Cell Adhesion and Development

Web sites worth visiting.

♦ Extra information on cell adhesion molecules: quite a good summary on cadherins,
selectins and an overview of integrin adhesion.
http://www.cytochemistry.net/Cell-biology/adhesion_molecules.htm
♦ Go to: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=books
and open the on-line version of “Molecular Biology of the Cell” Alberts et al. and look at
chapter 19 - key “cell-cell adhesion” into the search box will get you there.
Similarly look at “Developmental Biology” S.F. Gilbert
References:
Friedl et al. 2004. Collective cell migration in morphogenesis and cancer. Int. J. Dev.
Biol. 48:441-449.
Halbleib, J.M. & W.J. Nelson. 2006. Cadherins in development: cell adhesion, sorting and
tissue morphogenesis. Gene Dev. 20:3199.
Steinberg, M.S. 1996. Adhesion in development: An historical Overview. Devel. Biol.
180:377.
Danen, E.H.J. & A. Sonnenberg. 2003 Integrins in regulation of tissue development and
function. J. Pathol. 201:632.
Cell Adhesion and Development: Introduction
Without cell adhesion there would be no multicellular organisms.
In tissues cells are organised into very precise and distinctive patterns
controlled by specific cell adhesion. Cell adhesion is how cells talk to each other.
It is important for:
• tissue formation during morphogenesis
• cell migration
• regulation of : cell proliferation, gene expression and cell death
(apoptosis)
A large number of genes encode cell adhesion molecules, ~4-5% of human
genome is dedicated to adhesion.
Stable Adhesion: Multiple binding events frequently involving different adhesion
molecules binding their receptors (ligands).
Specificity: achieved by different combinations of adhesion molecules.
Cell-cell adhesion: very stable ⇓ long lasting (cells in tissues)
⇓
very transient ⇓ cells interacting with blood vessel walls
Cell Adhesion: Introduction
♦There are different families of adhesion molecules:
• vary in molecular structure, and
• perform different tasks.
♦ Cell-cell adhesion is the result of multiple, different adhesion molecule-ligand
binding events ⇒ Co-operation between different adhesion molecules.
♦ Considerable redundancy → adhesion is a fundamental process essential for
many physiological processes.
♦ Not all adhesion molecules are consistently expressed on cell surfaces.
Expression varies according to:
• differentiation state of the cell,
• stage of cell cycle,
• cytokine (growth factor) activation of a cell, or
• cell activation caused by signals arising from other adhesion molecules
having bound their ligands.
♦ During embryogenesis cell adhesion is dynamic → what regulates the functional
state & dynamics of adhesion molecules is as important as their expression.
Cell Adhesion: Introduction
Questions that underlie the study of morphogenesis:
1. How are tissues formed from a population of cells?
2. How are organs constructed from tissues?
3. How do organs form in particular locations and how do migrating cells
reach their destination?
4. How do cells and organs achieve polarity?
5. How do organs and cell grow and how is their growth coordinated?
Many of the answers involve the properties of the cell surface and the
molecules/proteins on these surfaces.
Differential cell affinity
How did this concept become understood?
In the mid 1950s - disassociate embryonic tissues into single cells & these cells
would reaggregate.
Cell suspensions from the 3 germ layers of the embryo behave remarkably when
recombined.
Differential cell affinity hypothesis

Upon reaggregation epidermal cells go to the outside and mesodermal cells

(neural cells) move inside. That is,
(1) cells sort out to reconstitute the tissue of origin,
(2) the position of the sorted populations reflect their positions in the
embryo. Interpreted in terms of selective affinity, and
(3) selective affinities change during development.
Differential cell affinity hypothesis
Tissue reconstruction from cell suspension

From Monroy and

Moscona 1979

Reconstruction of skin from a suspension of skin cells from a 15-day embryonic mouse. (A)
Section through intact embryonic skin, showing epidermis, dermis, and primary hair follicle. (B)
Suspension of single skin cells from both the dermis and the epidermis. (C) Aggregates after
24 hours. (D) Section through an aggregate, showing migration of epidermal cells to the
periphery. (E) Further differentiation of aggregates (72 hours), showing reconstituted
epidermis and dermis, complete with hair follicles and keratinized layer.
Differential cell affinity hypothesis

What forces direct cell movements during morphogenesis?

