Integrin-bound talin head inhibits actin filament barbed-end elongation

Abstract

Focal adhesions (FAs) mechanically couple the extracellular matrix (ECM) to the dynamic actin cytoskeleton, via transmembrane integrins and actin-binding proteins. The molecular mechanisms by which protein machineries control force transmission along this molecular axis, i.e. modulating integrin activation and controlling actin polymerization, remain largely unknown. Talin is a major actin-binding protein that controls both the inside-out activation of integrins and actin-filament anchoring and thus plays a major role in the establishment of the actin-ECM mechanical coupling. Talin contains three actinbinding domains (ABDs). The N-terminal head domain contains both the F3 integrin-activating domain and ABD1, while the C-terminal rod contains the actin-anchoring ABD2 and ABD3. Integrin binding is regulated by an intramolecular interaction between the N-terminal head and a Cterminal five-helix-bundle (R9). Whether talin ABDs regulate actin polymerization in a constitutive or regulated manner has not been fully explored. Here, we combine kinetics assays using fluorescence spectroscopy and single actin filament observation in TIRF microscopy, to examine relevant functions of the three ABDs of talin. We find that the N-terminal ABD1 blocks actin filament barbed end elongation while ABD2 and ABD3 do not show any activity. By mutating residues in ABD1, we find that this activity is mediated by a positively charged surface that is partially masked by its intramolecular interaction with R9. Our results also demonstrate that, once this intramolecular interaction is released, integrinbound talin head retains the ability to inhibit actin assembly.Cell adhesion to the extracellular matrix plays a critical role in many physiological functions such as cell migration, invasion or epithelial basement membrane attachment. Among the multiple adhesion structures, focal adhesions (FAs) play a major role (1,2). These multiprotein complexes couple various extracellular matrices to the actin cytoskeleton via the transmembrane heterodimeric αβ integrins and actin-binding proteins (ABPs) (3,4). The control of actin polymerization by ABPs is thought to play an important role to initiate the formation of the actomyosin stress fibers and control their tension by modulating their elongation. We showed previously that vinculin blocks actin filament barbed end elongation (5), while others reported that VASP promotes the elongation of actin filament barbed ends in a processive-like manner (6,7). Several formins may also play a role in the formation and elongation of Talin head inhibits actin assembly 2 stress fibers (8). However, despite these isolated characterizations, the respective roles of the multiple ABPs and their coordination in this process are poorly understood. The actin-binding protein talin plays a major role in FAs (9) ( Figure S1). First it acts very early to activate integrins. In this process the N-terminal PTB (phosphotyrosine binding) domain, located in the head domain of talin, also known as the F3 subdomain of the ...

Cellular Mechanotransduction: From Tension to Function

“…Talin is a 270 kDa protein composed of an N-terminal globular head, a flexible rod domain and C-terminal helices. While the helices are involved in protein dimerization ( Golji and Mofrad, 2014 ), the head interacts with both β-integrin cytoplasmic domain and F-actin through its FERM domain, recruiting protein 4.1, ezrin, radixin and moesin docking proteins ( Ciobanasu et al, 2018 ). Talin rod features an additional binding site for integrin, and two sites for actin ( Gingras et al, 2009 ).…”

Talin as a mechanosensitive signaling hub

“…Indeed, studies in Drosophila melanogaster strongly suggest that talin adopts distinct conformations in cell types with different cytoskeletal–ECM linkages (Klapholz et al, 2015). In cultured cells, the initial linkage is thought to involve the talin FERM domain bound to integrin and the C-terminal ABS3 bound to actin (Gingras et al, 2008; Kopp et al, 2010; Kumar et al, 2016; Ciobanasu et al, 2018). Adhesion maturation is accompanied by engagement of ABS2 with actin (Atherton et al, 2015), and mutational studies implicate ABS2 as the major site required for force transmission (Kumar et al, 2016).…”

The tale of two talins – two isoforms to fine‐tune integrin signalling

“…Talin contains three actin-binding sites (ABS1-3) [68]. ABS1 is in F2-F3 in the talin head [69] and has recently been shown to be important for capping actin filaments to block actin polymerisation [70].…”