InteractiveFly: GeneBrief

Vinculin: Biological Overview | References

Gene name - Vinculin

Synonyms -

Cytological map position - 1-0.8

Function - cytoskeletal interactor

Keywords - component of adherens and focal adhesion junctions - regulates cytoskeletal anchoring at the plasma membrane - regulates cell adhesion - mechanotransduction - triggers the formation of cytoplasmic adhesion complexes - interacts with α-Catenin - recruited to amniosersoa apical cell membranes during dorsal closure - regulates of cardiac function during aging

Symbol - Vinc

FlyBase ID: FBgn0004397

Genetic map position - chrX:2,214,347-2,222,348

NCBI classification - Vinculin family

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene

Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. This study examined how altering vinculin activity perturbs integrin function within the context of Drosophila development. Whereas loss of vinculin produced relatively minor phenotypes, gain of vinculin activity, through a loss of head-tail autoinhibition, caused lethality. The minimal domain capable of inducing lethality is the talin-binding D1 domain, and this appears to require talin-binding activity, as lethality was suppressed by competition with single vinculin-binding sites from talin. Activated Drosophila vinculin triggered the formation of cytoplasmic adhesion complexes through the rod of talin, but independently of integrin. These complexes contain a subset of adhesion proteins but no longer link the membrane to actin. The negative effects of hyperactive vinculin were segregated into morphogenetic defects caused by its whole head domain and lethality caused by its D1 domain. These findings demonstrate the crucial importance of the tight control of the activity of vinculin (Maartens, 2016).

Cell adhesion is mediated by multiprotein complexes that link transmembrane receptors to the cytoskeleton. These complexes are assembled at discrete sites of the membrane, and both loss and gain of adhesion protein activity causes cellular and developmental defects, which have pathological consequences (Maartens, 2016).

The first step in building a cell-matrix adhesion is the binding of transmembrane integrin receptors to extracellular matrix (ECM) components. This is followed by recruitment of cytoplasmic adhesion proteins, for example talin (also known as Rhea in flies), which occurs through the cytoplasmic tail of integrin. Talin is a crucial component of the link as it can simultaneously bind integrins (with its FERM-domain head) and actin (with an actin-binding site at the C-terminus of its long rod domain). Talin feeds back to promote integrin activation and is required for the recruitment of numerous cytoplasmic adhesion proteins. Of particular interest is the force-dependent recruitment of vinculin. In vitro work has established that stretching the rod of talin exposes previously hidden vinculin-binding sites (VBSs, single helices within the α-helical bundles that make up the rod) that can then bind vinculin (Papagrigoriou, 2004; del Rio, 2009). Consistent with this model, the recruitment of vinculin to adhesions in cell culture is particularly sensitive to myosin II inhibition (Riveline, 2001; Pasapera, 2010; Carisey, 2013; Maartens, 2016 and references therein).

A series of four-helical bundles (seven in vertebrates, six in invertebrates) make up the head domain of vinculin, which is linked by a partially disordered proline-rich region to the five-helical bundle of the tail (Bakolitsa, 2004; Borgon, 2004). Interaction sites for vinculin ligands have been mapped across the protein. A key ligand is talin, and the interaction has been narrowed to the first two four-helical bundles of the head, the D1 domain (also known as Vh1; Bois, 2006): the VBSs in talin bind to the first four-helical bundle of the D1 domain, transforming it into a five-helical bundle (Izard, 2004). This first bundle of D1 retains most of the VBS-binding activity of the D1 domain in a two-hybrid assay, suggesting it is the minimal talin-binding site, but the second bundle is also capable of binding some ligands, and the entire D1 domain is generally used as a minimal head domain. Vinculin is notable among integrin-associated proteins for also localising to cell-cell adhesions, and this is mediated through an interaction of the head with either α- or β-catenin. The flexible neck of vinculin binds proteins of the CAP and vinexin family, among other ligands, and the tail binds to actin, the scaffolding protein paxillin, and the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), and also promotes dimerisation. By simultaneously binding talin and actin, vinculin provides an additional link to the cytoskeleton, giving extra mechanical support to the adhesion. A strengthening role is consistent with the relatively milder effects of losing vinculin compared to losing talin in cells in culture and in developing animals (Maartens, 2016 and references therein).

Although vinculin has many binding partners, the full-length protein has little binding activity due to a head-tail interaction stabilising the inactive conformation. Constructs that relieve this head-tail autoinhibition are hyperactive, and dramatically increase the size and stability of focal adhesions associated with activated integrins, as well as making the recruitment of adhesion proteins no longer sensitive to myosin II inhibition. The talin-binding D1 domain alone is sufficient to produce these effects, and, reciprocally, reducing the ability of the D1 domain to bind talin eliminates them. The vinculin tail adds additional activity: it is required for hyperactive vinculin to produce traction forces (Dumbauld, 2013) and reorient adhesions in response to polarised forces (Carisey, 2013). A key aspect of vinculin function is therefore its activation status, and its effects on cell behaviour might be caused by its action on talin as well as direct or indirect recruitment of proteins to adhesions. Although the impact of hyperactive vinculin on cellular behaviour has been well documented, the impacts of these changes on cells within the organism have yet to be addressed. A mutant that produces hyperactive vinculin in mouse has a milder version of the defects caused by absence of vinculin, but vinculin levels are also strongly reduced, making it difficult to separate loss- and gain-of-function effects (Maartens, 2016).

To probe further how vinculin contributes to adhesion, this study has used Drosophila to compare loss- and gain-of-function effects during development. Vinculin hyperactivity was found to be far more deleterious to the organism than inactivity, and a new function was discovered for vinculin in bringing adhesion proteins together independently of the usual integrin cue. The D1-talin-rod interaction is crucial for the formation of these cytoplasmic adhesion subcomplexes, supporting a model where hyperactive vinculin ectopically activates talin in the cytoplasm by mimicking the effect of force on talin. Finally, this study dissected the negative effects of hyperactive vinculin into two discrete activities: morphogenetic defects caused by its head domain, and lethality caused by its D1 domain (Maartens, 2016).

Whereas flies can tolerate the loss of vinculin, this study has discovered that excessive vinculin activity is lethal, and causes defects in muscle development. Both of these deleterious effects appear to require binding a VBS-containing protein such as talin. Talin is also required for a new role of Drosophila vinculin: inducing the formation of cytoplasmic aggregates that are adhesion subcomplexes. These subcomplexes are not linked to integrins or the cytoskeleton, and demonstrate that adhesion protein complexes can form without any input from integrin (Maartens, 2016).

Flies lacking vinculin displayed defects in the adult musculature, similar to the mild defects in larval musculature reported by Bharadwaj (2013). Other tissues appeared normal, and attempts to identify additional impairment in the athletic abilities of the flies were not successful, so the fly phenotype remains weaker than the phenotypes observed in mice, zebrafish or nematodes lacking vinculin. Redundancy with other adhesion proteins (such as talin; Klapholz, 2015) might explain the relatively mild phenotype of this highly conserved protein (Maartens, 2016).

The contrast between the consequences of loss and gain of vinculin activity are striking. In general, overexpressed integrin-associated proteins do not induce lethality in Drosophila (for example, talin , tensin, and ILK). Two integrin-associated proteins cause lethality when the wild-type form is overexpressed: focal adhesion kinase and parvin. The lethality of vinculin relied on a reduction of its autoinhibition, but this does not seem general to Drosophila adhesion proteins: disruption of talin autoinhibition has mild effects, and whereas expression of tensin fragments does cause some phenotypes, expression of fragments of other adhesion proteins does not (see above references). The severe effects of hyperactive vinculin fit with the very strong intermolecular interactions that keep it in the closed state. Activating mutations of vinculin have not been reported in the human population, as expected if, as in flies, they cause dominant lethality (Maartens, 2016).

