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More than 250 proteins are associated with the formation of integrin adhesion complexes involving a vast number of complex interactions between them. These interactions enable adhesions to serve as dynamic and diverse mechanosignaling centers. Our laboratory focuses on the biochemical and structural study of these interactions to help unpick this complex network. Here, we describe the general pipeline of biochem- ical assays and methods we use. The chapter is split into two sections: (1) protein production and characterization and (2) biochemical assays for the characterization of binding between full-length proteins and/or specific regions of proteins with other proteins, peptides, and phospholipids. The suite of assays we use routinely includes circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy for sample quality assessment, prior to biochemical analysis using NMR, fluorescence polarization (FP), microscale thermophoresis (MST), size-exclusion chromatography multiangle light scattering (SEC-MALS), and pulldown/cosedimentation-based approaches. The results of our analysis feed into in vivo studies that allow for the elucidation of the biological role of each interaction.

Talin is a mechanosensitive adapter protein which couples integrins to the cytoskeleton and regulates integrin-mediated adhesion. Talin rod domain-containing protein-1 (TLNRD1) shares 22% homology with the R7R8 domains of talin, and is highly conserved throughout vertebrate evolution, however little is known about its function. Here we show that TLNRD1 is an α-helical protein which shares the same atypical topology as talin R7R8, but forms a novel antiparallel dimer arrangement. Actin co-sedimentation assays and electron microscopy reveal TLNRD1 is an actin-bundling protein that forms tight actin bundles. In addition, TLNRD1 binds to the same LD-motif containing proteins, RIAM and KANK, as talin, and thus may act in competition with talin. Filopodia are cell protrusions supported by tightly bundled actin filaments and TLNRD1 localises to filopodia tips, increases filopodia number and promotes cell migration in 2D. Together our results suggest that TLNRD1 has similar functionality to talin R7R8, serving as a nexus between the actin and microtubule cytoskeletons independent of adhesion complexes.