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

25
Paul Appleton Inke Näthke Cell & Dev. Biology University of Dundee

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

Page 1: Cell & Dev. Biology University of Dundee...N = 25 24 co n t A s i R N A A PC 260000 240000 220000 200000 180000 160000 140000 120000 =10 8 9 5 f l -cre 60000 50000 40000 30000 20000

Paul Appleton

Inke Näthke

Cell & Dev. BiologyUniversity of Dundee

Page 2: Cell & Dev. Biology University of Dundee...N = 25 24 co n t A s i R N A A PC 260000 240000 220000 200000 180000 160000 140000 120000 =10 8 9 5 f l -cre 60000 50000 40000 30000 20000

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 adhesionand transcriptional regulation.

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

• Direct and indirect cytoskeletal links

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β-catenin targeting/Wnt

Kap3 microtubules EB1

PDZ/DlgIQGAPASEF

F-actin

Microtubules

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GFP-APC tubulin DAPI

APC clusters correlate with cell migrationpromotes cellular protrusions stabilises microtubules in vivo and in vitrohelps to establish parallel microtubule arrays

in polarised cellsin mitotic spindle

J. Cell Biol. 1996Current Biology 2001

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

J. Cell Biol. 2007

AdenomatousPolyposis

Coli

APC tubulin

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driven by activecell migrationand division

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Accumulation in inappropriatelyproliferating and toxic environment

Loss of APC

Cell migration defect

Accumulation ofadditional mutations

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2425N =

cont s

iRNA

APC siRNA

260000

240000

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12000059810N =

fl-cre

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wt-cre

wt-ctrl

Karin Kroboth, Mol. Biol. Cell 2007

Migration of APC deficient cells is reduced

1814N =

APC-wt

APC-mutant

100000

80000

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GAPDH

APC

wt-cre

wt-ctrl

fl-cre

fl-ctrl

APC-wt

APC-mutant

APC siRNA

ctrl s

iRNA

T=0 T=24

∆A = At0- At24

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

WT + Cre Fl-APC + Cre

APC ex 14 ex 15loxP

loxP Hiroyuki Shibata et al.,

Science, 1997

Cre-recombinase

580 aa+ β-gal

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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?

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= Expression of

N-terminal

fragment

Accumulation

Loss of APC

Cell migration defect

F-actin/MTmotor Wnt pathway MT

Gain of function? Loss of function?

Zhuoyu Li

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N-terminal APC fragments have a dominanteffect on cell migration

Identify specific binding partners for the responsible domain

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

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Dominant effects

= Expression of

N-terminal

fragment

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/B56Wnt pathway

+

Phosphorylation?Other binding partners?

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WT

Late

Early

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Truncation of APC

Aneuploidy

Chromosomesegregation

Accumulation Transformation

Cellmigration

Deregulationof β-catenin

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Jason SwedlowKen KaplanNat. Cell Biol. 2001

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Dina Dikovskaya

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Dina D

ikovskaya/Ow

en Sansom

, J. Cell B

iol. 2007

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120-130nuclear area (um2)

cell

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wt/wtfl/flfl/fl p21 positive

nuclear area, µm2

cell

num

ber

Wt

Wt

∆APCp2

1

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APC inhibition leads to tetraploidy in vivo

Wt ∆APC

2n 4n 2n 4n

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loss of APC

spindle defects compromised spindle checkpoint

tetraploidy aneuploidy

segregation defects

“leaky” tetraploidcheckpoint

ApoptosisMT Checkpoint proteins

Dina Dikovskaya, J. Cell Biol. 2007

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

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Truncation of APC

Accumulation Aneuploidy Transformation

Cellmigration

Chromosomesegregation

Deregulationof β-catenin

APC

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Paul Appleton

Measure EARLY effects of APC mutations on:

Overall tissue architecture at different levels ofresolution

Subcellular organisation of the cytoskeletonmitotic spindle orientation/symmetrycell polarity/symmetry

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Paul Appleton

F-actinNuclei

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Paul Appleton

F-actin

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Paul Appleton

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Paul Appleton

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Jürg Zumbrunn

The Burroughs Wellcome FundRoyal Society

TenovusAssociation 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

BeatsonCardiff

Scripps

Owen SansomAlan Clarke

Clare Waterman-StorerKatsuhiro Kita Gaudenz Danuzer

David Schiffman

George PenmanAbby Oakley

Paul Appleton

Louie Leung

Sophia Lin

Aaron Quyn