Quantitative proteomics of the integrin adhesome show a myosin II‐dependent recruitment of LIM domain proteins

Abstract

A characteristic of integrins is their ability to transfer chemical and mechanical signals across the plasma membrane. Force generated by myosin II makes cells able to sense substrate stiffness and induce maturation of nascent adhesions into focal adhesions. In this paper, we present a comprehensive proteomic analysis of nascent and mature adhesions. The purification of integrin adhesion complexes combined with quantitative mass spectrometry enabled the identification and quantification of known and new adhesion-associated proteins. Furthermore, blocking adhesion maturation with the myosin II inhibitor blebbistatin markedly impaired the recruitment of LIM domain proteins to integrin adhesion sites. This suggests a common recruitment mechanism for a whole class of adhesion-associated proteins, involving myosin II and the zinc-finger-type LIM domain.

The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier

“…7D and 5T). To elucidate this phenomenon, we performed quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based focal adhesome proteomics (30), in which enriched and chemically cross-linked FA complexes are analyzed using mass spectrometry (30). Strikingly, we detected that loss of EPB41L5 modulated the composition of the focal adhesome, most prominently on the level integrin receptors such as INTEGRINbeta1 ( Fig.…”

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling

“…For affinity purification-mass spectrometry (AP-MS) of Jurkat T cells, proteins were eluted from the beads using urea buffer (6 M urea, 2 M thiourea, 10 mM HEPES [pH 8]), digested with trypsin (Promega) and Lys-C (WAKO, Osaka, Japan), and analyzed using electrospray ionizationliquid chromatography-tandem MS, as described earlier (26). Data were analyzed using MaxQuant 1.3.6.1 software (27), the Andromeda search engine (28), and the Perseus data analysis tool (http://www.perseus-framework.…”

Talin determines the nanoscale architecture of focal adhesions

“…The FA core consists of a membrane-proximal layer that contains signaling proteins such as FAK (focal adhesion kinase) and paxillin, an intermediate zone that contains force-transduction proteins such as talin and vinculin, and a stress fiber interfacial zone that contains actin-associated proteins such as VASP (vasodilatorstimulated protein) and α-actinin. Although such multilaminar architecture signifies a certain degree of compartmentalization within FAs that may serve to spatially constrain protein-protein interactions and dynamics, the structural connectivity, the molecular configuration and geometry of FA proteins, and the molecular basis of their higher-order organization remain unclear.Proteomic and interactome analysis of the integrin adhesome have uncovered several direct and multitier connections between integrins and actin (17)(18)(19)(20). This suggests that multiple highly interconnected protein-protein interactions could collectively selforganize into FA structures; such redundancy could also account for the remarkable mechanical robustness of FAs after cellular disruption or perturbation (21).…”

“…Proteomic and interactome analysis of the integrin adhesome have uncovered several direct and multitier connections between integrins and actin (17)(18)(19)(20). This suggests that multiple highly interconnected protein-protein interactions could collectively selforganize into FA structures; such redundancy could also account for the remarkable mechanical robustness of FAs after cellular disruption or perturbation (21).…”