Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.
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We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect"). (+info)
BACKGROUND: Foot process effacement and condensation of the glomerular epithelial cell (GEC) cytoskeleton are manifestations of passive Heymann nephritis, a model of complement-mediated membranous nephropathy. METHODS: To study the effects of complement on the actin cytoskeleton in this model, we have used an in vitro system in which GECs are sublethally injured using a combination of complement-fixing anti-Fx1A IgG and human serum as a source of complement. We examined the effects of this injury on the organization of the cytoskeleton and focal contacts using immunohistology and immunochemistry. RESULTS: By immunofluorescence, sublethal complement-mediated injury was accompanied by a loss of actin stress fibers and focal contacts but retention of matrix-associated integrins. Full recovery was seen after 18 hours. Western blot analysis showed no change in the cellular content of the focal contact proteins. Inhibition of the calcium-dependent protease calpain did not prevent injury. In addition, cycloheximide during recovery did not inhibit the reassembly of stress fibers or focal contacts. Injury was associated with a reduction in tyrosine phosphorylation of paxillin and a currently unidentified 200 kDa protein, but inhibition of tyrosine phosphatase activity with sodium vanadate did not prevent injury. Cellular adenosine triphosphate content was significantly reduced in injured cells. CONCLUSION: These results document reversible, complement-dependent disruption of actin microfilaments and focal contacts leading to the dissociation of the cytoskeleton from matrix-attached integrins. This may explain the altered cell-matrix relationship accompanying podocyte effacement in membranous nephropathy. (+info)
Molecular diversity of cell-matrix adhesions.
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In this study we have examined for molecular heterogeneity of cell-matrix adhesions and the involvement of actomyosin contractility in the selective recruitment of different plaque proteins. For this purpose, we have developed a novel microscopic approach for molecular morphometry, based on automatic identification of matrix adhesions, followed by quantitative immunofluorescence and morphometric analysis. Particularly informative was fluorescence ratio imaging, comparing the local labeling intensities of different plaque molecules, including vinculin, paxillin, tensin and phosphotyrosine-containing proteins. Ratio imaging revealed considerable molecular heterogeneity between and within adhesion sites. Most striking were the differences between focal contacts, which are vinculin- and paxillin-rich and contain high levels of phosphotyrosine, and fibrillar adhesions, which are tensin-rich and contain little or no phosphotyrosine. Ratio imaging also revealed considerable variability in the molecular substructure of individual focal contacts, pointing to a non-uniform distribution of phosphotyrosine and the different plaque constituents. Studying the quantitative relationships between the various components of the submembrane plaque indicated that the levels of vinculin, paxillin and phosphotyrosine in adhesion sites are positively correlated with each other and negatively correlated with the levels of tensin. Tyrosine phosphorylation of focal contacts was highly sensitive to cellular contractility, and was diminished within 5 minutes after treatment with the kinase inhibitor H-7, an inhibitor of actomyosin contractility. This was followed by the loss of paxillin and vinculin from the focal adhesions. Tensin-rich fibrillar adhesions were relatively insensitive to H-7 treatment. These findings suggest a role for contractility in the generation of matrix adhesion diversity. (+info)
Calreticulin affects focal contact-dependent but not close contact-dependent cell-substratum adhesion.
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We used two cell lines expressing fast (RPEfast) and slow (RPEslow) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPEfast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPEfast cells. After RPEfast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPEfast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPEfast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPEslow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPEslow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPEfast cells use close contacts to "stick" to it. (+info)
Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.
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Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL. (+info)
Domain analysis of supervillin, an F-actin bundling plasma membrane protein with functional nuclear localization signals.
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A growing number of actin-associated membrane proteins have been implicated in motile processes, adhesive interactions, and signal transduction to the cell nucleus. We report here that supervillin, an F-actin binding protein originally isolated from bovine neutrophil plasma membranes, contains functional nuclear targeting signals and localizes at or near vinculin-containing focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-length supervillin in these cells disrupts the integrity of focal adhesion plaques and results in increased levels of F-actin and vinculin. Localization studies of chimeric proteins containing supervillin sequences fused with the enhanced green fluorescent protein indicate that: (1) the amino terminus promotes F-actin binding, targeting to focal adhesions, and limited nuclear localization; (2) the dominant nuclear targeting signal is in the center of the protein; and (3) the carboxy-terminal villin/gelsolin homology domain of supervillin does not, by itself, bind tightly to the actin cytoskeleton in vivo. Overexpression of chimeras containing both the amino-terminal F-actin binding site(s) and the dominant nuclear targeting signal results in the formation of large nuclear bundles containing F-actin, supervillin, and lamin. These results suggest that supervillin may contribute to cytoarchitecture in the nucleus, as well as at the plasma membrane. (+info)
Rho controls actin cytoskeletal assembly in renal epithelial cells during ATP depletion and recovery.
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Actin cytoskeletal disruption is a hallmark of ischemic injury and ATP depletion in a number of cell types, including renal epithelial cells. We manipulated Rho GTPase signaling by transfection and microinjection in LLC-PK proximal tubule epithelial cells and observed actin cytoskeletal organization following ATP depletion or recovery by confocal microscopy and quantitative image analysis. ATP depletion resulted in disruption of stress fibers, cortical F-actin, and apical actin bundles. Constitutively active RhoV14 prevented disruption of stress fibers and cortical F-actin during ATP depletion and enhanced the rate of stress fiber reassembly during recovery. Conversely, the Rho inhibitor C3 or dominant negative RhoN19 prevented recovery of F-actin assemblies upon repletion. Actin bundles in the apical microvilli and cytosolic F-actin were not affected by Rho signaling. Assembly of vinculin and paxillin into focal adhesions was disrupted by ATP depletion, and constitutively active RhoV14, although protecting stress fibers from disassembly, did not prevent dispersion of vinculin and paxillin, resulting in uncoupling of stress fiber and focal adhesion assembly. We propose that ATP depletion causes Rho inactivation during ischemia and that recovery of normal cellular architecture and function requires Rho. (+info)
Polyphosphoinositides inhibit the interaction of vinculin with actin filaments.
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Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin's head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2. (+info)