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.

The integrin adhesome network at a glance

“…These methods relied on stabilisation of IACs using chemical cross-linkers and enrichment of IAC components by removal of the cell body and cytoplasmic proteins (Jones et al, 2015; Kuo et al, 2012). Mass spectrometry was then used to determine IAC compositions for several cell types under a variety of culture conditions (Ajeian et al, 2016; Byron et al, 2012, 2015; Horton et al, 2015, 2016a; Huang et al, 2014; Humphries et al, 2009; Kuo et al, 2011; Ng et al, 2014; Robertson et al, 2015; Salmela et al, 2016; Schiller et al, 2011, 2013; Yue et al, 2014). Seven of these mass spectrometry datasets, which were generated from different laboratories using diverse methods and from multiple cell types, were used to create a ‘meta-adhesome’ database of more than 2000 proteins that were enriched at fibronectin-induced IACs (Horton et al, 2015).…”

Small Heat Shock Protein αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress

“…Furthermore, it associates with the nuclear membrane [65], affects membrane and organelle dynamics with microtubule-associated motor proteins [66], alters mitochondrial function [67] [68] and is involved in stretch activation of regulatory subunits of NOX2 on transverse tubule (T-T) membranes of the heart muscle [69]. In addition, it has long been known that Rac1 functions with microtubules [70] [71], that membrane-associated Rac1 is a regulatory component of the NOX2 complex [72] and αB-crystallin participates in stretch-induced muscle contraction [12]. Thus, there is much more to be learned about αB-crystallin [1] [73] and its role in microtubule dynamics, mechano-biology and membrane physiology.…”

LIMD1 phase separation contributes to cellular mechanics and durotaxis by regulating focal adhesion dynamics in response to force

“…The purification of adhesion complexes combined with quantitative mass spectrometry has enabled the identification and quantification of known and new adhesion-associated proteins [22][23][24] . Blocking the adhesion maturation with the myosin II inhibitor blebbistatin markedly impaired the recruitment of LIM domain proteins to integrin adhesion sites 24 .…”

“…Force fluctuations within individual FAs have been observed to mediate ECM-rigidity sensing in guiding directed cell migration 15 . How the formation and dynamics of the complex protein assemblies at cell-ECM contact sites coordinate with force sensing and cell motility is yet to be thoroughly investigated.The purification of adhesion complexes combined with quantitative mass spectrometry has enabled the identification and quantification of known and new adhesion-associated proteins [22][23][24] . Blocking the adhesion maturation with the myosin II inhibitor blebbistatin markedly impaired the recruitment of LIM domain proteins to integrin adhesion sites 24 .…”