Expressing vinc-CO or vinc-Head in the developing musculature led to developmental defects. These could arise as a result of hyperactive vinculin in the aggregates or at the adhesion site. The defects are distinct from integrin loss phenotypes, and this might reflect recruitment of additional proteins contributing to muscle formation to the aggregates or the adhesion. Cytoskeletal machinery is crucial for muscle fusion and muscle pathfinding to tendon cell targets, and an interaction between hyperactive vinculin complexes and more general cytoskeletal factors might explain the muscle defects. Sequestration of Z-disc proteins to ectopic intracellular aggregates has been implicated in the muscle phenotypes associated with myofibrillar myopathy, and a similar effect may be stimulated by hyperactive vinculin. (Maartens, 2016).

Cytoplasmic aggregate formation appears to be unique to Drosophila vinculin. Gallus vinc-D1 did not generate cytoplasmic aggregates, even though it appeared to interact with Drosophila talin (coexpressing Drosophila VBSs blocked its recruitment and lethality). In vertebrate cell culture, hyperactive vinculin is recruited to integrin adhesions at the membrane, but cytoplasmic talin-containing aggregates have not been reported. The interaction between vertebrate vinc-Head and talin rod in vitro requires prior stretching of the rod by force (del Rio, 2009; Ciobanasu, 2014; Yao, 2014), consistent with the in vivo interaction relying on prior talin stretching at the adhesion. In contrast, mitochondrial targeting experiments indicate that activated Drosophila vinculin can bind to un-stretched talin in the cytoplasm. A prediction from these results is that vertebrate vinculin should not recruit talin to the mitochondria, whether active or not. However, in vertebrate cells, talin is recruited by mitochondrially targeted vinc-CO and even full-length vinculin (albeit very weakly). However, in these cases targeted vinculin constructs pull the mitochondria to the membrane, so that vinculin and talin become associated with integrins and actin (no such association was found in targeting experiments carried out in this study). Thus, it seems feasible that, in these experiments, the association of vinc-CO is with stretched talin at the adhesion site, rather than with cytoplasmic talin as occurs in Drosophila (Maartens, 2016).

Several lines of evidence show that hyperactive Drosophila vinculin formed aggregates by binding to cytoplasmic talin. In the absence of talin, no aggregates were formed, and the rod of talin was a sufficient platform for aggregation, with longer sections supporting more aggregates, presumably due to an increase in the number of VBSs available per talin molecule. VBS coexpression blocked aggregate formation, suggesting that direct binding between vinculin and talin was important, and indeed the minimal vinculin fragment capable of forming aggregates was the talin-binding D1 domain. Hyperactive, but not wild-type, vinculin was capable of recruiting talin to the mitochondrial surface. Vinc-CO recruitment of talin to the aggregates was not altered by the loss of integrins, ruling out an alternative hypothesis whereby an initial stretching of talin at the adhesion is a first step in the formation of the cytoplasmic aggregates (Maartens, 2016).

An interesting feature of the vinculin-talin interaction is its reciprocity: just as hyperactive vinculin appears to bind closed talin, isolated VBSs can bind closed vinculin on the mitochondria, consistent with the capacity of vertebrate VBSs to dislodge the head from the tail in vitro (Bois, 2006). Thus, the interaction between vinculin and talin in Drosophila need only require activation of one partner. An open question is whether there are normal signals, mimicked by the 'T12' mutation, that open Drosophila vinculin so that it can force talin into an extended conformation. Recently, it has been found that expression of a mutant talin with a deletion of domains R2-R3, which contain four VBSs, causes very similar effects to expressing vinc-CO. Binding of activated-vinculin thus alleviates some form of internal negative regulation within talin, which might in part be due to regulation of the central actin-binding domain encompassing R4-R8 (Maartens, 2016).

Gallus vinc-D1 demonstrated that hyperactive vinculin could induce lethality in Drosophila without forming aggregates. How it does so remains an open question, but the idea is favored that it is caused by the action of the D1 domain of vinculin on talin at the adhesion sites. The vinculin head stabilises talin into a stretched conformation in cells and in vitro (Margadant, 2011; Yao, 2014), and this relies on prior stretching of talin (Ciobanasu, 2014; Yao, 2014). Furthermore, vinculin is required for talin to extend fully away from the plasma membrane (Case, 2015; Klapholz, 2015). Thus, lethality could arise from hyperactive vinculin binding to stretched talin and the failure of vinculin to release when force is reduced. Cycles of stretching and relaxation might be crucial for normal talin function or relaxation of talin might be required for its dynamic turnover. Alternatively, hyperactive vinculin might stimulate too much adhesion, stabilising integrin adhesions and reducing turnover in dynamic morphogenetic events. Elevated integrin expression has been shown to hinder cell migration in the Drosophila ovary (Lewellyn, 2013), and vinculin stimulation of integrin activation might affect similar processes. The lethality caused by vinc-D1 constructs occurs without defects in muscle morphogenesis. Assessing whether the muscle defects of vinc-Head and vinc-CO also contribute to lethality would require a method to block the lethality of vinc-D1 without impairing the muscle phenotypes of vinc-Head or vinc-CO, which is currently lacking (Maartens, 2016).

Although this study has examined vinculin D1 from only two species, it is speculated that vertebrate vinculin has lost the ability to bind to closed talin, and might have become more tightly closed by the addition of an eighth four-helix bundle that occurred during the evolution of the deuterostome lineage. Thus, vertebrate cells might be even more sensitive to the consequences of aberrant association between vinculin and talin (Maartens, 2016).

The results suggest that certain proteins have the ability to act as a switch, triggering assembly of an integrin adhesion complex. Integrins are well known to have this switch ability: engagement with the ECM and clustering triggers the formation of adhesion sites. When Drosophila vinculin loses autoinhibition, it triggers the assembly of an adhesion complex, and this process can occur entirely independently of integrins. In contrast to integrins, however, the full complement of adhesion proteins is not recruited, suggesting that additional mechanisms are required (for instance, membrane proximity, application of force, or signalling). This raises the question of how the additional proteins are recruited to the cytoplasmic aggregates, and whether the pathways involved are similar to those utilised by constitutively active vinculin at adhesions and by integrins and talin in normal adhesions. Recruitment requires talin, but the relative contributions of vinculin and talin have yet to be established (Maartens, 2016).

Integrin-independent interactions of adhesion proteins have been demonstrated by fluorescence correlation analysis wherein adhesion components self-assembled in the cytosol. However, these 'building blocks' were composed of three or four protein species, never assembled into larger structures and did not include a talin-vinculin interaction. Nevertheless, the above work shows how interactions between the component parts of the adhesion need not necessarily rely on a direct or even indirect link to integrins, consistent with the current work. A key role of integrins might be to trigger the assembly of the cytoplasmic adhesion-complex-specific sites in the membrane, rather than being a necessary part of this link (Maartens, 2016).