Differential cell affinity hypothesis: cells interact so as to form an aggregate
with the smallest interfacial free energy - cells arrange themselves in the most
thermodynamically stable pattern.

Cells A & B have different patterns of molecules on their surfaces and hence
differences strengths of adhesion.

Sorting will occur with A

If A = A ≥ B= A or B= B becoming central

If A = A ≤ B= A Random mix - no sorting

If A = A >> B= A A and B will form separate aggregates.

This idea was tested experimentally and shown to be valid.

Differential cell affinity hypothesis
Hierarchy of cell sorting in order of decreasing surface
tensions. The equilibrium configuration reflected the
strength of cell cohesion, with the cell types having the
more cell cohesion segregating inside the cells with
less cohesion. (From Foty et al. 1996)

Simplest form of hypothesis: Cells could

have the same type of “glue” on their
surface. The amount of “glue” or its
differential distribution across the cell
surface could cause differences in the
number of stable contacts between cell
types.

Differences in strength of cohesion is

caused by differences in the types and
numbers of cell adhesion molecules on cell
surfaces.
Adhesion molecules

Different types of adhesion molecules exist to do different tasks.

All cell adhesion molecules spend some time at the cell surface.
All act by binding to particular ligands - these ligands may be:
• homophilic (i.e. adhesion molecules bind themselves on another cell
surface) or,
• heterophilic (i.e. bind to a molecule of a different structure).
May/may not be Ca++-dependent in their binding activity.
May link a cell to the extracellular matrix and have a role in cell migration
(the integrins), may form very stable cell-cell adhesions (cadherins) or
may be involved in very rapid transient interactions (selectins).
Main adhesion molecule families are:
• Cadherins
• Integrins
• IgCAMs (Immunoglobulin superfamily members)
• Selectins
Cadherins
Cadherins: Large family of cell
surface proteins (>100
members) that mediate Ca++ -
dependent cell-cell adhesion.
♦ All members have the
characteristic cadherin
extracellular repeat.
♦ They have a critical role in
morphogenesis.
- cell recognition & sorting
- boundary formation and maintenance
- coodinated cell movements
♦ In adults cadherins are responsible for tight cell-cell associations in tissues.
♦ They are intimately associated with the cytoskeleton, interacting via other
proteins with both microfilaments (actin) and intermediate filaments (keratins).
♦ Cells express multiple cadherins- specificity of adhesion is due to the
different combinations of cadherins expressed.
Cadherins
Cadherins join cells by binding cadherins on
another cell: cadherin zipper.
Cadherin-catenin complex links to actin
cytoskeleton.

• Classical cadherins are concentrated

at adherens junctions - modulate
adhesion through dynamic interactions
with actin cytoskeleton.
• Adherens junctions hold epithelial
cells together.
Adherens junctions, in the form of adhesion
belts, between epithelial cells in the small
intestine. The beltlike junction encircles each
of the interacting cells.
Cadherins
Blocking cadherin function can prevent the formation of tissues and cause cells
to disaggregate.

Depletion of EP-cadherin mRNA in the

Xenopus oocyte results in the loss of
adhesion between blastomeres and the
obliteration of the blastocoel. (A) control
embryo; (B) EP-cadherin-depleted
embryo. (From Heasman et al. 1994)

The importance of N-cadherin in the separation of

neural and epidermal ectoderm. At the 4-cell stage,
the blastomeres that form the left side of the
Xenopus embryo were injected with an mRNA for
N-cadherin that lacks the extracellular region of the
cadherin. This mutation blocks N-cadherin
function. During neurulation, the cells with the
mutant protein did not form a coherent layer
distinct from the epidermis. (From Kinter et al.,
1992).
Cadherins

