9 Cancer
normal hyperplastic dysplastic neoplastic

cytoplasm
nucleus
basal
membrane
Fig. 9.2 Different growth stages of a cells from benign (left) to malignant (right)
normal → hyperplastic → dysplastic → neoplastic → metastatic
Finding the origin of tumour cells is an important aspect to eradicate cancer. In

theory, the majority of the tumour mass could relate back to a single cell or multiple
different cells. Individual cells and their progeny are called “clones”, because they
are genetically very similar and link back to a common ancestor cell. So when
tumours are genetically homogeneous, it can be assumed that most of the tumour
cells originate from a single clone, and the tumour itself is thus monoclonal. If there
is more than a single dominating subset of tumour cells, the tumour is heterogeneous
and polyclonal (Fig. 9.3).
Tracing experiments have shown that most of the tumours are indeed of mono-
clonal origin. So a single cell outcompetes the others and soon dominates tumour
mass. But since cancer cells in general are rapidly dividing and evolving, new sub-
sets of tumour cells can emerge that deviate genetically and phenotypically from the
original clone [13], again highlighting cancer plasticity, which represents a major
challenge for the development of effective medications. How come, a disease such
as cancer is so diverse?
9.1.2 Causes of Cancer Development
Cancer is a multifactorial disease. The factors that can—directly or indirectly—lead