From an evolutionary perspective, certain cytoplasmic components like vinculin and talin predate the integrins. It is tempting to propose that integrins co-opted pre-existing cytoplasmic complexes, using them to strengthen their adhesion to the ECM at discrete sites along the cell surface. This evolutionary change may also have required mechanisms to restrict the spontaneous formation of adhesion-like complexes in the cytoplasm. The strong head-tail interaction of vinculin could be one such mechanism (Maartens, 2016).

Cell boundary elongation by non-autonomous contractility in cell oscillation

Throughout development, tissues exhibit dynamic cell deformation, which is characterized by the integration of cell boundary contraction and/or elongation. Such changes ultimately establish tissue morphology and function. In comparison to cell boundary contraction, which is predominantly driven by non-muscle myosin II (MyoII)-dependent contraction, the mechanisms of cell boundary elongation remain elusive. This study explored the dynamics of the amnioserosa, which is known to exhibit cell shape oscillation, as a model system to study the subcellular-level mechanics that spatiotemporally evolve during Drosophila dorsal closure. Cell boundary elongation is shown to occur through a combination of a non-autonomous active process and an autonomous process. The former is driven by a transient change in the level of MyoII in the neighboring cells that pull the vertices, whereas the latter is governed by the relaxation of junctional tension. By monitoring cell boundary deformation during live imaging, junctional tension at the specific phase of cell boundary oscillation, e.g., contraction or elongation, was probed by laser ablation. Junctional tension during boundary elongation is lower than during the other phase of oscillation. The tension measurements were extended to non-invasively estimate a tension map across the tissue, and a correlation was found between junctional tension and vinculin dynamics at the cell junction. It is proposed that the medial actomyosin network is used as an entity to both contract and elongate the cell boundary. Moreover, the findings raise a possibility that the level of vinculin at the cell boundary could be used to approximate junctional tension in vivo (Hara, 2016).

This study analyzed the apical cell oscillation of amnioserosa (AS) cells during Drosophila dorsal closure, which is a proven model system to study cell/tissue dynamics and wound healing. Live imaging of AS cells expressing Dα-cateninRFP showed complex dynamics in cell shape during the early stages of dorsal closure, which is the stage shortly after the edge of AS tissue is smoothed out. AS cells exhibited both apical area oscillations and cell boundary length oscillations. Moreover, cell boundary length oscillations were associated with little, if any, cell-cell junction rearrangement such as zipping or unzipping. These two oscillations did not necessarily synchronize, meaning that not all cell boundaries of shrinking cells were contracting, and vice versa. This suggested that cell area oscillation is in part an integrated result of complex cell boundary dynamics (Hara, 2016).

To investigate the mechanism of cell boundary oscillation, especially the relative contributions of both intrinsic and extrinsic contractility in cell boundary oscillation, the relative distribution was visualized of dynamic medial myosin II (MyoII), which was visualized by MyoII regulatory light chain (spaghetti squash, sqh) fused to GFP, hereafter referred to as sqhGFP. Medial MyoII distributes at the non-junctional cortical region of the apical side of the cell, and its level was higher compared to the level of junctional MyoII during the early stage of dorsal closure. To quantify the relative distribution of medial MyoII, the ratio was measured of MyoII levels between the two cells that shared the boundary of interest and the two neighboring cells that form vertices with the boundary of interest. The myosin ratio was then compared with the rates of boundary length deformation. Qualitatively, these two values were oscillatory and temporally anti-correlated. For instance, MyoII accumulated more in the neighboring cells (myosin ratio <1) when the cell junction was elongating, whereas MyoII accumulated more in the vicinity of the cell junction (myosin ratio >1) when the junction was shrinking. Temporal cross-correlation analysis showed that the rates of boundary length deformation coincided with the rate of the myosin ratio, suggesting that the change in the level of MyoII contributes to boundary length change. The myosin ratio reached its highest point 20 s after the peak of boundary length deformation. MyoII that appeared around the cell boundary during oscillation did not accumulate at cell junctions. This was different from MyoII dynamics in ratchet-like cell boundary contraction during Drosophila germband extension (GBE) (Hara, 2016).

To uncover the spatial and temporal propagation of junctional oscillation, temporal cross-correlation analysis was performed of the changes in boundary length between the nearest cell boundaries and between the next-nearest cell boundaries. The former showed anti-correlation with no time lag, and the latter showed no correlation. These results suggested that the effect of cell boundary oscillations remained localized and did not propagate throughout the tissue. This is consistent with a report showing that the mechanical perturbation in the AS does not affect the apical area of the neighboring AS cells. Together, these analyses suggested that the elongation of boundary length was correlated with the change of the level of MyoII in the neighboring cells (Hara, 2016).

To confirm that medial MyoII in the neighboring cells plays an active role in the elongation of the cell boundary, medial MyoII was perturbed by UV laser at the level of adherens junctions. A laser was targeted to disrupt the cluster of medial MyoII in a neighboring cell when a cell boundary was undergoing elongation. Still images and kymographs showed that the disruption of the MyoII cluster resulted in shorter cell boundaries. In contrast, similar laser ablation of the neighboring cell, where there was no notable MyoII cluster, caused no drastic change in the dynamics of the cell boundary. This showed that there were little, if any, effects on cell boundary length change by laser ablation without a MyoII cluster. It is important to point out that the myosin accumulation in two neighboring cells was not necessarily synchronized to cause junction elongation. Furthermore, laser ablation targeting a cluster of medial MyoII in a cell with a cell boundary undergoing contraction resulted in longer cell boundaries and in shortening of the neighboring cell boundaries. Statistical analyses of the relative cell boundary lengths 25 s after laser ablation (Dt25), compared to the length at the time of ablation (Dt0), further supported these observations. The laser ablations during cell boundary elongation and contraction made Dt25/Dt0 smaller or larger than 1, respectively. Two different control laser ablation experiments resulted in a Dt25/Dt0 that was close to 1, suggesting that there was little change in cell boundary length upon laser ablation (Hara, 2016).

These results support the idea that boundary elongation is a non-autonomous process where the transient accumulation of medial MyoII in the neighboring cell causes junction elongation by pulling the vertex that defines the end of the cell boundary (Hara, 2016).

To further investigate the mechanics behind cell boundary length oscillation, cell junctional tension was probed using UV laser ablation at different phases of cell boundary oscillation. To ablate the cell boundary at a specific phase of oscillation, the dynamics of cell boundaries was monitored during live imaging, and the laser was targeted to the boundary of interest. For all data, several parameters were quantified, including the instantaneous recoil velocity, cell boundary deformation rate, and ratio of medial MyoII prior to ablation. The instantaneous recoil velocity is proportional to the junctional tension and inversely proportion to the drag coefficient, and is widely accepted as a first approximation of the junctional tension before ablation. The dynamics of the cell boundary were classified into three categories based on its deformation rate, vdef: contraction (vdef ≤ -0.01 μm/s), stable (-0.01 < vdef < 0.01), and elongation (0.01 ≤ vdef). To take into account the geometry of the cell boundary, a further three classifications were added: low and high boundary waviness (Wn) as well as rosette, which is the cell boundary directly linked to a delaminating cell (Hara, 2016).

Yhe relationship between cell boundary dynamics and the medial myosin ratio measured from the laser ablation experiments was further analyzed. Consistent with the analysis of non-laser-ablated tissue, the medial myosin ratio was larger than 1 and smaller than 1 in the case of cell boundary contraction and expansion, respectively. When the cell boundary was stable, the medial myosin ratio was close to 1, meaning its distribution was balanced. Together, the boundary dynamics and the medial myosin ratio have a strong linear relationship, regardless of boundary waviness. Based on these two analyses, it was found that the recoil velocity and medial MyoII distribution are correlated, especially among the boundaries with low waviness. Moreover, the cell boundaries associated with delaminating cells, which were categorized as 'rosette,' did not follow the linear relationships. This is simply due to the fast recoil velocity and high accumulation of medial MyoII in delaminating cells (Hara, 2016).

