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.

Figure 1

Experimental workflow. (A) Immunostaining of paxillin and CD71 after treatment of cells with crosslinkers for 5, 15 and 30 min. DNA was stained with DAPI (blue). (B) Crosslinked cells were lysed, washed with high sheer flow water jetting and immunostained as in A. (C) The integrated total fluorescence intensities of CD71 and paxillin staining were quantified using Metamorph software (n>15 cells) and ratios were calculated. (D) Experimental workflow for identification and quantification of the myosin II-dependent adhesion-associated subproteome. DAPI, 4,6-diamidino-2-phenylindole; LC–MS/MS, liquid chromography–mass spectrometry; PAGE, polyacrylamide gel electrophoresis; TIRFM, total internal reflection microscopy.

Figure 2

Myosin II-dependent and -independent protein recruitment to integrin adhesions. (A) Cells were seeded on FN- or PLL-coated glass coverslips for 45 min in the absence or presence of blebbistatin, fixed and stained for nuclei (blue), actin (red) and paxillin (green). (B) Overall, 2,118 proteins were identified, quantified and grouped into clusters labelled A–P. Clusters D, F and G show FN enrichment and intensity reduction by blebbistatin and are marked with an asterisk. FN-enriched clusters C–G are shown with a black bar. (C) The depicted proteins were previously associated with integrin adhesions. A–P refer to the hierarchical cluster analysis in B. FN, fibronectin; PLL, poly-L-lysine.

Figure 3

Hierarchical PPI network around fibronectin-bound integrins. Integrin subunits are in the centre and the inner and outer circles show their direct and indirect interactors, respectively. Black lines between nodes indicate high confidence PPI, red arrows indicate activating and blue lines indicate inhibiting interactions. The nodes were labelled with gene symbols and colour-coded according to the log2 ratio of control over blebbistatin-treated cells (median; n=5). Red nodes were reduced relative to integrin-β1 on addition of blebbistatin. The LIM symbol marks proteins with LIM domains. FN, fibronectin; PPI, protein–protein interaction.

Figure 4

Differential dependence of migfilin and kindlin 2 on myosin II analysed by TIRFM. (A) Selected subnetwork. (B) The log2 ratio of protein intensities from control compared with blebbistatin-treated cells analysed by mass spectrometry (n=5). (C–E) Fibroblasts expressing (C) vasp–cherry, (D) migfilin–cherry and (E) kindlin 2–cherry monitored over 40 min (30 s time laps) by TIRFM. For acquiring a bleaching, baseline cells were either left untreated for 40 min, or 10 min followed by blebbistatin treatment for the remaining 30 min. Total integrated fluorescence intensities of (C) vasp–cherry (blebbistatin, n=6; untreated, n=4), (D) migfilin–cherry (blebbistatin, n=11; untreated, n=5) and (E) kindlin 2–cherry (blebbistatin, n=8; untreated, n=3) in the TIRFM field were quantified using the Metamorph program. (F) Fluorescence intensity values of blebbistatin-treated migfilin–cherry and kindlin 2–cherry cells were corrected for the bleaching and overlaid. TIRFM, total internal reflection microscopy.