Isolation of Integrin‐Based Adhesion Complexes

Abstract

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter‐receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin‐associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin‐associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry. © 2015 by John Wiley & Sons, Inc.

Proteomic analysis of integrin‐associated complexes from mesenchymal stem cells

“…Adhesion complexes were then isolated from MSCs spread on FN and PDL using a published method (Fig. B) . Western blotting of isolated complexes confirmed the specific recruitment of the integrin subunits β1 and α5, along with talin, a well‐characterised integrin‐binding cytoskeletal adaptor protein (Fig.…”

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).…”

Adhesion protein networks reveal functions proximal and distal to cell-matrix contacts

“…The development of biochemical methodologies to isolate integrin-associated proteins, coupled with advances in proteomics and informatics, has largely overcome the earlier major challenges, thus enabling the characterisation of adhesion complexes by quantitative mass spectrometry [10, 11, 12, 13, 14]. Computational integration of multiple adhesion-site proteomes yielded an experimentally defined ‘meta-adhesome’, from which a core set of 60 frequently identified proteins — a ‘consensus’ adhesome — was identified [15 •• ] (Figure 1).…”