Structural Dynamics of α-Actinin-Vinculin Interactions

115

Mutations in the ␣-actinin-4 gene ACTN4 cause an autosomal dominant human kidney disease. Mice deficient in ␣-actinin-4 develop a recessive phenotype characterized by kidney failure, proteinuria, glomerulosclerosis, and retraction of glomerular podocyte foot processes. However, the mechanism by which ␣-actinin-4 deficiency leads to glomerular disease has not been defined. Here, we examined the effect of ␣-actinin-4 deficiency on the adhesive properties of podocytes in vivo and in a cell culture system. In ␣-actinin-4-deficient mice, we observed a decrease in the number of podocytes per glomerulus compared with wild-type mice as well as the presence of podocyte markers in the urine. Podocyte cell lines generated from ␣-actinin-4-deficient mice were less adherent than wild-type cells to glomerular basement membrane (GBM) components collagen IV and laminin 10 and 11. We also observed markedly reduced adhesion of ␣-actinin-4-deficient podocytes under increasing shear stresses. This adhesion deficit was restored by transfecting cells with ␣-actinin-4-GFP. We tested the strength of the integrin receptor-mediated linkages to the cytoskeleton by applying force to microbeads bound to integrin using magnetic pulling cytometry. Beads bound to ␣-actinin-4-deficient podocytes showed greater displacement in response to an applied force than those bound to wild-type cells. Consistent with integrin-dependent ␣-actinin-4-mediated adhesion, phosphorylation of ␤1-integrins on ␣-actinin-4-deficient podocytes is reduced. We rescued the phosphorylation deficit by transfecting ␣-actinin-4 into ␣-actinin-4-deficient podocytes. These results suggest that ␣-actinin-4 interacts with integrins and strengthens the podocyte-GBM interaction thereby stabilizing glomerular architecture and preventing disease.

Section: Figure 2 Podocyte Number Per Glomerulus Is Decreased In Actn4mentioning

confidence: 49%

α-Actinin-4 Is Required for Normal Podocyte Adhesion

Dandapani

Sugimoto

Matthews

et al. 2007

115

“…The VBSs of the vinculin binding partners bind to the N-terminal seven-helix bundle domain of vinculin (Vh1) (16,23,24). Based on preliminary binding studies, 4 we suspected that the C-terminal sca4 domain (residues 411-1008) had two binding sites for Vh1.…”

Section: Identification Of Vbss In the Cellmentioning

confidence: 99%

“…Building on our findings in Shigella (16 - bundle head (Vh1) 3 domain with its five-helix bundle tail (Vt) domain (19 -22). Vinculin activation requires severing the head-tail interaction, and studies initially from our laboratory (23)(24)(25) and then by others (26 -30) have established that talin is a physiologic activator of vinculin, where it binds to the vinculin Vh1 domain via amphipathic ␣-helical vinculin binding sites (VBSs) present in its central talin rod domain. Notably, the VBSs of talin are normally buried within helix bundle domains (26,29,31), and talin needs to be force-activated via integrin receptors to release these VBSs so they can bind to and activate vinculin (26,30,32).…”

mentioning

confidence: 99%

The Rickettsia Surface Cell Antigen 4 Applies Mimicry to Bind to and Activate Vinculin

Park

Lee

Gouin

et al. 2011

Self Cite

Background:Rickettsiae infect the host cell by co-opting the actin cytoskeleton. Results: The Rickettsiae invasin sca4 harbors two ␣-helices that bind and activate vinculin; the binding mode of one of these ␣-helices is unique. Conclusion: Rickettsiae use mimicry of talin to subvert vinculin functions. Significance: The unique nature of the sca4-vinculin interaction suggests it can be targeted by small molecules.

“…Structural studies have revealed that binding of VBS peptides to Vhd1 (residues 1-258) induces a large conformational change that disrupts a major binding site for Vt (18). Moreover, VBS3 peptide has been reported to bind with similar affinity to either Vhd1 or full-length vinculin (residues 1-1066) when these proteins are adsorbed onto a solid-phase substrate (22). In addition, VBS3 peptide alters the protease sensitivity of full-length vinculin in solution (22).…”

mentioning

confidence: 99%

A Conformational Switch in Vinculin Drives Formation and Dynamics of a Talin-Vinculin Complex at Focal Adhesions

Cohen

Kutscher

Chen³

et al. 2006

153

188

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrincytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or ␣-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to K d ϳ 10 ؊7 is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.Integrins mediate transmembrane connections between the actin cytoskeleton and extracellular matrix (1, 2). These connections are organized into discrete clusters such as focal complexes (3) and focal adhesions (3, 4) in adherent cells. Focal adhesions serve dual, opposing functions in cell motility, acting both in transmission of traction forces to generate displacement of the cell body and as anchors that resist detachment from the substratum (5). Thus, dynamic regulation of focal adhesion structure, and the integrin-cytoskeleton associations contained therein, plays a central role in balancing adhesive and migratory stimuli in the cell.Two key proteins implicated in the physical connections between integrins and F-actin are the actin-binding proteins talin and vinculin. In vitro, talin binds to ␤ integrin tails through its FERM domain (6) and induces conformational changes in integrin associated with increased binding to the extracellular matrix (7). In cells, exposure of activated epitopes on ␤ 1 and ␤ 3 integrins and fibronectin binding are strongly inhibited by knockdown of talin expression (8). Moreover, talin-1 null cell lines are deficient in the formation of mechanical linkages between fibronectin, ␤ 3 integrin, and the cytoskeleton as shown by loss of a transient molecular slip bond that can sustain up to 2 piconewtons of force (9).Interestingly, the seminal report that talin binds directly to ␣ 5 ␤ 1 integrin also demonstrated that integrin, talin, and vincu...