to cancer include:
A A A A A
A b A A A mono-
clonal
a A A A c A
normal
cells cancer
b
of evolution
origin B C C a B
c
B B B B B poly-
clonal
C C C C C
Fig. 9.3 Normal cell clones start to multiply unrestrictedly, cancer starts evolving. While a mon-
oclonal tumour is dominated by a single-cell clone, polyclonal tumours exhibit more than one
dominating clone
9.2 Pro et Contra Cancer 107
• Genetics The presence or absence of certain gene variants can alter the risk to
develop certain types of cancers. An example is the gene BRCA1, which encodes
for the breast cancer type 1 susceptibility protein. Mutations in BRCA1 represent
a heritable genetic predisposition to develop breast cancer, which can be tested
for [70].
• Life-style Whether we expose ourselves to cancer-inducing (“carcinogenic”) sub-
stances, such as tobacco smoke, is a question of lifestyle. On the other hand, phys-
ical activity and a well-balanced diet can serve as preventive measures lowering
the risk to develop cancer [55].
• Environment The effect of UV light from the sun, which increases skin cancer
susceptibility is well documented [30]. Besides, other environmental factors such
as pollutants can contribute as one out of many factors to the development of
cancer.
How much do these individual factors weigh in the formation of malignant tumours
is hard to quantify. There are interdependencies between genetics, lifestyle and envi-
ronment. You may live in an area with high UV exposure but your lifestyle features
extensive use of sun protection or interior activities. Or your genetics exhibit a muta-
tion in the melanocortin 1 receptor (MC1R) gene, which renders you even more
sensitive to UV light [30]. These examples highlight the complex interplay of multi-
ple factors. Each factor alone can either increase or decrease the risk to develop cancer
but they also affect one another. Adapting your lifestyle aids cancer prevention.
UV light can cause cancer because it induces mutations. The consequences of
those changes in the genetic code depend on where they occur. If mutations affect the
DNA repair machinery (see Sect. 2.4.2), the risk increases that errors are propagated.
The effect of mutations in other genes depends on the role of those genetic regions
in homeostasis under physiological conditions.
9.2 Pro et Contra Cancer
There is a fine balance between pro- and contra- (or anti-) carcinogenic signalling.
Genes that are linked to the promotion of cell growth and multiplication are the
origin of tumour formation, because if they are hyperactive, proliferation becomes
unrestricted. Genes that have been linked to the generation of cancer are called
oncogenes. A prime example for an oncogene is Ras. Ras stands for “Rat sarcoma”
and was discovered as a leukaemia virus gene capable of transforming normal cells
from rodents (e.g. rats and mice) into cancer cells [110]. The original (“wild-type”)
version of the gene encoding Ras is not yet considered an oncogene because it
requires the alterations present in the viral gene to become hyperactive. Therefore,
the original Ras is referred to as a proto-oncogene. Mutations in the original Ras
gene ortholog were soon after found in human tumours. These mutations render the
protein hyperactive and lead to uncontrolled proliferation (see Sect. 7.2.3). Negative
regulators of oncogene signalling can suppress tumour formation. These genes are
tumour suppressors. Prime examples are the dual-specificity phosphatase (DUSP)
108 9 Cancer
and the phosphatase and tensin homolog (PTEN), which are both negative regula-
tors of Ras signalling. Other tumour suppressor genes are involved in DNA repair
(see Sect. 2.4.2), such as BRCA1. Through various ways, mutations rendering tumour
suppressors dysfunctional contribute to erroneous cell-cycle control, continuous pro-
liferation and tumour formation.
9.2.1 Immortality and Programmed Cell Death
Once cells proliferate continuously, they will steadily do so in a dish in the lab and
can be utilized as “immortal” cell lines (see Sect. 7.1.1). Immortal does not mean
that individual cells never die, it just means that cells never cease to proliferate,
and the population is maintained. In other words, these cell populations can become
super old. How is this achieved? A common feature of tumours and immortal cell
lines seems to be an overexpression of telomerases (see Sect. 2.4.1). Telomerases can
counteract the shortening at the ends of chromosomes during DNA replication. A lot
of telomerase activity is needed when cells replicate their DNA rapidly to progress
through the cell cycle repeatedly [15]. Hyperactive telomerases in cancer cells are
of course bad for our health. In normal cells, hyperactive telomerases are associated
with healthy ageing and longevity.
On the contrary, a way to prevent a cell from proliferating uncontrollably and
forming malignant tumours is the induction of programmed cell death. This physio-
logical process is called apoptosis and has to be tightly balanced for the maintenance
of tissue integrity. Too little killing can cause malignant outgrowth, whereas too much
killing can affect healthy cells and cause lesions. Apoptosis is controlled in two ways:
• Extrinsically through ligands from the outside of the cell (to be killed). Extra-
cellular death-inducing signals include tumour necrosis factor (TNF) and the
TNF-related apoptosis-inducing ligand (TRAIL).
• Intrinsically through intracellular stress during DNA replication or during ATP
synthesis at mitochondria.
Both, extrinsic and intrinsic signals serve as upstream regulators of apoptosis cul-
minating in the activation of downstream effectors. Most prominent among those
effectors are proteases of the caspase family. Proteins have to be degraded in the
cell to be killed, and caspases do that job. Apoptotic cells are full of granules and
vesicles as they are partially digesting themselves. Remaining chunks are eaten up
by neighbouring cells some of them specialized phagocytes from the immune system
(see Sect. 8.2.1).
There are also negative regulators of apoptosis that prevent programmed cell
death, such as the Bcl2 family. Most of them function through binding and suppres-
sion of pro-apoptotic triggers at the outer membrane of mitochondria. If integrity
of mitochondria is preserved, there is no release of cytochrome c, and in turn no
activation of caspases. Without the activation of downstream effector caspases, pro-
grammed cell death is prevented. Cranking up the expression of negative regulators
of apoptosis is therefore commonly observed in cancer cells—no wonder Bcl2 is

named after B cell lymphoma 2.
There is a more splatter-like version of cell death called “necrosis”. While the
corpses of apoptotic cells are fragmented and eaten up by neighbouring cells, necrotic
cells swell up and explode thereby scattering their inner contents to the local sur-
roundings of the tissue microenvironment. Released substances can attract additional
immune cells with various (also promoting) consequences for tumour growth.
If cells are not dying but just partially digesting themselves in intracellular
lysosomes due to the lack of nutrients as energy sources, the process is called
“autophagy”—self-eating. Conceptually, autophagy also involves upstream regu-
lators and downstream effectors. These molecules are not the same as for apoptosis,
but there is some notable crosstalk [80]. While a starved tumour (i.e. deprived from
nutrients and growth factors) cannot hyperactively proliferate, it may become therapy
resistant [112].
9.2.2 Molecular Complexity Underlying Cancer
The concept of proto-/oncogenes and tumour suppressors as well as the regulation