Together, the laser ablation experiments at specific phases of cell boundary oscillation clearly demonstrated that the recoil velocity, and in turn the cell junctional tension, correlates with boundary dynamics. This suggests that the state of tension at the boundary changes during cell boundary oscillation. Interestingly, the junctional tension becomes weak when the boundary is elongating. This weakening could emerge from the lower level of medial MyoII in the vicinity of the boundary. The results raise the possibility that cell boundary elongation arises from the combination of junctional tension relaxation, which is represented by the transition from the high junctional tension during contraction to the low tension during elongation, and an active pulling by the neighboring cells (Hara, 2016).

Knowing the relative recoil velocity of the cell boundaries, with respect to cell boundary dynamics and junctional geometries, allowed estimation of the tension distribution across the AS non-invasively. To investigate this, five categories of junctional tension were established based on magnitude, where category I represents rosette boundaries, which exhibit the highest tension (relative tension 4.2), category II represents the contracting low Wn boundaries (2.7), category III represents the stable low Wn boundaries (2.0), category IV represents the contracting high Wn boundaries (1.5), and category V represents the elongating and stable high Wn boundaries (1). Around 90% of the boundaries in category V were elongating boundaries. These categories were applied to time-lapse images, and the tension across AS tissue at the early stage of dorsal closure was estimated. To validate estimations of cell junctional tension, the spatial distribution of the average tissue tension was estimated by integrating the estimated junctional tension. Each estimate of junctional tension (T) was split into two components: a tension parallel to the medio-lateral (M-L) axis (TML), and a tension parallel to the anterior-posterior (A-P) axis (TAP). The area of AS tissue from which the junctional tensions were estimated was then split into six sections along the A-P or M-L axis, and the average tension of each component, and , in each section was subsequently computed. The spatial distribution of the computed average tissue tension was uniform across the AS tissue, except for the section with rosette boundaries. This uniform distribution of tissue tension was compared with the tissue tension mapped by tissue-level laser ablation. Particle image velocimetry was applied to two DE-cadGFP images, right before and 11 s after the laser ablation, and vector fields of tissue relaxation were generated. The spatial distributions of the tissue relaxation speed, which is proportional to tissue stress, were uniform across AS tissue. This was in good agreement with the distribution of the computed average tissue tension. Together, it is concluded that the non-invasive tension estimation is valid (Hara, 2016).

It has been shown that the binding or unbinding of vinculin to α-catenin within adherens junctions is dependent on the tension exerted on α-catenin through cytoskeletal contractility. To investigate the tension-dependent dynamics of vinculin at adherens junctions in vivo, embryos expressing hs-vinculinGFP (heat shock-vinculinGFP) ubiquitously under a heat shock promoter were imaged. it was first confirmed that a high level of vinculinGFP was found at loci known to associate with endogenous vinculin, such as the wound edge and the leading edge of the lateral epidermis during dorsal closure. Then the dynamics of vinculin during dorsal closure were monitored, and it was found that the level of vinculin at the cell boundary changed during cell boundary oscillation. The level of vinculin was further compared with the rates of boundary length deformation. Qualitatively, these two values were temporally anti-correlated. For instance, vinculin accumulated more when the cell junction was shrinking, whereas there was less vinculin when the junction was elongating. Temporal cross-correlation analysis showed that the change in the boundary length coincided with the change in the level of vinculin at the cell junctions. This correlation was not observed between the change in the boundary length and the change in the fluorescence intensity of the plasma membrane, which was visualized with a pleckstrin homology domain-containing plasma membrane marker, PLCγ-PH-GFP (PH domain of phospholipase C-γ fused with GFP). Together with a few junctional rearrangements during boundary oscillation, these results suggest that the change in the level of vinculin at the cell junctions was attributable to the accumulation or dissociation of vinculin. Using non-invasive tension estimation, the relationship was examined between vinculin dynamics, and junctional tension was estimated. The rate at which the vinculin level changed was correlated with junctional tension. The rate of vinculin change was positive when the tension was high, whereas the rate of vinculin change was negative when the tension was low. Temporal cross-correlation analysis showed that the rates of change in vinculin level coincided with the junctional tension. Together, these analyses suggested that the accumulation or dissociation of vinculin at the cell boundary was correlated with the dynamics of the cell boundary and, in turn, the junctional tension (Hara, 2016).

This study has described how cell boundaries elongate and how junctional tension evolves during the cell boundary oscillation of AS cells during Drosophila dorsal closure. Using quantitative image analysis and laser ablation, it was shown that cell boundary elongation occurs through a combination of a non-autonomous active process, which is driven by the transient change of the level of medial MyoII in the neighboring cells, and a relaxation of junctional tension, which could be governed by the reduction of the level of medial MyoII in the vicinity of a cell boundary. Recently, it has been reported that cell boundary elongation, which occurs during cell intercalation, and in particular the type 2-to-type 3 transition in GBE, is powered by medial MyoII in the neighboring cells. Moreover, Bardet reported that reduction of MyoII and, in turn, a downregulation of junctional tension are required for cell junction growth during the type 2-to-type 3 transition in Drosophila pupal wing development (Bardet, 2013). This takes place through a mechanism that is distinct from that in GBE. It is proposed that the active pulling by neighboring medial MyoII accumulation and/or the junctional tension relaxation is a general mechanism of cell boundary elongation. The junctional tension measurements were extended to non-invasively estimate a tension map across the tissue, and the vinculin dynamics at cell boundaries were found to be correlated with junctional tension. However, the molecular basis of vinculin accumulation and dissociation at the cell junction, and whether these vinculin dynamics are a cause or an effect of junction deformation and tension, remains an open question. Although a Drosophila vinculin mutant did not exhibit notable phenotypical defects, and thus the functions of vinculin in Drosophila development remain elusive, this finding raises the possibility that the level of vinculin at the cell boundary could be used to approximate junctional tension (Hara, 2016).

α-Catenin stabilises Cadherin-Catenin complexes and modulates actomyosin dynamics to allow pulsatile apical contraction

This study investigated how cell contractility and adhesion are functionally integrated during epithelial morphogenesis. To this end, the role of α-Catenin, a key molecule linking E-Cadherin-based adhesion and the actomyosin cytoskeleton, was analyzed during Drosophila embryonic dorsal closure, by studying a newly developed allelic series. α-Catenin was found to regulate pulsatile apical contraction in the amnioserosa, the main force-generating tissue driving closure of the embryonic epidermis. α-Catenin controls actomyosin dynamics by stabilising and promoting the formation of actomyosin foci, and also stabilises DE-Cadherin (Drosophila E-Cadherin, also known as Shotgun) at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, a genetic interaction was uncovered between α-Catenin and Vinculin, and a tension-dependent recruitment of Vinculin to amniosersoa apical cell membranes, suggesting the existence of a mechano-sensitive module operating in this tissue (Jurado, 2016).

