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Transcript of Cell & Dev. Biology University of ... N = 25 24 co n t A s i R N A A PC 260000 240000 220000...

  • Paul Appleton

    Inke Näthke

    Cell & Dev. Biology University of Dundee

  • Adenomatous Polyposis Coli = APC

    • Mutated in Familial Adenomatous Polyposis (FAP) • Lost early in most (> 80%) sporadic colon cancers • Large cytoplasmic protein (2843 amino acids) • Involved in Wnt-regulated phosphorylation and subsequent

    degradation of β-catenin, an important protein for cell adhesion and transcriptional regulation.

    • Multiple binding partners include signalling molecules, transcriptional regulators, structural proteins.

    • Direct and indirect cytoskeletal links

  • β-catenin targeting/Wnt

    Kap3 microtubules EB1

    PDZ/DlgIQGAPASEF

    F-actin

    Microtubules

  • GFP-APC tubulin DAPI

    APC clusters correlate with cell migration promotes cellular protrusions stabilises microtubules in vivo and in vitro helps to establish parallel microtubule arrays

    in polarised cells in mitotic spindle

    J. Cell Biol. 1996 Current Biology 2001

    J. Cell Biol. 2002 Mol. Biol. Cell 2004 Mol. Biol. Cell 2006 Mol. Biol. Cell 2007

    J. Cell Biol. 2007

    Adenomatous Polyposis

    Coli

    APC tubulin

  • driven by active cell migration and division

  • Accumulation in inappropriately proliferating and toxic environment

    Loss of APC

    Cell migration defect

    Accumulation of additional mutations

  • 2425N =

    co nt

    siR NA

    AP C s

    iRN A

    260000

    240000

    220000

    200000

    180000

    160000

    140000

    120000 59810N =

    fl-c re

    60000

    50000

    40000

    30000

    20000

    10000

    fl-c trl

    wt- cre

    wt- ctr

    l

    Karin Kroboth, Mol. Biol. Cell 2007

    Migration of APC deficient cells is reduced

    1814N =

    AP C-

    wt

    AP C-

    mu tan

    t

    100000

    80000

    60000

    40000

    20000

    0

    -20000

    100 120

    90 80 70 60

    40 50

    GAPDH

    APC

    wt- cre

    wt- ctr

    l fl-c

    re fl-c

    trl

    AP C-

    wt

    AP C-

    mu tan

    t

    AP C s

    iRN A

    ctr l s

    iRN A

    T=0 T=24

    ∆A = At0- At24

  • Inactivating APC in intestinal tissue of mice inhibits cell migration Owen Sansom/Alan Clarke, Genes & Devel.

    WT + Cre Fl-APC + Cre

    APC ex 14 ex 15lo xP

    lo xP Hiroyuki Shibata et al., Science, 1997

    Cre- recombinase

    580 aa + β-gal

  • Lack of APC –> decrease in cell migration –> fewer and shorter protrusions –> fewer post-translationally

    modified MTs (reduced life time) –> less stable MTs –> altered MT dynamics

    Are cells without APC more sensitive to MT poisons? Can we exploit this “Achilles Heel” clinically?

  • = E xpr

    ess ion

    of

    N-t erm

    ina l

    fra gm

    ent

    Accumulation

    Loss of APC

    Cell migration defect

    F-actin/MTmotor Wnt pathway MT

    Gain of function? Loss of function?

    Zhuoyu Li

  • N-terminal APC fragments have a dominant effect on cell migration

    Identify specific binding partners for the responsible domain

    Determine if the effect of N-APC is on EMT or cytoskeleton directly F-actin polarisation is altered by N-APC in Dictyostelium

  • Dominant effects

    = E xpr

    ess ion

    of

    N-t erm

    ina l

    fra gm

    ent

    Accumulation

    Loss of APC

    Cell migration defect

    F-actin/MTmotor Wnt pathway MT

    Gain of function Loss of function

    Asef/Kap3/PP2 Wnt pathway MT

    Zhuoyu Li, Cancer Res. 2005

    Asef/ Kap3

    /B56 Wnt p

    athwa y

    +

    Phosphorylation? Other binding partners?

  • WT

    Late

    Early

  • Truncation of APC

    Aneuploidy

    Chromosome segregation

    Accumulation Transformation

    Cell migration

    Deregulation of β-catenin

  • Jason SwedlowKen Kaplan Nat. Cell Biol. 2001

  • Dina Dikovskaya

  • D ina D

    ikovskaya/O w

    en S ansom

    , J. C ell B

    iol. 2007

    0

    20

    40

    60

    80

    100

    120

    0-10

    20 20-30

    30-40

    40-50

    50-60

    60-70

    70-80

    80-90

    90-100

    100-110

    110-120

    120-130nuclear area (um2)

    ce ll

    nu m

    be r

    wt/wt fl/fl fl/fl p21 positive

    nuclear area, µm2

    ce ll

    nu m

    be r

    Wt

    Wt

    ∆APC p2

    1

    ∆APC

    APC inhibition leads to tetraploidy in vivo

    Wt ∆APC

    2n 4n 2n 4n

  • loss of APC

    spindle defects compromised spindle checkpoint

    tetraploidy aneuploidy

    segregation defects

    “leaky” tetraploid checkpoint

    ApoptosisMT Checkpoint proteins

    Dina Dikovskaya, J. Cell Biol. 2007

    –> Mitotic defects in APC-deficient cells are largely independent of changes in Wnt signaling

  • Truncation of APC

    Accumulation Aneuploidy Transformation

    Cell migration

    Chromosome segregation

    Deregulation of β-catenin

    APC

  • Paul Appleton

    Measure EARLY effects of APC mutations on:

     Overall tissue architecture at different levels of resolution

     Subcellular organisation of the cytoskeleton mitotic spindle orientation/symmetry cell polarity/symmetry

  • Paul Appleton

    F-actin Nuclei

  • Paul Appleton

    F-actin

  • Paul Appleton

  • Paul Appleton

  • Jürg Zumbrunn

    The Burroughs Wellcome Fund Royal Society

    Tenovus Association for International Cancer Research

    Human Frontiers Scientific Programme

    Sam Swift

    Dundee

    Xuesong Yang/Kees Weijer

    Team OME

    Dina Dikovskaya

    Ian Newton

    Zhuoyu Li

    Katie Schumm

    Karin Kroboth

    Current lab members:

    Former lab members:

    Songül Bekir

    Elsewhere

    Beatson Cardiff

    Scripps

    Owen Sansom Alan Clarke

    Clare Waterman-Storer Katsuhiro Kita Gaudenz Danuzer

    David Schiffman

    George Penman Abby Oakley

    Paul Appleton

    Louie Leung

    Sophia Lin

    Aaron Quyn