Cell & Dev. Biology University of Dundee · 2017-10-13 · 200000 180000 160000 140000 120000 =10 8...
Transcript of Cell & Dev. Biology University of Dundee · 2017-10-13 · 200000 180000 160000 140000 120000 =10 8...
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Paul Appleton
Inke Näthke
Cell & Dev. BiologyUniversity of Dundee
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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
220000
200000
180000
160000
140000
12000059810N =
fl-cre
60000
50000
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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
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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
0
20
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20 20-30
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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
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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