Focal adhesions (FAs) are specialized integrin-dependent adhesion complexes, which mediate cell anchoring to the extracellular matrix (ECM) (Winograd-Katz et al., 2014). FAs also function as mechano-transducing machineries perceiving and integrating diverse physical and (bio)chemical environmental cues, and transducing them into intracellular signaling pathways (Zaidel-Bar et al., 2007a; Wehrle-Haller, 2012; Yu et al., 2012). Indeed, FAs control cellular programs as diverse as cell adhesion, migration, survival, growth, proliferation, and differentiation (Wehrle-Haller, 2012; Winograd-Katz et al., 2014). To accomplish these diverse regulatory functions, αβ heterodimeric integrin receptors (Hynes, 2002) cluster in the plasma membrane, recruit numerous proteins to their cytoplasmic tails, and give rise to a highly dynamic intracellular protein network which has been termed the “integrin adhesome” (Zaidel-Bar et al., 2007a; Winograd-Katz et al., 2014). The tight regulation of its protein composition ensures FAs functioning as mechanical anchoring points, as well as signaling platforms (Wozniak et al., 2004; Zaidel-Bar et al., 2007a; Geiger and Yamada, 2011; Winograd-Katz et al., 2014).

The extensive implication of integrins and FAs-dependent signaling in pathological conditions (Bachmann et al., 2019) pushes current research towards a better understanding of their functioning and spatiotemporal regulation (Wu et al., 2019; Su et al., 2020). A few years ago, interferometric photoactivated localization microscopy (iPALM) has revealed a layered organization of integrin-containing FAs (Kanchanawong et al., 2010; Case et al., 2015). This model, proposing the spatial segregation of specific adhesome components between a integrin signaling layer (closest to the membrane), a force transduction layer, and an actin regulatory layer (innermost), has been endorsed by studies making advantage of diverse techniques, such as single protein tracking microscopy, superresolution microscopy, proximity biotinylation, and bimolecular fluorescence complementation (Dong et al., 2016; Chastney et al., 2020; Legerstee and Houtsmuller, 2021; Orre et al., 2021; Ripamonti et al., 2021). Despite these great advances, the characterization of several structural and mechanical aspects of the sophisticated integrin-dependent protein network, a comprehensive understanding of the FA machinery is still far from being accomplished (Chastney et al., 2020; Legerstee and Houtsmuller, 2021). The decoding of how specific cellular responses can be provoked by a given physiological, pathological, or pharmacological stimulus is challenged by the interdependency of FA players, regulatory systems, including the plasma membrane and its composition, and the tension across integrin receptors (Vogel, 2006; Gauthier and Roca-Cusachs, 2018). In addition, a wide range of post-translational modifications and the expression of many FA protein splice variants and isoforms generate additional layers of complexity that need to be understood to identify specific versus more general functions of FAs (Anthis et al., 2010; Choi et al., 2011; Soto-Ribeiro et al., 2019).

This review offers an overview of the molecular basis of the mechano-sensitivity and mechano-signaling capacity of the core FA proteins β3 integrin, talin1, and kindlin (Figure 1) that enable mechano-transduction. The focus is on β3 integrins as a paradigm for paxillin- and mechano-dependent mechanisms that may be extended to other classes of integrins. We will first address β3 integrin receptors, their link to talin1 and kindlin, and how paxillin is recruited to this complex for further mechanical stabilization, as well as to elicit diverse signaling pathways. We will highlight the role of paxillin and its central position to integrate the structural changes of the β3 integrin-talin1-kindlin complex, and offer evidence that paxillin is not only a scaffold or signaling protein as previously described, but also a key component of the mechano-transducing machinery (Figure 2).

Integrins come in different flavors, ranging from diversity in ligand binding and exhibiting specific structural features (Hynes, 2002; Bachmann et al., 2019), which makes it impossible to cover the entire family in this review. For historical reasons the αvβ3 integrin is one of the best studied receptors, with implication in many pathophysiological settings (Zhu C. et al., 2019), representing a typical example of many integrin-dependent functions.

