title: Microtubule-dependent modulation of adhesion complex composition.
abstract: The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α5β1 integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α5-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α5-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.
authors.forename: Daniel H J
authors.initials: DH
authors.lastname: Ng
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Humphries
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.
authors.forename: Adam
authors.initials: A
authors.lastname: Byron
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.
authors.forename: Angélique
authors.initials: A
authors.lastname: Millon-Frémillon
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D002448:Cell Adhesion; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism*; D052816:Foreskin / Q000166:cytology; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods; D050257:Tubulin Modulators / Q000494:pharmacology*; D016596:Vinculin / Q000378:metabolism
chemical_list: D039081:Integrin alpha5beta1; D050257:Tubulin Modulators; C488955:VCL protein, human; D016596:Vinculin; D015739:Nocodazole