Different cadherins are expressed on different tissues both in the adult and in
the embryo. For example,
• E-cadherin: on all mammalian embryos, in adults restricted to epithelia
• P-cadherin: on placenta, sticks placenta to uterus.
• N-cadherin: on cells of developing nervous system- is first seen on mesodermal
cells in the gastrulating embryo as they lose their E-cadherin expression.
• R-cadherin: critical in formation of the retina
• B-cadherin: on many nerual structures
• EP-cadherin (C-cadherin): maintains adhesion between the blastomeres of the
Xenopus blastula and is required for the normal movements of gastrulation
• Protocadherins differ from the classic cadherins by lacking connections to the
cytoskeleton through catenins. Are very important in separating the notochord
from the other mesodermal tissues during Xenopus gastrulation.
Cadherins can engage in homophilic adhesion, or Different cadherins may bind
to each other - strength of cadherin-cadherin binding that mediates formation
of many embryonic structures.
Cadherins
Cadherins can engage in homophilic adhesion, or different cadherins may bind to
each other.
- strength of cadherin-cadherin binding is determined by both the quantity and
type of cadherin expressed.

A & B Cells transfected with

different levels of N-cadherin.
Red cells have 50% more N-cad.
on their surface than green cells.
C. Cells expressing B-cad.
(green) and R-cad (red) - clear
segregation.

P-cad cells (red); E-cad cells

(green).
If more P-cad than E-cad, P-cad
cells are internal, if equal levels
no sorting, if more E-cad than P-
cad E-cad internal
Pictures from Duguay et al. 2003.
NB: the Differential cell affinity hypothesis. Devel. Biol. 253:309.
Integrins

Integrins: Involved in cell-extracellular matrix

adhesion and cell-cell adhesion.
• Structure: heterodimer consisting of two
transmembrane glycoprotein subunits (α and
β), which are non-covalently bound.
• Functional integrins always have: one α subunit
and one β subunit.
• Both subunits contribute to ligand binding.
• About 18 α subunits and 8 β subunits have been
identified, giving ~24 unique integrins.
→ A large number of possible specificities.
• Ligand binding is divalent cation dependent
(Ca ++ , Mg++ and Mn++)
• Common ligands are: the ECM proteins
fibronectin, vitronectin, collagen and laminin
(recognised by multiple integrins) or members
of the Ig superfamily.
Integrins
Integrins are found
in invertebrates as
well as vertebrates.
Greatest diversity is
in mammalian
integrins.
Red: laminin binding
Blue: RGD binding
(fibronectin & vitronectin)
Integrins connect the actin cytoskeleton to
extracellular matrix proteins outside the
cell. They bind talin and α-actinin which
connect to the actin cytoskeleton.
Dual binding enables the cell to move by
contracting the actin microfilaments
against a fixed extracellular matrix.
Key integrin function: cell migration

The clustering of integrins - formation of focal adhesions

Integrins

Cells in culture labeled with antibodies against both actin (green)

and vinculin (red). Vinculin is located at focal adhesions, which is
where bundles of actin filaments terminate at the plasma
membrane. (B) Some of the proteins that form focal adhesions.
(from Geiger et al. 1983.)
Signals generated at focal adhesions help to
regulate cell division, growth and survival, as well
as being important for cell migration.
The local activation of focal adhesion kinase (FAK) and
other protein kinases is shown as a red stain (mAb to
phosphotyrosine). Note the red stain is concentrated and
overlaps with the actin filaments terminating at focal
adhesions. Picture (Courtesy of Keith Burridge
Integrins

♦ Integrins are key molecules during early development, having roles in

fertilization, gastrulation, implantation, placentation, nervous system
formation, myogenesis and blood vessel formation.
e.g. β1 integrin knock-out mice develop normally until the blastocyst stage
but die soon after initiating implantation → delayed growth of inner cell
mass and absence of primitive endoderm migration.
e.g. injection of mAbs directed against β1 integrins or RGD peptides at the
beginning of neurulation → interfere with neural crest cell migration,
and…… injection of antisense oligonucleotides against integrin α chains
induce neural crest/neural tube abnormalities ⇒ importance of both α and
β chains.

♦ Integrins also regulate extracellular matrix (ECM) assembly →proper

interactions of cells with ECM is critical for correct development.
e.g. integrin α5β1 (fibronectin receptor) is involved in fibronectin assembly,
and α3β1 (laminin receptor) is involved in the formation/maintenance of
basememt membranes.
 Integrins

Fibronectin
and amphibian
gastrulation.

