In 1979, Benjamin Geiger isolated a 130 kD protein from chicken gizzard that localized to the ventral surface of cells and to areas of cell-cell contact (Geiger 1979). About the same time, Keith Burridge and colleagues isolated a 130 kD, also from chicken gizzard (Burridge and Feramisco 1980). Burridge observed fluorescently labeled versions of this protein extensively co-localized with fibronectin and actin, suggesting a possible role in the organization of actin filaments at membrane attachment sites. Based on this information, the novel protein was named vinculin from the Latin word vinculum, which means a “bond” or “link.”
After its identification, many early studies reinforced the initial idea that vinculin links actin to attachment sites. Key among these was the identification of vinculin as a direct binding partner for actin and for talin – a new protein found to interacted with the integrins (Burridge and Mangeat 1984; Horwitz et al. 1986). These early studies also identified vinculin as the first substrate for Src kinase, though the significance of this phosphorylation event would not become evident for many years (Sefton et al. 1981).
By the mid-1980s, recombinant DNA techniques were routinely employed to characterize actin-binding proteins. These efforts resulted in the publication of partial sequences for vinculin in 1987 by the laboratory of David Critchley (Price et al. 1987). Subsequently, the full-length chick sequence was published in 1988 by Susan Craig (Coutu and Craig 1988). The amino acid sequence later revealed that vinculin was mostly alpha helical and was composed of a globular head domain, a proline-rich linker, and a tail domain. The identification of the vinculin sequences from other species indicated a remarkable conservation, demonstrating an indispensable cellular function that continues to be the subject of study to this day.
Crystal structures of the vinculin tail and full-length protein confirm early studies that claimed vinculin was predominantly alpha-helical protein (Bakolitsa et al. 1999, 2004; Borgon et al. 2004). According to these crystal structures, the vinculin head domain is made up of three alpha-helical bundles, known as domains 1–3 and a small portion of domain 4. The remainder of domain 4 contains the proline-rich linker. Domain 5, also an alpha-helical bundle, composes the tail domain. In the closed, “inactive” conformation, the head domains form pincers that bind and hold the tail in an autoinhibited conformation via a series of high-affinity intramolecular interactions. How the autoinhibited confirmation is released has been the subject of much debate. It is generally agreed that, in the autoinhibited conformation, vinculin localizes to the cytoplasm. At adhesion sites, binding of a combination of talin (or alpha catenin) and actin triggers vinculin to adopt an open conformation. In this open conformation, vinculin is largely extended which and exposes binding sites for actin and other actin-binding proteins (reviewed in Peng et al. 2011).
Vinculin-Binding Partners and Posttranslational Modifications
In the open conformation, vinculin binds a diverse array of proteins (Peng et al. 2011). Talin, alpha catenin, and beta catenin interact with the vinculin head domain. These molecules largely play a role in activating and/or localizing vinculin to sites of adhesion. In parallel, the Shigella invasin IpaA binds to the vinculin head domain and is required for entry into host cells. The linker domain of vinculin coordinates a series of actin-binding proteins, including the Arp2/Arp3 complex (an actin nucleator), VASP (an anti-capping protein), ponsin, and vinexin. These binding partners suggest that vinculin might rearrange the actin structures it binds. The vinculin tail binds F-actin, linking the actin cytoskeleton to sites of adhesion. Finally, the tail domain binds paxillin, which, under some conditions, recruits vinculin to cell-matrix adhesions. Phosphoinositide 4,5-bisphosphate (PIP2) is currently believed to facilitate the release of vinculin from focal adhesions. This diverse repertoire of proteins suggests that vinculin has the capacity to dynamically modulate linkage of adhesion plaques with the actin cytoskeleton.
The interaction of vinculin with its binding partners is regulated, in part, by phosphorylation. Vinculin is phosphorylated by protein kinase C (PKC) – a serine/threonine kinase. The significance of this phosphorylation event is not well understood but may regulate the vinculin binding to phospholipids. In contrast, more is known about vinculin tyrosine phosphorylation. Vinculin has three tyrosine residues that are highly phosphorylated. These residues include Y100 in the vinculin head domain, Y822 in the proline-rich linker, and Y1065 in the tail domain. Residues Y100 and Y1065 are phosphorylated by Src. Recently, Bays et al. identified Y822 as a substrate for Abelson kinase (Bays et al. 2014). Disruption of vinculin phosphorylation of Y100 and Y1065 prevents recruitment of the Arp2/Arp3 complex. Similarly, vinculin mutants that cannot be phosphorylated at Y822 fail to bind beta-catenin and do not efficiently localize to sites of cell-cell adhesion. Future work is needed to better understand how phosphorylation at distant residues regulates vinculin structure and/or conformational changes.