of the different flavours of cell death highlight the molecular complexity underlying
cancer formation and its counteraction. There is a gradual process of a normal cell
transforming into a cancer cell ultimately giving rise to a malignant tumour. A single
gene, such as mutated Ras or BRCA1, can be sufficient to radically change multiple
properties of a cell and its progeny. Altered cell size, shape, metabolism and division
times indicate loss of proliferation control leading to the accumulation of tumour
mass.
Despite this observation, cancer does not fit the one-gene-one-disease paradigm.
An example for a disease, which is caused by a mutation in both alleles of a single
gene, is cystic fibrosis. The affected gene in cystic fibrosis is CFTR. Cancer is not
a single disease, it is multiple diseases, and it is not necessarily caused by the mal-
functioning of a single gene. Instead, multiple genes can be involved; in fact, entire
networks of genes [63]. And it is not always the same gene(s) that initiate the process.
Mutated BRCA1 can lead to tumour formation, but by itself, it is not necessarily the
cause of breast cancer. By hampering DNA repair, BRCA1 mutant most likely con-
tributes to erroneous other genes through which entire molecular machineries start
to go wrong leading ultimately to uncontrolled cell proliferation.
Due to tumour type diversity and complex gene networks involved, researchers
have long been striving to identify common features - the so-called hallmarks of
cancer—for the development of therapeutic interventions tackling those hallmarks.
110 9 Cancer
9.2.3 Hallmarks of Cancer
There are different hallmarks of cancer, which are commonly found during the estab-
lishment of a tumour [43]. As noted above, there are many individual exceptions to
those rules. Regardless, the hallmarks of cancer can be seen as the common denom-
inator of cancer:
Pro proliferation signalling

The information to grow and divide needs to be sustained for a tumour to form.
This is achieved through positive feedback loops of mitogen signalling stimu-
lating the source and neighbouring cells to progress in cell cycle (see Sect. 7).
Pathways that induce proliferation are constitutively (i. e. “constantly”) active
through mutations in the MAPK pathway. In addition, negative regulators such as
DUSP become dysfunctional through mutations. All these events lead to excessive
pro-proliferative signalling.
Contra growth suppression
There are several safety measures that keep a cell from proliferating too fast. Over-
activation of pro-proliferative signalling can trigger a cellular growth arrest known
as “senescence” [25]. Cancer cells adapt to sub-maximal mitogen signalling for
optimal proliferation avoiding the induction of growth arrest. Molecularly, this
can be achieved by a loss of function of tumour suppressors such as Rb or p53.
Pro invasion and metastases
If cells grow too densely, further proliferation is prevented by a phenomenon
called “contact inhibition”. In simple terms: Cells stop to multiply once they start
touching each other. Cancer often overcomes such inhibition, for instance by inter-
fering with anchoring junctions through cadherin (see Sect. 1.4.2), thereby also
loosening the extracellular matrix. In such cases, the tumour suppressor NF2 is
mutated [84]. Another important factor in this context is the transforming growth
factor beta (TGF-β), which is usually responsible to shut down cell prolifera-
tion. In the cancer context, TGF-β is known to induce epithelial-mesenchymal
transition (EMT) as a precondition for metastases.
Contra senescence and cell death
For cancer cells to become immortal, two barriers to proliferation need to be
overcome. Senescence and apoptosis. Upregulation of telomerases [15], and anti-
apoptotic genes such as Bcl2 [44], have been named as two measures for tumour
cells to proliferate continuously. It is worth noting that expression levels of telom-
erases can vary dynamically throughout the process of cancer development as pre-
malignant lesions in the human breast exhibited reduced telomerase expression
levels and shortened chromosome ends [22].
Pro blood vessel formation
For a sufficient supply of the growing tumour with oxygen and nutrients, blood
vessels need to penetrate the malignant tissue. The process of blood vessel forma-
tion is known as “angiogenesis”, which mostly happens during the development
of the embryo and in the adult organism predominantly in wound healing, besides
tumours. Some human cancers such as those in the kidney exhibit a very high
density of newly formed blood vessels [115]. Of note, already pre-malignant