The β3 integrin receptors comprise two heterodimers originating from the pairing of the β3 subunit with either αIIb or αv chains, creating the αIIbβ3 and the αvβ3 heterodimers, respectively (Hynes, 2002; Bachmann et al., 2019). The αIIbβ3 integrin complex is a platelet-specific receptor which is activated by multiple signaling cascades to trigger platelet activation and aggregation (Ye et al., 2012; Huang et al., 2019). In contrast, the αvβ3 integrin has a wider expression and physiological functions related to tissue repair and inflammation in osteoclasts, platelets, megakaryocytes, kidney, vascular smooth muscles, endothelium, and placenta (Horton, 1997). In addition, αvβ3 is upregulated in endothelial cells undergoing tumor-induced angiogenesis (Mahabeleshwar et al., 2007), as well as in many tumor cells (Horton, 1997; Vonlaufen et al., 2001). The αvβ3 integrin recognizes the Arg-Gly-Asp (RGD)-tripeptide-containing sequence present in different ECM ligands (Pytela et al., 1985; Horton, 1997; Humphries et al., 2006), and preferentially binds to vitronectin and osteopontin, especially under low force conditions (Bachmann et al., 2020). Changes in mechanical cues of the microenvironment enlarge the ligand preference of αvβ3 integrin, and can induce mis-regulation of integrin-dependent signaling pathways (Bachmann et al., 2020), as in the case of pathological ECM stiffening in the tumor niche (Attieh et al., 2017). The αvβ3 receptor also plays a role in tumor progression and metastasis formation: by controlling the actin cytoskeleton (Havaki et al., 2007); by supporting tumor cell binding to, and transmigration across activated endothelia (Saalbach et al., 2005); by synergizing with VEGF-dependent pathways to promote angiogenesis (Mahabeleshwar et al., 2007); and by sustaining the activation of the Src kinase (Huveneers et al., 2007). Importantly, αvβ3 FAs localize proteolytically active matrix metalloproteinases at the cell surface (Brooks et al., 1996) and support efficient directed cell migration to promote metastasis formation (Ballestrem et al., 2001).

The assembly of FAs requires the conformational switch of the β3 integrin receptor from a bent-close low-affinity, to an extended-open high-affinity state (Figure 1). This activation can be triggered by the binding of the intracellular adapter proteins talin1 and kindlin to the cytoplasmic tail of the β3 integrin (Hytonen and Wehrle-Haller, 2014; Bachmann et al., 2019; Huang et al., 2019). These integrin activators are essential for integrin-dependent attachment and spreading: talin-null cells and kindlin-null cells display a non-adherent phenotype (Bottcher et al., 2017), suggesting a lack of transmission of mechanical signals and of cellular responses.

The progression from first integrin-adapter interactions and ECM-ligand binding toward FA maturation involves integrins clustering and their mechanical connection to the intracellular actin network (Thievessen et al., 2013; Hytonen and Wehrle-Haller, 2014) (Figure 1; Figure 2A). During this process, force is a key player acting at several steps. For example, catch bonds (i.e. force-dependent bonds strengthened by the force applied along the receptor) are formed at the level of the integrin-ligand interaction (Gauthier and Roca-Cusachs, 2018), while the mechanical tension along the integrin-adapters-actin axis leads to the exposure of cryptic binding sites in the talin C-terminal rod domain (Del Rio et al., 2009; Rahikainen et al., 2017), thus favoring the interaction with adapter proteins (e.g., paxillin, vinculin, and FAK) and the assembly of multiprotein signaling complexes (Hytonen and Vogel, 2008; Hytonen and Wehrle-Haller, 2014; Goult et al., 2018).

Integrin-mediated adhesions are described as mechano-sensitive because of their changes in response to mechanical stimuli (Hytonen and Wehrle-Haller, 2016; Gauthier and Roca-Cusachs, 2018). However, adhesions also fulfil the role of mechano-transducer, transmitting physical and mechanical signals from the ECM to the cytoskeleton, and converting them into cellular responses (Wehrle-Haller, 2012; Stutchbury et al., 2017). This function of FAs relies on the presence of intracellular proteins capable of sensing force-induced conformational changes, as observed for the talin and kindlin adapters (Stutchbury et al., 2017; Bachmann et al., 2019).

Several reports described the mechano-sensitivity of paxillin, although the molecular basis of its force sensing capacity is at the present not fully understood. The presence of talin and kindlin for the arrival of paxillin at nascent adhesions is necessary but not sufficient, since the development of force across the adhesion complex is also required (Cluzel et al., 2005; Hytonen and Wehrle-Haller, 2014) (Figure 2A). Along this line, it was shown that paxillin exhibits a stretch-dependent binding to the cytoskeleton (Sawada and Sheetz, 2002), as well as a remarkable ability of its LIM domains to detect mechanically strained stress fibers (Smith et al., 2013). Furthermore, several LIM domain-containing proteins that cluster at FAs (Kadrmas and Beckerle, 2004) are recruited in a myosin II-dependent fashion, suggesting that LIM domains could function as tension sensors of a strained F-actin network (Schiller et al., 2011; Sun et al., 2020; Winkelman et al., 2020).

The tension-dependent binding of paxillin to adhesions, combined to its hub function, suggests a major role of this protein in integrating signals from the integrin complex and in activating molecular pathways shaping cell behaviour (Green and Brown, 2019) (Figure 2B). The versatility of paxillin in the selection of binding partners is supported by the nature of its LD domains (Alam et al., 2020) that generally establish poorly selective, transient interactions, which require multiple layers of regulation (Alam et al., 2014). Due to the low binding affinity of single LD motifs, multiple simultaneous interactions are required to achieve stable complexes and elicit cellular responses (Alam et al., 2014). For example, opposite faces of the four-helix bundle in the FAT (Focal Adhesion Targeting) domain of FAK and in vinculin tail associate to paxillin LD2 and LD4 (Hoellerer et al., 2003).

