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    Nuclear Trans Lec 23 Tjian

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    abhishekbsnlbsnl
    The document discusses nucleocytoplasmic transport, including the role of the nuclear pore complex in facilitating the bidirectional transport of molecules and macromolecular complexes between the nucleus and cytoplasm. Specific signals on transport cargoes are recognized by importins and exportins to mediate their translocation through the nuclear pore complex. The GTPase Ran plays a key role in regulating the binding and release of transport cargoes by importins and exportins depending on whether Ran is bound to GTP in the nucleus or GDP in the cytoplasm.

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    Nuclear Trans Lec 23 Tjian

    Uploaded by

    abhishekbsnlbsnl
    9 views23 pages
    The document discusses nucleocytoplasmic transport, including the role of the nuclear pore complex in facilitating the bidirectional transport of molecules and macromolecular complexes between the nucleus and cytoplasm. Specific signals on transport cargoes are recognized by importins and exportins to mediate their translocation through the nuclear pore complex. The GTPase Ran plays a key role in regulating the binding and release of transport cargoes by importins and exportins depending on whether Ran is bound to GTP in the nucleus or GDP in the cytoplasm.

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    _Nuclear Trans lec 23 Tjian

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    The document discusses nucleocytoplasmic transport, including the role of the nuclear pore complex in facilitating the bidirectional transport of molecules and macromolecular complexes between the nucleus and cytoplasm. Specific signals on transport cargoes are recognized by importins and exportins to mediate their translocation through the nuclear pore complex. The GTPase Ran plays a key role in regulating the binding and release of transport cargoes by importins and exportins depending on whether Ran is bound to GTP in the nucleus or GDP in the cytoplasm.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    9 views23 pages

    Nuclear Trans Lec 23 Tjian

    Uploaded by

    abhishekbsnlbsnl
    The document discusses nucleocytoplasmic transport, including the role of the nuclear pore complex in facilitating the bidirectional transport of molecules and macromolecular complexes between the nucleus and cytoplasm. Specific signals on transport cargoes are recognized by importins and exportins to mediate their translocation through the nuclear pore complex. The GTPase Ran plays a key role in regulating the binding and release of transport cargoes by importins and exportins depending on whether Ran is bound to GTP in the nucleus or GDP in the cytoplasm.

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    of 23

    THE NUCLEUS & NUCLEOCYTOPLASMIC TRANSPORT

    1. Introduction
    a. The nucleus
    b. The nuclear envelope
    c. Transport cargoes

    2. The solid phase: the nuclear pore complex (NPC)

    3. Signals
    a. Nuclear localization signals (NLSs)
    b. Nuclear export signals (NESs)

    4. The soluble phase: nuclear transport factors


    a. Importins and exportins
    b. The GTPase Ran

    5. Mechanism of transport through the NPC

    6. Segregation of nuclear components during cell division


    1. Introduction a. The Nuclear Envelope
    b. Transport Cargoes

    5SrRNAs
    Ribosomal
    Subunits
    Nuc le olus Ribosomal
    mRNAs Proteins
    rRNAs

    Proteins
    snRNAs
    snRNAs snRNPs
    Proteins

    snRNPs
    tRNAs

    Karyophilic protei
    ns
    (NLS)
    Ions
    Small molecules
    (<60kD)
    2. The solid phase: the nuclear pore complex (NPC)

    Cytoplasmic side

    Nuclear side
    The nuclear pore has a 9nm wide aqueous diffusion channel
    3. Signals: how were they identified?
    4. The soluble phase a. Importins and Exportins

    IMPORT EXPORT

    IMPORT EXPORTIN
    IMPORTIN EXPORT
    CARGO
    CARGO

    CYTOPLASM CYTOPLASM

    NUCLEUS NUCLEUS

    EXPORT
    EXPORTIN
    IMPORTIN CARGO
    IMPORT
    CARGO
    4. The soluble phase b. The GTPase Ran

    CYTOPLASM
    Ran-GDP
    RanGEF:
    Pi GTP
    RanGAP 1 RCC1
    H2O GDP

    Ran-GTP
    NUCLEUS
    4. The soluble Ran regulates substrate binding and release
    phase

    IMPORT EXPORT

    IMPORT EXPORTIN
    IMPORTIN EXPORT GDP
    CARGO CARGO
    GDP

    RanGTP-hydrolysis and export cargo release

    CYTOPLASM CYTOPLASM

    NUCLEUS NUCLEUS

    RanGTP-binding and import cargo release

    IMPORTIN GTP EXPORT


    EXPORTIN GTP
    IMPORT CARGO
    CARGO
    5. A model for translocation through the NPC
    Compartmentalisation allows the
    regulation of gene expression
    Example: Reversible nuclear accumulation of NF-AT
    SUMMARY

    I. Signals target proteins into and out of the nucleus


    a. active transport
    b. signals are necessary and sufficient

    II. Transport occurs through the nuclear pore complex (NPC),


    a large multi-protein complex
    a. NPC has aqueous channel
    b. no unfolding of cargoes is required
    c. transport is bi-directional

    III. Signals are recognized by soluble receptors:


    Importins and exportins
    a. bind specific classes of cargo
    b. shuttle between cytoplasm and nucleus
    c. interact with nucleoporins

    IV. Ran-GTP defines the nucleoplasm and the perichromatin space


    a. Ran-GTP is asymmetrically distributed
    b. Ran regulates cargo binding and release
    Visualization of Transcription in Cells or Tissue Sections

    •3H-Uridine Incorporation
    •Br-UTP Incorporation
    •Fluorescence In Situ Hybridization
    Visualizing Gene Expression in Living Cells

    Janicki et al. (2004) Cell 116, 683-698


    2.5 hrs. post-transfection (-) Dox

    CFP-Lac MS2 Binding Merge


    Repressor Protein-YFP

    Janicki et al. (2004) Cell 116, 683-698.


    2.5 hrs. post-transfection + 2.5 hrs. after the
    addition of Dox

    CFP-Lac Repressor MS2 Binding Merge


    CFP-SKL Protein-YFP

    Janicki et al. (2004) Cell 116, 683-698.


    The RNA polymerase II large subunit is
    recruited to the active locus

    CFP-lac repressor YFP-RNA pol II Merge


    CFP-SKL

    Janicki et al. (2004) Cell 116, 683-698.


    Fluorescence Recovery after photobleaching (FRAP)
    FRAP analysis of a pre-mRNA splicing factor
    in the cell nucleus

    SF2/ASF has a half-time of fluorescence recovery of approximately


    1.8 (+/- 0.6) seconds.
    Bubulya et al. (2004) J. Cell Biol. 167, 51-63.
    SUMMARY

    I. Single Cell Imaging of Gene Regulation

    II. Development of in vivo fluorescent tags

    III. New ways to amplify gene loci and detect


    transcripts

    IV. Clever ways of using RNA binding proteins


    to localize RNA products

    V. Using laser to bleach and measure


    recovery of proteins to a locus

    VI. Fluorescence in situ hybridization (FISH) and multi-


    copy gene arrays to localize specific genes

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