tumours promote the formation of blood vessels [74], which was first believed to
be a property of macroscopically visible tumours only.
Not all the hallmarks are necessary or even essential to the generation of cancer.
Tumour blood supply alters local metabolic properties within and around the malig-
nant tissue, which changes also the immune landscape. Such overarching principles
are commonly observed:
Tumour metabolism
For a tumour cell to proliferate continuously, the cellular metabolism needs to be
reprogrammed. Normal cells use glucose as an energy source and first process it
to pyruvate in a reaction chain called “glycolysis” (i.e. the degradation of glucose).
If enough oxygen is present, normal cells then paste pyruvate to mitochondria (see
Sect. 1.3) for oxidation and further energy production in the form of ATP. Conversely,
cancer cells mostly stick to glycolysis even in the presence of sufficient amounts of
oxygen. What seems like an inefficient energy production strategy turns out to be
advantageous to fuel tumour growth. Cancer cells adapted to efficiently take up
glucose from their surrounding through high-affinity glucose transporters such as
GLUT1 [52]. Glucose uptake by the tumour is so high that it can be visualized by
a positron emission tomography (PET) scan with a radioactively labelled form of
glucose.
Once cancer cells start to hyperactively proliferate, oxygen supply in the tis-
sue might become limiting, which is a problem for normal cells, because they rely
on the oxygen for their energy metabolism but cancer cells are already adapted
and more independent from oxygen. In addition, low oxygen conditions result in
stress responses and apoptosis of normal cells but therapy resistance of metaboli-
cally adapted cancer cells [51].
Of note, tumour metabolism first and foremost shapes the (tumour) microen-
vironment of the respective tissue, which is referred to as the local surrounding
spanning only a few layers of cells around the growing tumour. This spatial vicinity
is directly altered by the cancer cells, e.g. in terms of glucose or oxygen concentration
or abundance of signalling molecules.
Tumour immunology
The metabolic properties of a tumour render the malignant tissue a niche for immune
cells. In fact, human tumours are infiltrated by cells of both the innate and the adap-
tive arm of the immune system to various degrees [36]. Ideally, for the human host,
immune cells recognise cancer cells as something foreign and kill them, e.g. through
cytotoxic CD8 T cells (see Sect. 8.3.3). Fortunately for the tumour, it has evolved
from normal cells of the body and therefore escapes immune surveillance or weakens
the immune response by means like exhaustion of adaptive T cells [111]. Among the
innate immune cells, tumour-associated macrophages have been described, interact-
ing with cancer cells, reciprocally stimulating one another thereby fuelling metastatic
dissemination of breast cancer [113].
112 9 Cancer
Intuitively, promoting inflammation of the tumour should aid the immune-

mediated eradication of cancer cells. But the example of the macrophages above
exemplifies that tumour cells recruit particularly cells of the innate immune system
to exploit them, e.g. as sources of growth factors and nutrients like in prostate cancer
[72].
These examples highlight the dual role of the immune system, which can both
suppress and enhance cancer development.
Tumour plasticity
The presence of immune cells within the tumour underlines the fact that malignant
tissues are no uniform array of cells. Even among cancer cells exists a considerable
heterogeneity. Reasons for those differences between cancer cells within the same
tumour are mostly due to genetics and the microenvironment [73].
• Genetics as a reason for tumour plasticity: In general, chromosomal instability