Vinculin Function in Cells
Volumes of evidence suggest that vinculin regulates the stability and the dynamics of adhesion plaques. Indeed, high levels of vinculin expression in fibroblasts are accompanied by increased cell adhesion, increased cell spreading, and decreased cell motility (Rodriguez Fernandez et al. 1992). In contrast, fibroblasts lacking vinculin form smaller and fewer focal adhesions. These cells fail to form stable cell-matrix adhesions and consequently migrate more rapidly than cells expressing vinculin (Xu et al. 1998). A similar phenotype is observed in neuronal cells lacking vinculin (Varnum-Finney and Reichardt 1994). Decreased expression of vinculin in this cell type hampers neurite outgrowth and slows growth cone progression. Comparably, when vinculin expression is inhibited in endothelial and epithelial cell lines, decreases in cell adhesion to neighboring cells and increases in paracellular permeability are observed (Birukova et al. 2016).
How vinculin regulates cell-cell and cell-matrix adhesion is becoming more evident. Vinculin regulates the clustering, turnover, and activation of integrins – the major cell-matrix adhesion receptor. Similarly, vinculin regulates retainment of the cell-to-cell adhesion receptor, E-cadherin, at the plasma membrane. In addition to directing regulating adhesion receptor function, vinculin transmits mechanical forces experienced by the adhesion receptors and orchestrates their mechanical signaling events. Indeed, in response to force, vinculin is actively recruited to regions where cells adhere to the substratum and where cells adhere to neighboring cells. These forces lock vinculin into an open conformation, allowing it to bear the force and recruit additional binding partners. These vinculin-associated events are critical for the cell to reinforce its actin cytoskeleton and grow its adhesions in response to force. How vinculin facilitates reinforcement of the actin cytoskeleton and cell adhesion is not completely understood. Binding to F-actin is clearly important. Additionally, vinculin may increase the bundling and/or polymerization of actin filaments. Alternatively, emerging evidence indicates that vinculin may recruit a number of actin regulatory proteins, such as Arp2/Arp3 and VASP, which could subsequently rearrange the actin cytoskeleton (DeMali et al. 2014).
Physiological Function in Organisms
In light of vinculin’s role in stabilizing cell-cell and cell-matrix adhesions, it is not surprising that its deletion interferes with proper development. Indeed, loss of the C. elegans vinculin homolog halts development at the L1 larval stage and disrupts muscle formation and function (Barstead and Waterston 1991). Mice lacking vinculin die at embryonic day 10 of gestation (Xu et al. 1998). The embryos from vinculin null mice are smaller size, have neural tube closure defects, and contain a malformed heart. Interestingly, deletion of vinculin in flies has no effect. However, this observation may warrant reexamination now that vinculin function in other model organisms and cellular processes are better understood.
The dramatic effect of vinculin deletion in the heart led to intense research into vinculin function in this vital organ. A tissue-specific deletion of vinculin in caroinmyoctes results in mice that die suddenly from ventricular tachycardia and disruptions in electrical conductance. These heart disturbances result from detachment of the intercalated disk from myofibrils. Along similar lines, Kaushik et al. recently found that flies genetically programmed to over-express vinculin live longer (Kaushik et al. 2015). This increase in longevity is attributed to an improvement in the contractile function of the hearts. Hence, vinculin is critical for the heart’s contractile function, further supporting a role for vinculin in bearing and transmitting mechanical force.
Implication of Vinculin in Disease States
Given its role in stabilizing cell-cell and cell-matrix adhesions and transmitting force, it seemed likely that vinculin should be altered in disease states. Indeed, vinculin expression is frequently lost in cancer such as rhabdomyosarcoma, breast carcinomas, and squamous carcinomas (reviewed in Peng et al. 2011). Moreover, colorectal and breast cancer patients experience a poor prognosis when their tumors express low vinculin levels. Hence, vinculin antagonizes the development and progression of cancer. Unlike cancer where its expression is lost, vinculin is mutated in many cardiomyopathies. Missense mutations in the vinculin head domain are found in patients with hypertrophic cardiomyopathy (Vasile et al. 2006). This mutation lies in the N-terminal lobe of vinculin head domain 2 which makes extensive contacts with head domain 3. Therefore, mutations at this residue could affect vinculin stability and/or conformational changes.
Since its identification and characterization as a key component of focal adhesions and adherens junctions over 35 years ago, vinculin has emerged as a critical modulator of adhesion receptor stability. This critical role for vinculin is most evident in embryonic development and in contractile tissues where vinculin bears the forces experienced by the adhesion receptors and transmits this force to the actin cytoskeleton. To serve this critical role, vinculin is recruited to adhesion receptors via binding partners such as talin, alpha catenin, and beta catenin. At the adhesion receptor, it forms a direct link with filamentous actin and a number of actin-binding partners, which may reorganize the actin cytoskeleton. Future studies will allow the interactions between these proteins and others to be dissected so that a complete picture emerges for how vinculin orchestrates cell-cell and cell-matrix adhesion and mechanotransduction.