{"id":{"repo_id":"east-anglia","oai_identifier":"oai:ueaeprints.uea.ac.uk:47984"},"canonical_url":"https://search.dev.ndltd.org/etd/east-anglia/oai:ueaeprints.uea.ac.uk:47984","repository":{"repo_id":"east-anglia","name":"University of East Anglia","base_url":"https://ueaeprints.uea.ac.uk/cgi/oai2"},"display":{"title":"Microtubule Organisation in Normal and Colon Cancer Cells","abstract":"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.]","abstract_html":"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.]","abstract_has_math":false,"creators":["Gadsby, Jonathan"],"institution":"University of East Anglia","degree_name":"phd","degree_level":"doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-01","date_published":"2014-01","updated_at":"2026-07-24T02:11:59Z","subjects":[],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Gadsby, Jonathan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2014-01"]},{"key":"dc:date.issued","label":"Date","values":["2014-01"]},{"key":"dc:publisher.department","label":"Dc Publisher Department","values":["School of Biological Sciences"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of East Anglia"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://ueaeprints.uea.ac.uk/id/eprint/47984/"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["phd"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://ueaeprints.uea.ac.uk/id/eprint/47984/1/2014GadsbyJRPhD.pdf"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.]"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Microtubule Organisation in Normal and Colon Cancer Cells"]}]}],"canonical_facts":{"dc:creator":["Gadsby, Jonathan"],"dc:date":["2014-01"],"dc:date.issued":["2014-01"],"dc:description.abstract":["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.]"],"dc:format":["application/pdf"],"dc:identifier.uri":["https://ueaeprints.uea.ac.uk/id/eprint/47984/1/2014GadsbyJRPhD.pdf"],"dc:language":["en"],"dc:publisher.department":["School of Biological Sciences"],"dc:publisher.institution":["University of East Anglia"],"dc:relation.isreferencedby":["https://ueaeprints.uea.ac.uk/id/eprint/47984/"],"dc:title":["Microtubule Organisation in Normal and Colon Cancer Cells"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["doctoral"],"dc:type.qualificationname":["phd"]},"updated_at":"2026-07-24T02:11:59Z"}