Proteomic analysis of integrin‐associated complexes from mesenchymal stem cells

Abstract

PurposeMultipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decision‐making. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force sensing, we sought to catalogue MSC integrin‐associated adhesion complex composition.Experimental designAdhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by MS. Identified proteins were interrogated by comparison to a literature‐based reference set of cell adhesion‐related components and using ontological and protein–protein interaction network analyses.ResultsAdhesion complex‐specific proteins in MSCs were identified that comprised predominantly cell adhesion‐related adaptors and actin cytoskeleton regulators. Furthermore, LIM domain‐containing proteins in MSC adhesion complexes were highlighted, which may act as force‐sensing components.Conclusion and clinical relevanceThese data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention.

High content image analysis of focal adhesion-dependent mechanosensitive stem cell differentiation

“…Current estimates implicate as many as 232 different components, of which 148 are intrinsic and 84 are transient 49 , as a common signature of adhesions. Recent analyses of focal adhesions have even identified more than 1300 distinct proteins within isolated adhesion complexes 50 , suggesting exceedingly complex adhesion-based mechanisms for cells that must actively sense their surroundings. Focal adhesion composition and structure have also recently been shown to be relatively stable to external perturbation, including siRNA knockdown or chemical inhibition of components, suggesting that signaling transduction occurs independently of structural integrity 51 .…”

Mechanosensitivity of integrin adhesion complexes: role of the consensus adhesome

“…To provide an unbiased view of IAC composition and function [27], the global composition of IACs has recently been characterised using protocols to isolate the adhesion nexus biochemically coupled with mass spectrometry (MS)-based proteomic analysis [28], [29]. These methods have been used to analyse IAC composition from a variety of cell types and conditions, including mesenchymal stem cells [30], [31], and the effects of different ECM ligands or integrin heterodimers [32], [33], [34], [35], microtubule polymerisation inhibition by nocodazole [36], [37], and myosin-II inhibition by blebbistatin [35], [38], [39] on IAC composition. In addition, the phosphorylation profile of isolated IACs has been reported [40], which identified novel phosphorylation sites on IAC components and regulators of adhesion signalling.…”

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).…”