How adhesion and actomyosin contractility are integrated at junctions is a fundamental question in morphogenesis. To tackle this, the role of α-Catenin, a key protein linking adherens junctions and the actin cytoskeleton, was analyzed in the context of Drosophila embryogenesis and in particular during dorsal closure. α-Catenin was found to regulates pulsatile actomyosin dynamics in apically contracting cells by stabilising and promoting actomyosin contractions. α-Catenin also stabilises DE-Cadherin at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, the results reveal an interaction between α-Catenin and Vinculin that could be important for DE-Cadherin stabilisation (Jurado, 2016).

Live imaging of mutant embryos shows a strong requirement for α-Catenin in the migration of the dorsal ridge primordia towards the dorsal midline, preventing the formation of the dorsal ridge and thus affecting both dorsal closure and head involution. These results reveal that the dorsal ridge is particularly sensitive to the levels of α-Catenin and suggest it is a key region that could mechanically coordinate both processes. Although it is clear that some of the defects observed during dorsal closure are a consequence of the defective dorsal ridge morphogenesis, this analysis shows that other cellular processes more specific to dorsal closure are affected. In particular, it was observed that the actin cable is disorganised and that the pulsatile apical contraction of the amnioserosa is abnormal (Jurado, 2016).

The defects observed at the level of amnioserosa apical cell oscillations could be a consequence of a defective actin cable, which would be acting as a ratchet and thus progressively restricting the expansion of apical cell area. However, several lines of evidence suggest that a ratchet mechanism stabilising the contracted state of amnioserosa cells is acting at the level of individual cells. In particular, the analysis performed in this study of actin oscillatory dynamics in α-Cat mutants suggests that the increase in the expansion half-cycle of amnioserosa apical cell oscillations could be due to an increase in the time interval between the appearance of consecutive foci. Thus, the results favour the idea that the Cadherin-Catenin complex has a role in promoting actomyosin oscillatory dynamics. How α-Catenin promotes actomyosin contractility remains to be elucidated, but it is likely to involve both direct and indirect (through other actin-binding proteins) interactions with the actin cytoskeleton. For example, an antagonistic interaction between α-Catenin and the Arp2/3 complex has been observed both in cell systems and in Drosophila embryos, raising the possibility that the actin-bundling activity of α-Catenin at adherens junctions, rather than the formation of Arp2/3-dependent networks, could be important for apical contraction (Jurado, 2016).

Interestingly, it was found that with the α-Cat2049 allele, adhesion dynamics are also defective, suggesting that medioapical actomyosin dynamics promote adherens junction stabilisation. In contrast, with the α-Cat421 allele, which would bind constitutively to Vinculin in a context of defective medioapical actomyosin dynamics, DE-Cadherin stabilisation is recovered. This result suggests that the stabilisation of DE-Cadherin could be mediated by the binding of Vinculin to α-Catenin. This is in agreement with what has been observed in cell systems, where forms of α-Catenin that constitutively bind to Vinculin have decreased mobility. It was further shown that, although DE-Cadherin is stabilised in α-Cat421 mutants, possibly due to the Vinculin-α-Catenin interaction, this stabilisation is not able to rescue normal medioapical actin dynamics. Thus, it is suggestd that direct binding of α-Catenin to actin through its actin-binding domain promotes the formation of medioapical actomyosin foci, whereas indirect binding to actin through Vinculin would promote junction stabilisation. Taken together, these data suggest that α-Catenin domains, through their interactions with other actin-binding proteins and actin, might differentially regulate actin dynamics (Jurado, 2016).

Finally, the results show that there is a tension-dependent recruitment of Vinculin at the membranes of amnioserosa cells, which could be mediated by α-Catenin. Interestingly, it has recently been found, in experiments using a heat-shock inducible Vinculin reporter, that the rate of change of Vinculin levels correlates with junctional tension (Hara, 2016). The results also suggest that Vinculin is able to perform an adhesive function when α-Catenin function is compromised. This could result from an α-Catenin-independent binding of Vinculin to E-Cadherin or from an interaction between Vinculin and other junctional proteins such as ZO-1 (also known as TJP1), which has been shown to recruit Vinculin to VE-cadherin junctions and increase cell-cell tension (Tornavaca, 2015). However, given that ZO-1 can also interact with α-Catenin, it remains to be investigated whether the mechano-sensitivity of Vinculin is completely dependent on α-Catenin. Thus, it is likely that Vinculin is able to perform different functions depending on its developmental context. Interestingly, different mechanisms for Vinculin binding to Talin in integrin-mediated adhesion have recently been uncovered in different morphogenetic processes, meaning that Talin can sense different force vectors (Klapholz, 2015). Given that a role for Talin and integrin-mediated adhesion during dorsal closure has been uncovered, it would be interesting to investigate whether Vinculin is also involved in integrin-mediated adhesion at this stage. The results suggest that a tension-dependent module involving Vinculin is present in amnioserosa cells. An exciting avenue will be to identify the mechanisms and function of such module in the context of morphogenesis (Jurado, 2016).

Alternative mechanisms for Talin to mediate integrin function

Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors (see Myospheroid) to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. This study shows that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. These alternatives were confirmed by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, it was shown that vinculin and actomyosin activity help change talin's orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures (Klapholz, 2015).

This study has presented key findings that change the view of talin function: (1) talin is needed for every integrin adhesion event in fly development, each with variable dependence on individual talin interaction sites; (2) the IBS2 (integrin-binding site 2) of talin is separated from integrins in muscle but not in wing, and this partly requires myosin activity and vinculin; and (3) even though the absence of vinculin is tolerated, vinculin is required for certain mutant talins to retain their residual function (Klapholz, 2015).

Vinculin's maintenance through evolution in Drosophila was at odds with the lack of a mutant phenotype (Alatortsev, 1997), especially as vinculin mutants are lethal in other organisms. However, vinculin mutants have recently been observed to cause mild muscle detachment in late-stage fly larvae (Bharadwaj, 2013), and this study shows that vinculin is required for the partial activity of talin mutants. Thus, vinculin supports normal functions of talin by adding additional actin/membrane-binding sites. Activated vinculin increases focal adhesion size, slows talin turnover, and maintains stretched talin in an unfolded conformation, and so vinculin may also increase the stability of mutant talins at adhesion sites. The ability of vinculin to aid mutant talin function is somewhat paradoxical if stretch between head and ABD is required to expose VBSs: how therefore do talins that lack the C-terminal ABD recruit vinculin? Possible explanations include: (1) some VBSs are exposed in unstretched talin; (2) other interactions stretch and expose VBSs; (3) truncation exposes VBSs; and (4) activation of vinculin drives binding to truncated talins, because artificially activated vinculin can recruit talin (Klapholz, 2015).

The finding that the C terminus of vinculin was in close enough proximity to talin to show FRET was surprising, because the talin-binding domain of vinculin is at its N terminus and therefore the actin-binding C terminus would be expected to extend away from talin. In all other ongoing experiments, fluorescence lifetime imaging (FLIM) is achieved only if the tag is adjacent to the interaction site. The close proximity therefore suggests that vinculin becomes aligned with talin. In muscle and wing, this alignment would be in the same direction, with vinculin binding a VBS N-terminal to IBS2, resulting in vinculin's C terminus in close proximity to the mCherry inserted C-terminal to IBS2. This is consistent with actin-mediated forces pulling the C-terminal ABDs of talin and vinculin away from integrins and talin head, respectively. The FRET indicates that some vinculin is pulled in the opposite direction in wings but not muscles, bringing vinculin's C terminus near talin's N terminus. This difference fits talin's parallel orientation in the wing, where the cortical actin meshwork could pull vinculin in a variety of directions. It is also possible that talin's head and vinculin's C terminus are brought into proximity by membrane binding (Klapholz, 2015).