(A) Immunofluorescence reveals a network of fibronectin on the basal surface of the prospective
ectodermal cells lining the blastocoel roof in the salamander embryo. Scanning electron
micrographs of normal (B, C) and abnormal (D, E) salamander gastrulation. The blastocoel in (D)
and (E) was injected with the cell-binding fragment of fibronectin (RGD). (B) Section during mid-
gastrulation. (C) The yolk plug toward the end of gastrulation. (D, E) Final stages of arrested
gastrulation: the mesodermal precursors, having bound RGD peptides, cannot recognize the
normal fibronectin-lined migration route. The archenteron fails to form, and the noninvoluted
mesodermal precursors remain on the surface. (A from Boucaut et al. 1985; B-E from Boucaut et
al. 1984)
IgCAMs
Ig-superfamily includes over 100
members many of which are cell
Fig (B) N-CAM
recognising adhesion molecules. All have one or
another N-CAM more immunoglobulin (Ig)-like domain.
molecules on a
different cell • Ig-like domains are β-sheets stabilised
(homophilic by di-sulphide bonding.
ligand).
• Ig domains are resistant to proteases
and are adaptable for the presentation
of recognition domains.
• FNIII repeat, in most IgCAMs, is a ~90 amino acid motif
related to domains in fibronectin
• IgCAMs recognise both homophilic and heterophilic ligands.
• Integrins are frequently heterophilic ligands for Ig-
superfamily members, e.g. ICAM binds to β2-integrins on
blood cells;
• Ca++ dependence for ligand binding is variable.
• N-CAM is the most abundant Ca++ -independent cell-cell
adhesion molecule in vertebrates.
 IgCAMs
• N-CAM exists in multiple isoforms generated by
alternative splicing of an RNA transcript from one
gene.
• N-CAM180: neurons late in development,
• N-CAM140: on neurons and glia, mediates neurite
outgrowth (on migratory growth cones & axon shafts of
developing neurons)
• N-CAM120: predominantly on glial cells.

• Some N-CAMs carry chains of sialic acid -

these chains prevent adhesion rather than
cause it. Polysialylation is regulated
developmentally, decreasing in the adult.
• N-CAM knock out mice develop with defects
in neural development and neural tube
closure.
• A number of other Ig-super family members
are implicated in neuron growth and
Four of the different forms of N-CAM
guidance - L1 being the best studied.
IgCAMs

L1 binds β1-integrins & αvβ3 (there is a RGD

motif in the 6th Ig-domain) and other IgCAMs
as well as binding itself.
N-CAM blocking antibody studies show a role
in neurite outgrowth, neuroblast migration and
guidance of retinal cell axons.
L1 mutations: variety of neural defects
including mental retardation. L1 knock-out
mice show axon guidance errors in the
corticospinal tract.

IgCAMs mainly act by fine tuning adhesive

interactions that initially involve cadherins.
e.g. in rodent pancreas formation of islets of
Langerhans, blocking cadherin function → no
cell aggregation, blocking N-CAM function →
The main neural IgCAMs no sorting so disorganised islets form.
Selectins
The Selectins: There are three selectins-
• E-selectin, found exclusively on endothelia
• L-selectin, found on all circulating leukocytes
except activated T-lymphocytes
• P-selectin, found in secretory granules of platelets
and endothelial cells.
• All are structurally closely related having, at their
N-termini a carbohydrate recognition (C-type
lectin) domain and variable numbers of repeats
related to complement regulatory proteins.
• Their ligands are carbohydrates presented on
glycoproteins, their ligands are,
• E-selectin: sialylated Lewis X (SLeX) & Slea
• P- & L-selectin: SLeX, Slea & sulfated
polysaccharides.
• The way these carbohydrates are presented on the glycoprotein helps to
regulate selectin binding.
Selectins

Selectins are noted for

their role in white blood
cell (leukocyte) migration
into the tissues during
inflammation.
Leukocyte migration is
controlled by the
interactions that occur
between the leukocyte and
the vascular endothelia.
Different adhesion
molecules are involved at
“Inflammation: The Leukocyte Adhesion Cascade”
each stage.
http://bme.virginia.edu/ley/index.html

L-selectin also functions outside of vasculature - role in embryo implantation.