of cancer cells is achieved through mutations in key mediators of genomic integrity
such as BRCA1 involved in DNA repair, or mutations in the tumour suppressor
p53, the original “guardian of the genome” [65]. Systemic defects in genome
maintenance are key for cancer cell-to-cell variability and tumours to develop.
• Microenvironment as a reason for tumour plasticity: The physical and chem-
ical properties at the core of a tumour are very different from the properties at
the outer margins. The proximity around blood vessels is different from hubs
of cytokine-secreting immune cells. This diversity gives rise to cancer cells of
various shades, e.g. with respect to their metabolic capacity [96].
Consequence of cancer cell heterogeneity is plasticity, which means the ability to

adapt. If there are many different cancer cells within a tumour, the chance is high
that one (clone) of them is well adapted to a certain condition such as a treatment.
Metabolic changes for instance can induce cancer therapy resistance. Nutrient star-
vation may lead to autophagy in cancer cells and concomitant growth arrest—a state
called “dormancy” eventually resulting in resistance to clinical interventions [56].
The dormancy state is reversible meaning that cancer cells can re-enter hyperactive
proliferation once conditions permit.
Reversibility of functional states is an important aspect of tumour plasticity. A
prime example is the epithelial-mesenchymal transition (EMT), which cancer cells
transiently undergo for metastatic dissemination. Once these cells reached a distant
tissue to colonize, they need to reverse the EMT process and perform mesenchymal-
epithelial transition (MET). In the case of lung cancer, the full spectrum between
EMT and MET phenotype was revealed by single-cell mRNA sequencing [54]. Such
analyses hint to a huge challenge in cancer therapy. As tumours evolve and adapt
quickly and diversely, it is very hard to treat them.
9.3 Combat Cancer 113
9.3 Combat Cancer
The fight against cancer begins before its actual occurrence by raising awareness
and cancer prevention. Early detection is an important indicator of a good progno-
sis. Regular check-ups have to be performed routinely. Diagnosis means the actual
detection of a tumour, be it benign or malignant, using technologies like histology
(i.e. inspection of the tissue’s structure and composition) or PET scans as mentioned
above. Besides detection of cancer cells in respective organs, the cancer patients need
to be stratified. Stratification means that the type and stage of cancer are defined,
which determines an efficient treatment.
9.3.1 Current Therapeutic Approaches
There are different ways to treat cancer:
• Surgery: The malignant tissue is surgically removed. The biggest problem is that
it is hard to ensure that all cancer cells were cut out and nothing is growing back.
• Chemotherapy: Dose of chemical drugs like Cisplatin, which targets rapidly
dividing cells by binding DNA thereby inhibiting DNA replication. Those drugs
are not very specific and affect also other cells that divide quickly such as within
hair follicles. This is a reason why chemotherapy patients lose their hair. Other
drugs are small molecules and usually kinase inhibitors targeting growth-factor
signalling.
• Irradiation: High doses of locally confined radiation aim at killing tumour cells.
Most challenging is to specifically hit only cancer cells and as little as the possible
surrounding tissue, particularly if the target is moving as in breathing lung cancer
patients.
• Immunotherapy: Engineering mostly the adaptive immune system for the recog-
nition and targeted eradication of cancer cells. A prime example involves the
blockade of negative regulators of the immune response, e. g. CTLA-4 [67].
9.3.2 the Future of Personalized Medicine
As molecular networks are part of the aetiology (i.e. origin and cause) of the disease,
they can be part of the solution to cure cancer. The entire genome, transcriptome
or proteome can now be quantitatively assessed (see Sect. 3.4.2). These “omics”
data become more and more available for individual patients even at multiple time
points throughout the course of the disease. Single-cell information depicts cancer
cell heterogeneity. What needed now, are mathematical formalisms to integrate those
big data into innovative, interdisciplinary approaches to offer a new generation of
personalized medicine.
Each cancer patient has an individual history of the disease. Powerful machine
learning approaches can be utilized to address these in a patient-specific manner
114 9 Cancer
[78]. And therefore, we need you, dear reader. With your expertise in mathematics,
computer sciences, physics and engineering, biomedical research will benefit largely
from your conceptual input supporting us on our ultimate quest to combat cancer.
Summary
• Cancer is not just one disease, but there are many different types and subtypes
depending on when and where tumours are initiated in the body.
• Cancer occurs individually in every patient depending on genetics, lifestyle
and environment.
• Despite common hallmarks of cancer, combating it requires a personalized
approach of prevention, diagnosis and treatment.
Glossary
antigen Agent that can evoke an immune reaction.