The results provide additional support for binding of IBS2 to integrins, consistent with results showing that mutating IBS2 and the IBS2-binding site on the βPS integrin subunit cytoplasmic domain have similar phenotypes. Continued interaction between IBS2 and integrins is context dependent, with lack of IBS2 proximity to integrins at muscle attachment sites (MASs), as in focal adhesions, and retention of proximity in the wing. The finding that IBS2 was not required in the embryo for the residual function of talin lacking ABD, or talin/PINCH maintenance in this mutant, seems inconsistent with the defects caused by an IBS2 site-directed mutation, including muscle detachment and separation of talin and PINCH from integrins. Furthermore, it is necessary to to explain how IBS2 can be required for talin to remain bound to integrins but not remain in close proximity. One explanation is to hypothesize that IBS2-integrin binding strengthens the interaction of talin's head with another integrin or the plasma membrane, so that it can resist the pulling forces on ABD and vinculin that separate IBS2 away from integrins. When IBS2 is mutated the interaction between talin head and integrins/membrane is weakened, such that the full-length protein is pulled off, but a protein lacking ABD remains attached sufficiently to provide some function. This suggests that IBS2 should be in close proximity to integrins during early stages of adhesion formation in muscles, but no FRET was detected. It could therefore be a transient interaction or IBS2 may bind another protein in muscles (Klapholz, 2015).

Three distinct models for the mechanisms adopted by talin to mediate integrin adhesion are proposed, and these explain all the findings. (1) In muscle, talin dimers bind to integrins or membrane with their heads and to actin directly with the C-terminal ABD and indirectly with vinculin. Actomyosin activity and vinculin likely exert force on the rod of talin, each separating a fraction of the IBS2s from integrins. (2) In the wing, talin is oriented parallel to the membrane, with each talin dimer binding four integrins using all IBSs. Alternatively, talin heads are bound to the membrane or cortical actin, and the IBS2s are bound to two integrins. Actin is bound directly with the C-terminal ABD and indirectly with vinculin. (3) During germband retraction (GBR), it is suggested that talin dimers are bound to cortical actin or membrane directly with the head and indirectly with vinculin. Because IBS2 is critical for GBR, it is further suggested that talin dimers bind to integrins with IBS2s and to actin with the C-terminal ABD. These models have opted for the simplest explanation where IBS2 binds directly to integrins, but intermediate adaptor proteins have not been ruled out (Klapholz, 2015).

In the wing, the proximity between IBS2 and integrins could result from insufficient actomyosin activity perpendicular to the membrane, but such a 'passive' mechanism could not explain why IBS2 was critical in some tissues. The requirement for both talin head and IBS2 in the wing and during GBR suggests new parallel orientations of talin that could sense stretching forces within the adhesion plane, similar to EPLIN at cell-cell adhesions. In the wing, stretch would occur between integrins, and between integrin and membrane or actin in GBR. It is also possible that talin senses stretch between the membrane and cortical actin, as organisms lacking integrins have talin. The different orientations will also impact on integrin density and integrin:talin stoichiometry. In the wing, the distance between integrins can be fixed by talin, whereas in the muscle, integrin density would vary, depending on the flexibility of the talin dimer. It will be of interest to find whether parallel orientation of talin is found in epithelia of other organisms (Klapholz, 2015).

Finally, the results emphasize that when mutant versions of a protein are found to work better in some cell types than others, this may be indicating different mechanisms of action, a possibility that could resolve apparently contradictory findings (Klapholz, 2015).

Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart
The human heart is capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. However, identification of compensatory remodeling events in the aging heart remains elusive. This study presents the cardiac proteomes of young and old rhesus monkeys and rats, from which it was shown that certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. Cardiac-restricted vinculin overexpression reinforced the cortical cytoskeleton and enhanced myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosin-deficient fly hearts. Moreover, cardiac-specific vinculin overexpression increased median life span by more than 150% in flies. A broad array of potential therapeutic targets and regulators of age-associated modifications, specifically for vinculin, are presented. These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity, which may be assisted by therapeutic intervention to ameliorate the decline of function in aging patient hearts (Kaushik, 2015).

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. This study demonstrates that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. This study investigated the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function (Bharadwaj, 2013).

Interactions between cells and the extracellular matrix (ECM) are crucial for many biological processes. These include cell migration, directed process outgrowth, basement membrane-mediated support of tissues and maintenance of cell shape. Communication between cells and ECM proteins often occurs through the action of α/β-integrin heterodimers, a receptor complex that forms adhesive contacts, including focal adhesions, hemiadherens junctions, costameres and myotendinous junctions. In response to extracellular forces, focal adhesions undergo structural changes and initiate signaling events that allow adaptation to tensile stress. Vinculin is thought to be the primary force sensor in the integrin complex, mediating homeostatic adaptation to external forces (Bharadwaj, 2013 and references therein).

Vinculin-binding partners include proteins belonging to the CAP (Cbl-associated protein) protein family. However, the physiological significance of this association is unknown. Mammalian CAP proteins are components of focal adhesions in cell culture. In myocytes, CAP localizes to integrin-containing complexes called costameres that anchor sarcomeres to muscle cell membranes. There are three mammalian CAP protein family members: CAP, Vinexin and ArgBP2. CAP associates in vitro with many proteins, including the cytoskeletal regulators Paxillin, Afadin and Filamin, vesicle trafficking regulators such as Dynamin and Cbl, and the lipid raft protein Flotillin. In vitro studies demonstrate that CAP regulates the reassembly of focal adhesions following nocodazole dissolution. However, despite extensive studies on CAP, little is known about its functions in vivo. Cap (Sorbs1) mutant mice are defective in fat metabolism, and targeted deletion of the vinexin gene results in wound-healing defects. Drosophila CAP binds to axin and is implicated in glucose metabolism. Analysis of CAP function in mammals is complicated by potential functional redundancy of the three related CAP proteins. Therefore, the function of Drosophila CAP, the single CAP family member in Drosophila, was examined in vivo (Bharadwaj, 2013).

The Drosophila muscle attachment site (MAS) is an excellent system for studying integrin signaling. Somatic muscles in each segment of the fly embryo and larva are connected to the body wall through integrin-mediated hemiadherens junctions. Somatic muscles in flies lacking integrins lose their connection to the body wall. Surprisingly, flies lacking Vinculin, a major component of cytosolic integrin signaling complexes, are viable and show no muscle defects (Alatortsev, 1997). Thus, unlike its mammalian counterpart, Drosophila Vinculin is apparently dispensable for the initial assembly of integrin-mediated adhesion complexes at somatic MASs (Bharadwaj, 2013).

The fly MAS is structurally analogous to the fly chordotonal organ. These organs transduce sensations from various stimuli, including vibration, sound, gravity, airflow and body wall movements. The chordotonal organ is composed of individual subunits called scolopidia, each containing six cell types: neuron, scolopale, cap, ligament, cap attachment and ligament attachment cells. Chordotonal neurons are monodendritic, and their dendrites are located in the scolopale space, a lymph-filled extracellular space completely enveloped by the scolopale cell. Within the scolopale cell, a cage composed of actin bars, called scolopale rods, facilitates scolopale cell envelopment of the scolopale space. Thus, like the MAS, the actin cytoskeleton plays a specialized role in defining chordotonal organ morphology. Similarities between MASs and chordotonal organs include the requirement during development in both tendon and cap cells for the transcription factor Stripe. Furthermore, both of these cell types maintain structural integrity under force and so are likely to share common molecular components dedicated to this function (Bharadwaj, 2013).

