Paul Appleton

Inke Näthke

Cell & Dev. BiologyUniversity 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 adhesionand 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 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

driven by activecell migrationand division

Accumulation in inappropriatelyproliferating and toxic environment

Loss of APC

Cell migration defect

Accumulation ofadditional mutations

2425N =

cont s

iRNA

APC siRNA

260000

240000

220000

200000

180000

160000

140000

12000059810N =

fl-cre

60000

50000

40000

30000

20000

10000

fl-ctrl

wt-cre

wt-ctrl

Karin Kroboth, Mol. Biol. Cell 2007

Migration of APC deficient cells is reduced

1814N =

APC-wt

APC-mutant

100000

80000

60000

40000

20000

0

-20000

100120

90 80 70 60

4050

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

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

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?

= 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

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

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?

WT

Late

Early

Truncation of APC

Aneuploidy

Chromosomesegregation

Accumulation Transformation

Cellmigration

Deregulationof β-catenin

Jason SwedlowKen KaplanNat. Cell Biol. 2001

Dina Dikovskaya

Dina D

ikovskaya/Ow

en Sansom

, J. Cell 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)

cell

num

ber

wt/wtfl/flfl/fl p21 positive

nuclear area, µm2

cell

num

ber

Wt

Wt

∆APCp2

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” tetraploidcheckpoint

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

Cellmigration

Chromosomesegregation

Deregulationof β-catenin

APC

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

Paul Appleton

F-actinNuclei

Paul Appleton

F-actin

Paul Appleton

Paul Appleton

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