Microtubule-dependent modulation of adhesion complex composition

PLoS One. 2014 Dec 19;9(12):e115213. doi: 10.1371/journal.pone.0115213. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Adhesion
  • Cells, Cultured
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Focal Adhesions / metabolism*
  • Foreskin / cytology
  • Humans
  • Integrin alpha5beta1 / metabolism*
  • Male
  • Mass Spectrometry / methods
  • Microtubules / drug effects*
  • Microtubules / metabolism
  • Nocodazole / pharmacology*
  • Proteomics / methods
  • Tubulin Modulators / pharmacology*
  • Vinculin / metabolism

Substances

  • Integrin alpha5beta1
  • Tubulin Modulators
  • VCL protein, human
  • Vinculin
  • Nocodazole