apoptosis Programmed cell death including cell shrinkage, fragmentation and coor-
dinated decay with remaining debris being eaten up by neighbouring cells.
autophagy Cellular self-digestion to remove excessive contents through degrada-
tion and recycling.
biopsy Taking cells out of the living organism.
carcinogenesis Process of cancer development induced by a carcinogenic substance.
chaperone Proteins that guide correct folding of other proteins or preventing them
from the formation of aggregates.
clone Cell with very similar genetic code and shared ancestry among other clones.
consensus A sequence, e.g. of DNA, which is very similar in different species and
thus evolutionary conserved.
cyclin D2 Molecule that is up- and down-regulated during the progression of the
cell cycle.
cyclin G2 Molecule that is up- and down-regulated during the progression of the
cell cycle.
cytokine Secreted protein that affects nearby cells that carry a cognate cytokine
receptor.
dysplasia Dysplastic growth of a tissue results in abnormal cells, for instance, with
larger nuclei.
endomembrane system Highly interconnected network of membrane-enclosed
organelles including endoplasmatic reticulum, Golgi apparatus, lysosomes, per-
oxisomes and endosomes of eukaryotic cells.
enzyme A molecular machine that catalyses a certain chemical reaction.
epithelium Thin tissue sheet forming the outer surface of our body and lining inner
cavities such as the gut.
gene Section of DNA that is transcribed to an RNA molecule that either exerts a
task by itself or is translated to a protein with a specific function.
HeLa Cervix cancer cell line derived from the patient Henrietta Lacks.

https://doi.org/10.1007/978-3-662-65357-9
116 Glossary
helicase An enzyme that catalyses the unwinding of a helix, e.g. the DNA double
helix.
homoeostasis Maintenance of a physiological process under steady-state condi-
tions.
hyperplasia Hyperplastic growth of a tissue results in an overall increase in cell
number such that there are more of all cells.
kinase An enzyme catalysing the addition of a phosphate group to its substrate.
lesion Damage or partial loss of tissue resulting from injury or disease.
ligand Molecule recognized by a receptor.
malignancy Presence of a malignant tumour, synonymous for cancer.
metaplasia Almost normally looking cells are found in disparate places.
mitogen Extracellular molecule that stimulates cell-cycle progression and division.
mutation Change in the genetic code deviating from the original version.
Myc Origin: Myelocytomatosis, a transcription factor involved in cell-cycle pro-
gression.
necrosis Cell death induced by a cellular injury leading to spillage of cytoplasmic
contents to the surroundings.
neoplasia A process leading to the formation of new tissue as cell types are found
in disparate places creating a neoplasm.
non-homologous end joining Mechanism of DNA repair.
nucleus Membrane-enclosed compartment within a cell storing and organizing her-
itable information as DNA.
oncogene A gene that encodes for a protein responsible for uncontrolled growth and
division of cells. Overexpression or hyperactivation of those genes often leads to
cancer. E.g. Ras.
ortholog Gene version in different species, such as the Ras gene in humans and
mice encoding for a protein with the same function but slightly different DNA
sequence.
p53 Tumour suppressor, “guardian of the genome” as the main regulator of DNA
damage response, aka tumour protein TP53.
pathogen A substance, such as a bacterium or a virus, that can cause a disease.
phosphatase An enzyme catalysing the subtraction of a phosphate group from its
substrate.
proliferation Process of repeated cell divisions upon completed cell cycles to
increase cell numbers.
protease An enzyme that degrades proteins by cleaving peptide bonds between
certain amino acids.
proteasome A cellular machinery that degrades proteins.
pSTAT5 Phosphorylated and thus activated signal transducer and activator of tran-
scription 5.
quiescent Cell stopped in cell cycle and thus not dividing.
Raf Origin: rapidly accelerated fibrosarcoma, family of cancer-related serine/
threonine protein kinases.
Ras Origin: Rat sarcoma, small molecule capable of GTP hydrolysis, involved in
signal transduction.
Glossary 117
receptor Protein structure incorporated into the cellular membrane to bind extra-
cellular ligands and activate intracellular processes.
senescence Cellular ageing and deterioration leading to dysfunction and eventually
growth arrest.
SNARE Transmembrane proteins that face the cytoplasmic side of vesicles and
target membranes and wind up to catalyse fusion.
syncytium A cell containing multiple nuclei, usually at an early stage of embryonic
development.
transcription factor Molecule capable of DNA binding for control of gene expres-
sion.
tumour suppressor A cellular factor preventing cells from uncontrolled growth
and division thereby blocking tumour formation, e.g. p53, often found mutated or
inactive in cancer. ubiquitination Addition of a single or several small molecules
of ubiquitin to a protein that is thereby labelled for degradation.
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3.4 Measuring Cellular Content. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

