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.
Highlights
Adhesion complexes (ACs) serve as hubs to integrate and convey mechanical and chemical signals intracellularly and extracellularly [1, 2]
We found that upon microtubule disruption, there was an intracellular force-dependent switch in the maturation state of ACs towards focal adhesions, such that a5-integrin is redistributed to focal adhesions from other ACs, and is accompanied by an overall increase in the abundance of AC components
In nocodazole-treated cells, there was an increase in avb3-integrin staining at the cell periphery in large focal adhesions, and a5-integrin and vinculin were found at centrally located focal adhesion-like structures (Fig. 8c). These results suggest that microtubule disruption causes a switch in the maturation state of ACs towards focal adhesions, such that a5-integrin is redistributed to focal adhesions and is accompanied by an increase in the abundance of AC components
Summary
Adhesion complexes (ACs) serve as hubs to integrate and convey mechanical and chemical signals intracellularly and extracellularly [1, 2]. In turn, is activated by the application of force via actin contractility and promotes the recruitment of AC proteins [18] Together, these proteins act as a mechanosensing module that allows cells to respond rapidly to their environment by directly modulating the state of ACs in response to intra- or extracellularly applied forces. In contrast to focal adhesions, the formation of fibrillar adhesions is thought to occur via lowtensional forces due to the high translocation of a5b1-integrin complexes from the distal ends of FAs [19] These a5b1-integrin complexes are rich in tensin, but lack other AC components such as avb3-integrin, vinculin and paxillin, and display low levels of phosphotyrosine (pTyr) [20, 21]. It is clear that while tensional forces affect the different AC states, compositional differences play an important role in determining the nature of the different AC states and their responses to tensional forces
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