 Selectins
What makes an embryo stick? ….L-selectin does!!
L-selectin initiates interactions of human
blastocysts with the uterine lining.
Carbohydrates on the surface of the receptive
uterus are up-regulated. Trophoblast cells of
blastocyst begin to express L-selectin. Under
sheer stress cytotrophoblasts bind to the
epithelial portion of the receptive uterus by a L-
selection mediated mechanism.

A. Before hatching
little L-selectin
expression on human
trophoblast, B & C.
After hatching bright Cytotrophoblast cells adhere to
L-selectin staining. D. endometrial biopsies and stain with
Phase contrast L-selectin (A & B). Adhesion was
image of C. blocked with a mAb directed against
L-selectin (C - F). Controls C & E;
with mAb D & F.

Pictures from: Genbacev et al. 2003 Science 229:405.

 Collective cell migration
During morphogenesis on many occasions cells move as sheets, e.g. neural crest
migration: membrane ruffling at the free edge, whilst cell-cell junctions are intact.

Emigration of One leading cell guides the group, while rear

multicellular retraction is executed by several cells.
epithelial cell (Carcinoma cells moving in a collagen matrix)
strands into a Pictures from Friedl et al. 2004 Int. J. Dev. Biol.
collagen matrix. 48:441

Collective cell migration: cells keep their cell-cell contacts and migrate as
chords or strands into the tissues.
→both cell-cell adhesion and cell-matrix adhesion, strength of both types of
adhesion need to be balanced for movement of the cell cluster.
Collective cell migration
Single Cell movement requires:
• an interaction with the substrate
via adhesion molecules (integrins)
binding extracellular matrix
proteins
• cell extensions (lamellipodia)
propelled by actin polymerisation in
the cell cortext,
• shortening of membrane tethered
actin filaments causes cell
contraction and
• retraction of the rear of the cell
relaxes the tension caused by
extension of the lamellipodia.
• focal contacts may be resolved at the trailing edge by affinity modulation of
integrins → in migrating cells focal adhesions are dynamic structures that are
constantly being remodeled in size and shape.
Collective cell migration
β1 & β3 integrins
cluster at ruffling
edges.
Leading cells
generate actin and
integrin-mediated
traction.
Cadherin mediated
cell-cell adhesion
with IgCAMs aiding from Friedl et al. 2004 Int. J. Dev. Biol. 48:441
in interactions →
a cortical actin network extends along cell-cell junctions into the cell cluster.
Shortening of membrane tethered actin filaments causes the cell cluster to
retract.
Surface proteases (members of the MMP family) execute local proteolysis →
space for forward moving cell body to penetrate ECM scaffolds.
For cell detachment integrins are either released into the matrix or
internalised following affinity modulation which decreases adhesion.
Collective cell migration

Differential expression and

distribution of E-cadherin and β1-
integrins in a multicellular cluster
from a melanoma explant (A & B).
(cadherin: red; integrin: green
Collagen fibres :blue)
∗ Region of partial matrix
degradation and remodelling at the
trailing edge.
from Friedl et al. 2004 Int. J. Dev.
Biol. 48:441

Maintenance of front rear

asymmetry requires specific
mechanisms of cell-cell
communication.
↑cell-cell adhesion converts dispersed cells towards collective cell migration.
↓ cell-cell adhesion by blocking cadherins causes the disruption of cell
collectives → individual cell migration.
Adhesion molecules: Summary
Very stable cell-cell ⇓ Cadherin-cadherin
interactions mediated
⇓
Fine-tuning of cadherin ⇓ IgCAMs engaged in
mediated cell-cell ⇓ homophilic binding
interactions
⇓
Interactions that ⇓ Integrins/IgCAMs
allow cell movement binding their ligands
⇓
⇓
Very transient cell- ⇓ Mediated by selectins
cell interactions binding their ligands
⇓
Adhesion molecules play essential roles in the developing embryo.
Different adhesion molecules have different roles and multiple adhesion
molecules are important for many of the developmental pathways.
Different adhesion molecules act co-operatively to perform very complex
processes.