Owing to its extraordinary connection with a plethora of adhesome components, paxillin is regarded as a unique protein capable of integrating the diverse functions of FAs (Green and Brown, 2019; Chastney et al., 2020). In other words, paxillin fulfils the crucial linker function connecting the core actin, the cell cortex, the signaling, and the regulatory modules constituted by subsets of FA proteins (Figure 2B) (Green and Brown, 2019; Chastney et al., 2020). For a complete understanding of paxillin interactions and functions, precise analyses considering FA protein isoforms and their post-translational modifications should be considered as well. The analysis of these aspects goes beyond the goal of this review, yet we can provide as an example the cell adhesion-triggered paxillin phosphorylation at Tyr³¹ and Tyr¹¹⁸ (Burridge et al., 1992) which modulates its binding to β3 integrin adhesions (Ripamonti et al., 2021), possibly by increasing paxillin affinity for FAK and vinculin (Zaidel-Bar et al., 2007b; Choi et al., 2011; Case et al., 2015). Noteworthy, the described paxillin-dependent nanoscale (re-)localization of vinculin within the FA architecture (Case et al., 2015) suggests that paxillin functions as a FA organizer beside its linker function (Figure 2B) (Green and Brown, 2019).

How tension and the assembly of the described β3 integrin-talin1-kindlin-paxillin complex at FAs is dynamically regulated during cell motility remains an open question. Adhesion remodelling directly and positively correlates with the ability of cells to migrate (Deakin and Turner, 2011), which physically relies on adhesion formation at the leading edge and adhesion disassembly at the cell rear (Webb et al., 2004; Cluzel et al., 2005). The latter was proposed to be under the control of Src-mediated phosphorylation of paxillin Tyr^31/118 (Cortesio et al., 2011). Accordingly, Tyr-to-Phe mutations of these residues hampered adhesion turnover (Webb et al., 2004; Zaidel-Bar et al., 2007b) and inhibited tumor cell invasion (Mekhdjian et al., 2017). Consistent with these findings, sustained paxillin binding to FAs, phosphorylation of Tyr^31/118, and FAK signaling can result in FA disassembly and turnover of its components (Webb et al., 2004). On the other hand, loss of paxillin phosphorylation was proposed to be responsible of hindering FA disassembly and support FA maturation toward fibrillar adhesions and their translocation to the cell center (Zaidel-Bar et al., 2007b; Bachmann et al., 2019).

Paxillin may be involved in the regulation of tension at the cell edge during migration on ECM ligands. In this direction, the complex between the ArfGAP and scaffold protein GIT1 (G-protein-coupled receptor-kinase interacting protein-1) and the guanine nucleotide exchange factor for Rac1 βPix has been implicated in the regulation of FAs and cell migration (Turner et al., 1999; Premont et al., 2000). GIT1 is recruited to FAs by direct binding of its FA-targeting domain to paxillin LD2 and LD4 motifs (Schmalzigaug et al., 2007; Zhang Z. M. et al., 2008; Wehrle-Haller and Bastmeyer, 2014). Recently, evidence has been provided for the formation of protein condensates of the GIT1/βPix complex driven by liquid-liquid phase separation (Zhu et al., 2020), a process involved in the organization and compartmentalization of several events occurring in the cytoplasm and the nucleus of eukaryotic cells (Banani et al., 2017; de Curtis, 2021). The results from the study of the Zhang’s group indicate that the formation of GIT1/βPix condensates and their targeting at FAs by paxillin are required to regulate cell migration (Zhu et al., 2020). Paxillin is shown to promote the formation of GIT1/βPix condensates, and one intriguing hypothesis is that paxillin-mediated formation and recruitment of GIT1/βPix condensates at FAs may modulate F-actin polymerization and tension within adhesions to modulate FAs turnover (Figure 2B). Also, dominant-active Rac1-transfected cells presented slower integrin turnover than control cells, indicating that βPix activity may locally stabilize the turnover of integrins, and arrest retrograde sliding adhesions (Ballestrem et al., 2001). These mechanisms could explain the paxillin-mediated rescue of unstable and rapidly sliding adhesions, indicating a role of paxillin in the control of the F-actin feedback loop (Ripamonti et al., 2021).

The gathering of structural and positional data led to the proposal of the slanted fence model of FAs, in which connections among neighbouring integrin-talin1-kindlin-paxillin units stabilize the complex (Sun et al., 2016; Bachmann et al., 2019; Ripamonti et al., 2021) (Figure 1). The proposed layered organization of FAs potentially bears the secret how integrin receptors support mechanical load and create intracellular signaling in response to changes in tensional forces (Kanchanawong et al., 2010; Bachmann et al., 2019). The detailed characterization of this key biological mechanism, tightly related to paxillin recruitment and binding to FAs (Figure 2) (Cluzel et al., 2005; Hytonen and Wehrle-Haller, 2014, 2016; Ripamonti et al., 2021), will help in the development of efficient integrin-targeting anti-cancer therapies, so far challenged by the complexity of the integrin system which has caused unexpected side effects (Su et al., 2020; Li et al., 2021).