6.6 Emergent Properties as Output . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

4E-BP 4E-binding protein

GFP green fluorescent protein

STAT5 signal transducer and activator of transcription 5

© Springer-Verlag GmbH Germany, part of Springer Nature 2022 1

1.1 Cells and Their Biochemical Composition

• carbohydrates (“sugars”, “carbs”),

These macromolecules are assembled from building blocks: monosaccharides, fatty

Table 1.2 Size scales of cellular components. 1 nm = 10−9 m, 1 µm = 10−6 m

1.2 The Cell’s Skeleton

1.2.1 Actin Filaments Form Flexible Meshes

Actin filaments with a diameter of ≈ 7 nm are chains of actin monomers. Actin

1.2.2 Intermediate Filaments for Robustness

1.2.3 Microtubules as Tracks for Intracellular Transportation

1.2.4 Motor Proteins to Position Cellular Cargo

1.3 Organelles of a Cell

A useful representation of these organelles can be found under https://smart.servier.

1.4 Cell-To-Cell Contacts

1.4.1 Occluding Junctions as Diffusion Barriers

Occluding junctions, as the name implies, close up cellular layers. An epithelium is

1.4.2 Anchoring Junctions for Adhesion

1.4.3 Gap Junctions for Communication

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2.1 Structural Elements

basei basei+1 basei+2 basei+3

sugar P sugar P sugar P sugar P

The sequence of nucleosides is linked by phosphodiesters creating a regular “back-

2.2 Organization of Genetic Information

2.2.1 Gene Elements, Histones and Chromatin

The heterochromatin revealed a higher level of a structural organization unit of the

2.2.2 Chromosomes, Genes and Heredity

Fig. 2.3 Schematic

Fluorescence in situ Hybridization aka. FISH

Another method to investigate distinct sites of a chromosome is called chromo-

2.3 The Genetic Code

• Diversity of amino acids required.

2.4 DNA Replication

For maintenance of DNA integrity and genomic stability throughout generations,

1. Licensing: The origin of replication is recognized by an origin recognition com-

Fig. 2.4 DNA double helix

DNA double helix

2.4.1 Molecular Components Involved in DNA Replication

• Helicase unwinds double-stranded DNA under ATP hydrolysis.

a single round of replication of the cellular genome. To prevent these mismatches

2.4.2 DNA Recombination to Secure Integrity and Diversity

• Base excision-repair: Most common in humans is a point mutation changing the

1. Homologous recombination: Overhangs are created by cutting out opposing

Fig. 2.6 A DNA

6.6 Emergent Properties as Output . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73