Cell & Dev. Biology University of Dundee...N = 25 24 co n t A s i R N A A PC 260000 240000 220000...
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...
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
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iRNA
APC siRNA
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Karin Kroboth, Mol. Biol. Cell 2007
Migration of APC deficient cells is reduced
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APC siRNA
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iRNA
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∆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
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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
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iol. 2007
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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