This study shows that the Drosophila CAP protein is selectively localized to both muscle attachment sites and chordotonal organs. In Drosophila CAP mutants morphological defects are observed that are indicative of actin disorganization in both larval MASs and the scolopale cells of Johnston's organ in the adult. The morphological defects in scolopale cells result in vibration sensation defects in larvae and hearing deficits in adults. It was also found that, like its mammalian homologues, Drosophila CAP interacts with Vinculin both in vitro and in vivo. These results reveal novel CAP functions required for actin-mediated organization of cellular morphology, lending insight into how CAP mediates muscle and sensory organ development and function (Bharadwaj, 2013).

Integrin-based adhesion complexes are crucial for cell attachment to the extracellular matrix. These complexes change their composition and architecture in response to extracellular forces, initiating downstream signaling events that regulate cytoskeletal organization. This study has investigated the role played by the CAP protein in two stretch-sensitive structures in Drosophila: the MAS and the chordotonal organ. CAP mutants exhibit aberrant junctional invaginations at the MAS and collapse of scolopale cells in chordotonal organs. This study highlights a crucial integrin signaling function during development: the maintenance of membrane morphology in stretch-sensitive structures (Bharadwaj, 2013).

The morphological defects observed in CAP mutants could result from an excessive integrin signaling, or possibly accumulation of additional membranous components related to integrin signaling, in CAP mutants, owing to defects in endocytosis at the MAS. This is consistent with known interactions between CAP family members and vesicle trafficking regulators, including Dynamin and Synaptojanin, which are required for internalization of transmembrane proteins. Alternatively, CAP may be required for proper organization of the actin cytoskeleton at MASs, and the aberrant membrane invaginations that were observe are a secondary consequence of these cytoskeletal defects. This idea garners support from known interactions between CAP and various actin-binding proteins, including Vinculin, Paxillin, Actinin, Filamin and WAVE2. A third possibility is that CAP and Vinculin are regulators of membrane stiffness at the MAS, and aberrant junctional infoldings observed in CAP and vinculin mutants derive from diminished membrane rigidity in the presence of persistent myofilament contractile forces. Biophysical studies demonstrate that Vinculin-deficient mammalian cells in vitro show reduced membrane stiffness. Interestingly, the CAP protein ArgBP2 interacts with Spectrin, a protein important for cell membrane rigidity maintenance. These models for CAP function at MASs, however, are not mutually exclusive. Interestingly, disruption of the ECM protein Tiggrin leads to MAS phenotypes similar to CAP. Future studies on CAP interaction with Tiggrin and other CAP-interacting proteins will shed light on mechanisms underlying CAP function. Nevertheless, this study demonstrates in vivo the importance of CAP in stretch-sensitive organ morphogenesis, and it will be interesting to determine whether this function is phylogenetically conserved (Bharadwaj, 2013).

Apart from the MAS, CAP is also expressed at high levels in chordotonal organ scolopale cells, and this study has found that CAP mutants are defective in vibration sensation, a hallmark of chordotonal organ dysfunction. However, only the initial fast hunching response to vibration is disrupted in CAP mutant larvae. This may result from a partial loss of chordotonal function in these organs in the absence of CAP. A functional defect was also observed in the adult Johnston's organ; CAP mutant flies show diminished sound-evoked potentials. Importantly, the scolopale cells in CAP mutants appear partially collapsed. The extracellular space within the scolopale cell is lined by an actin cage, and CAP may influence the proper assembly of this actin cage or its association with the scolopale cell membrane. Ch organs are mechanosensory detectors and are constantly exposed to tensile forces. Thus, CAP apparently influences cytoskeletal integrity in two actin-rich structures: the MAS and the chordotonal organ, both of which are involved in force transduction (Bharadwaj, 2013).

Mammalian and Drosophila CAP bind to Vinculin (Kioka, 1999; Kioka, 2002; Zhang, 2006). Vinculin is required for the recruitment of the mammalian CAP protein vinexin to focal adhesions in NIH3T3 cells in vitro. Consistent with this observation, a dramatic decrease was seen in CAP levels at MASs in vinculin mutants, but residual levels of CAP protein remain. Furthermore, CAP localization at the muscle fiber Z-lines is completely unaltered in vinculin mutants. These observations indicate that Vinculin is not the sole upstream regulator of CAP localization. vinculin mutants show some of the phenotypic defects observed in CAP mutants; however, these defects are less pronounced. Therefore, the residual CAP pool that is recruited to MASs in a Vinculin-independent manner is apparently sufficient for partial CAP function. Assessment of CAP and Vinculin function at the larval MAS shows that these proteins are required for maintaining the integrity of junctional membranes in the face of tensile forces. CAP proteins may serve as scaffolding proteins at membrane-cytoskeleton interfaces and facilitate the assembly of protein complexes involved in cytoskeletal regulation and membrane turnover (Bharadwaj, 2013).

Mutations in the CAP-binding protein filamin cause myofibrillar myopathy. This, in combination with data showing a crucial role for CAP in regulation of muscle morphology, sets the stage for investigating how loss of CAP protein function might influence the etiology of myopathies (Bharadwaj, 2013).

Functions of Vinculin orthologs in other species

Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions

Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, this study found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function (Case, 2015).

Force-dependent conformational switch of alpha-catenin controls vinculin binding

Force sensing at cadherin-mediated adhesions is critical for their proper function. alpha-Catenin, which links cadherins to actomyosin, has a crucial role in this mechanosensing process. It has been hypothesized that force promotes vinculin binding, although this has never been demonstrated. X-ray structure further suggests that alpha-catenin adopts a stable auto-inhibitory conformation that makes the vinculin-binding site inaccessible. By stretching single alpha-catenin molecules using magnetic tweezers, this study shows that the subdomains MI vinculin-binding domain (VBD) to MIII unfold in three characteristic steps: a reversible step at ~5 picoNewtons (pNs) and two non-equilibrium steps at 10-15 pN. 5 pN unfolding forces trigger vinculin binding to the MI domain in a 1:1 ratio with nanomolar affinity, preventing MI domain refolding after force is released. The findings demonstrate that physiologically relevant forces reversibly unfurl alpha-catenin, activating vinculin binding, which then stabilizes alpha-catenin in its open conformation, transforming force into a sustainable biochemical signal (Yao, 2014).

Actomyosin-dependent formation of the mechanosensitive talin-vinculin complex reinforces actin anchoring

The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin-vinculin complex and the resulting activity of the complex are not known. This study developed a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, it was shown that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin-talin-vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton (Ciobanasu, 2014).

