Microtubule Organisation in Normal and Colon Cancer Cells
Abstract+ ApicoNbasal" polarisation" of" epithelial" cells" is" essential" for" normal" function," with" the" rearrangement" of" the"microtubule" cytoskeleton" on" differentiation" critical" for" the" establishment" of" polarity." "During" this" process,"microtubules" reorganise" to" form"a"stabilised"array"of"apicoNbasal"bundles"via"a"release"and"capture"mechanism."" Disruption"of"this"array"can"cause"a"loss"of"epithelial"polarity,"which"can"contribute"to" diseases" such"as" cancer." "Microtubule" rearrangements" are"driven"by"their" inherent" dynamics,"which" are" regulated"by"+TIP"proteins." "This"project" aims" to"examine" the" mechanisms" behind" apicoNbasal" array" formation," and" how" they" may" be" compromised" in" colorectal" cancer." " It" has" focussed" on" two" +TIPs;" APC" (which" is" mutated" in" most" colorectal" cancers)," and" EB2" (which" has" also" been" observed" upregulated"in"cancer)." The"role"of"APC"at"the"cortex"was"examining"using"cell"lines"containing"wild" type"or"APC" truncation"mutations." "Microtubule"orientation" at" junctional" sites"was" determined,"with"Nocodazole"regrowth"assays"used"to"determine"cortical"approach."" Additionally,"the"angle"of"the"mitotic"spindles"was"analysed"in"the"different"cell"lines." The" impact" of" EB2" loss" on" microtubule" organisation" was" determined" by" immunolabelling" following" siRNA" treatment." "A" fluorescently" tagged" EB2" construct" was"created"and"used"to"rescue"depleted"phenotypes.""The"effect"of"EB2"depletion" on"microtubule"stability"was"assessed"by"analysing"live"microtubule"dynamics.""The" impact"of"EB2"overexpression"on"cell"migration"was"assessed"by"creating"a"stable"EB2" overexpressing" cell" line" and" live" imaging" sparsely" seeded" cells" and" scratch" assays."" Overexpressing"cells"were"grown"in"3D"culture"to"study"any"effects"on"polarisation." The" TC7" cell" line" containing" truncated" APC" possessed" fewer" microtubules" orientated"perpendicular"to"the"cortex"than"wildNtype"expressing"cells.""This"could"be" rescued"with"full"length"GFPNAPC.""Nocodazole"regrowth"showed"microtubules"in"TC7" cells"would"approach"the"cortex"perpendicularly,"and"then"curl"away.""TC7"cells"also" had"an"increased"proportion"of"misorientated"spindles." " EB2"depletion" caused" a"dramatic" increase" in" cell" size,"and" redeployment"of" EB1" and" the" microtubuleNactin" crosslinking" protein" ACF7" along" the" microtubule" lattice.""These"effects"could"be"rescued"by"transfection"with"mCherryNEB2.""Analysis" revealed" EB2" depleted" cells" possessed" less" dynamic" microtubules." " The" large" depleted"cells"were"found"capable"of"division,"although"some"appeared"to"fail"during" the"process,"with"cell"cycle"analysis"revealing"suggesting"the"potential"induction"of"a" cell"death"pathway." "Overexpressed"EB2" could"be" located" at" the"migratory" leading" edge,"and"was" found" to"promote" cell"migration,"with" cells"moving" at"an" increased" rate" and"wounds" healing"more" quickly." " There"was" also" evidence" of"compromised" tissue"integrity"in"overexpressing"cells"grown"in"3D"culture" The" results" gathered" identified" a" potential" role" for" full" length" APC" in" stabilising" cortical" microtubule" contacts," and" helped" identify" EB2" as" a" central" regulator" of" microtubule"dynamics"and"organisation"during"polarisation.""EB2"overexpression"was" found"to"increase"cell"migration,"and"may"be"able"to"induce"EMT." *[N.B.: Movie quicktime files were attached to this thesis at the time of its submission. Please refer to the author for further details.]
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