Vinculin regulates the recruitment and release of core focal adhesion proteins in a force-dependent manner

Cells sense the extracellular environment using adhesion receptors (integrins) linked to the intracellular actin cytoskeleton through a complex network of regulatory proteins that, all together, form focal adhesions (FAs). The molecular basis of how these sensing units are regulated, how they are implicated in transducing mechanical stimuli, and how this leads to a spatiotemporal coordination of FAs is unclear. This study shows that vinculin, through its links to the talin-integrin complex and F-actin, regulates the transmission of mechanical signals from the extracellular matrix to the actomyosin machinery. The vinculin interaction with the talin-integrin complex drives the recruitment and release of core FA components. The activation state of vinculin is itself regulated by force, as underscored by the observation that vinculin localization to FAs is dependent on actomyosin contraction. Using a variety of vinculin mutants, it was established which components of the cell-matrix adhesion network are coordinated through direct and indirect associations with vinculin. Moreover, using cyclic stretching, it was demonstrated that vinculin plays a key role in the transmission of extracellular mechanical stimuli leading to the reorganization of cell polarity. Of particular importance is the actin-binding tail region of vinculin, without which the cell's ability to repolarize in response to cyclic stretching is perturbed. Overall these data promote a model whereby vinculin controls the transmission of intracellular and extracellular mechanical cues that are important for the spatiotemporal assembly, disassembly, and reorganization of FAs to coordinate polarized cell motility (Carisey, 2013).

How vinculin regulates force transmission

Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, this study demonstrated that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (VH) and tail domains. VH enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; VH has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or VH, correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer (Dumbauld, 2013).


Search PubMed for articles about Drosophila Vinculin

Alatortsev, V. E., Kramerova, I. A., Frolov, M. V., Lavrov, S. A. and Westphal, E. D. (1997). Vinculin gene is non-essential in Drosophila melanogaster. FEBS Lett 413(2): 197-201. PubMed ID: 9280281

Bakolitsa, C., Cohen, D. M., Bankston, L. A., Bobkov, A. A., Cadwell, G. W., Jennings, L., Critchley, D. R., Craig, S. W. and Liddington, R. C. (2004). Structural basis for vinculin activation at sites of cell adhesion. Nature 430(6999): 583-586. PubMed ID: 15195105

Bardet, P. L., Guirao, B., Paoletti, C., Serman, F., Leopold, V., Bosveld, F., Goya, Y., Mirouse, V., Graner, F. and Bellaiche, Y. (2013). PTEN controls junction lengthening and stability during cell rearrangement in epithelial tissue. Dev Cell 25(5): 534-546. PubMed ID: 23707736

Bharadwaj, R., Roy, M., Ohyama, T., Sivan-Loukianova, E., Delannoy, M., Lloyd, T. E., Zlatic, M., Eberl, D. F. and Kolodkin, A. L. (2013). Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila. Development 140: 627-638. PubMed ID: 23293294

Bois, P. R., O'Hara, B. P., Nietlispach, D., Kirkpatrick, J. and Izard, T. (2006). The vinculin binding sites of talin and alpha-actinin are sufficient to activate vinculin. J Biol Chem 281(11): 7228-7236. PubMed ID: 16407299

Borgon, R. A., Vonrhein, C., Bricogne, G., Bois, P. R. and Izard, T. (2004). Crystal structure of human vinculin. Structure 12(7): 1189-1197. PubMed ID: 15242595

Carisey, A., Tsang, R., Greiner, A. M., Nijenhuis, N., Heath, N., Nazgiewicz, A., Kemkemer, R., Derby, B., Spatz, J. and Ballestrem, C. (2013). Vinculin regulates the recruitment and release of core focal adhesion proteins in a force-dependent manner. Curr Biol 23(4): 271-281. PubMed ID: 23375895

Case, L. B., Baird, M. A., Shtengel, G., Campbell, S. L., Hess, H. F., Davidson, M. W. and Waterman, C. M. (2015). Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions. Nat Cell Biol 17(7): 880-892. PubMed ID: 26053221

Ciobanasu, C., Faivre, B. and Le Clainche, C. (2014). Actomyosin-dependent formation of the mechanosensitive talin-vinculin complex reinforces actin anchoring. Nat Commun 5: 3095. PubMed ID: 24452080

del Rio, A., Perez-Jimenez, R., Liu, R., Roca-Cusachs, P., Fernandez, J. M. and Sheetz, M. P. (2009). Stretching single talin rod molecules activates vinculin binding. Science 323(5914): 638-641. PubMed ID: 19179532

Dumbauld, D. W., Lee, T. T., Singh, A., Scrimgeour, J., Gersbach, C. A., Zamir, E. A., Fu, J., Chen, C. S., Curtis, J. E., Craig, S. W. and Garcia, A. J. (2013). How vinculin regulates force transmission. Proc Natl Acad Sci U S A 110(24): 9788-9793. PubMed ID: 23716647

Hara, Y., Shagirov, M. and Toyama, Y. (2016). Cell boundary elongation by non-autonomous contractility in cell oscillation. Curr Biol 26: 2388-2396. PubMed ID: 27524484

Izard, T., Evans, G., Borgon, R. A., Rush, C. L., Bricogne, G. and Bois, P. R. (2004). Vinculin activation by talin through helical bundle conversion. Nature 427(6970): 171-175. PubMed ID: 14702644

Jurado, J., de Navascues, J. and Gorfinkiel, N. (2016). α-Catenin stabilises Cadherin-Catenin complexes and modulates actomyosin dynamics to allow pulsatile apical contraction. J Cell Sci 129: 4496-4508. PubMed ID: 27831494

Kaushik, G., et al. (2015). Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart. Sci Transl Med 7: 292ra299. PubMed ID: 26084806

Kioka, N., Sakata, S., Kawauchi, T., Amachi, T., Akiyama, S. K., Okazaki, K., Yaen, C., Yamada, K. M. and Aota, S. (1999). Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization. J Cell Biol 144: 59-69. PubMed ID: 9885244

Kioka, N., Ueda, K. and Amachi, T. (2002). Vinexin, CAP/ponsin, ArgBP2: a novel adaptor protein family regulating cytoskeletal organization and signal transduction. Cell Struct Funct 27: 1-7. PubMed ID: 11937713

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Papagrigoriou, E., Gingras, A. R., Barsukov, I. L., Bate, N., Fillingham, I. J., Patel, B., Frank, R., Ziegler, W. H., Roberts, G. C., Critchley, D. R. and Emsley, J. (2004). Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle. EMBO J 23(15): 2942-2951. PubMed ID: 15272303

Pasapera, A. M., Schneider, I. C., Rericha, E., Schlaepfer, D. D. and Waterman, C. M. (2010). Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation. J Cell Biol 188(6): 877-890. PubMed ID: 20308429

Riveline, D., Zamir, E., Balaban, N. Q., Schwarz, U. S., Ishizaki, T., Narumiya, S., Kam, Z., Geiger, B. and Bershadsky, A. D. (2001). Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J Cell Biol 153(6): 1175-1186. PubMed ID: 11402062

Tornavaca, O., Chia, M., Dufton, N., Almagro, L. O., Conway, D. E., Randi, A. M., Schwartz, M. A., Matter, K. and Balda, M. S. (2015). ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation. J Cell Biol 208(6): 821-838. PubMed ID: 25753039

Yao, M., Qiu, W., Liu, R., Efremov, A. K., Cong, P., Seddiki, R., Payre, M., Lim, C. T., Ladoux, B., Mege, R. M. and Yan, J. (2014). Force-dependent conformational switch of alpha-catenin controls vinculin binding. Nat Commun 5: 4525. PubMed ID: 25077739

Zhang, M., Liu, J., Cheng, A., Deyoung, S. M., Chen, X., Dold, L. H. and Saltiel, A. R. (2006). CAP interacts with cytoskeletal proteins and regulates adhesion-mediated ERK activation and motility. EMBO J 25: 5284-5293. PubMed ID: 17082770

Biological Overview

date revised: 26 March 2017

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