05-386 Sigma-AldrichAnti-Vinculin Antibody, clone V284

Vimentin, type III intermediate filament, has stage-specific localization in the Sertoli cell. In the rat, during stages I-V and XI-XIV of the seminiferous epithelium, vimentin is localized in the perinuclear area with filaments projecting into the apical region toward the developing germ cells. These filaments decrease in length at stages VI-VII with perinuclear staining in stages VIII-IX, when spermiation occurs. Our earlier studies following 17β-estradiol treatment to adult male rats demonstrated an increase in germ cell apoptosis, spermiation failure and disruption of Sertoli cell microfilaments and microtubules. The present study was undertaken to determine the stage-specific distribution of vimentin and its involvement in spermiation failure and germ cell apoptosis. Immunofluorescence studies revealed that in contrast to the perinuclear localization with small extensions in control stages VII-IX, long extensions radiating apically to the spermatids in deep recess were observed in the treated group. Immunoprecipitation studies showed marked absence of phosphorylated vimentin in stages VII-VIII in the treated group. Further, localization of plectin, cytoskeletal linker protein, showed decrease in all the stages of spermatogenesis following estradiol treatment. Interestingly, for the first time the localization of plectin in the tubulobulbar complex was observed. In conclusion, the study suggests that estradiol treatment leads to an effect on vimentin phosphorylation, which could have inhibited the disassembly of vimentin leading to retention of apical projection in stages VII-VIII. These effects could be presumably due to a decrease in plectin, affecting the reorganization of vimentin and therefore the apical movement of spermatids, leading to spermiation failure.

Flightless I (Flii) is an actin-remodeling protein that influences diverse processes including cell migration and gene transcription and links signal transduction with cytoskeletal regulation. Here, we show that Flii modulation of focal adhesions and filamentous actin stress fibers is Rac1-dependent. Using primary skin fibroblasts from Flii overexpressing (Flii(Tg/Tg)), wild-type, and Flii deficient (Flii(+/-)) mice, we show that elevated expression of Flii increases stress fiber formation by impaired focal adhesion turnover and enhanced formation of fibrillar adhesions. Conversely, Flii knockdown increases the percentage of focal complex positive cells. We further show that a functional effect of Flii at both the cellular level and in in vivo mouse wounds is through inhibiting paxillin tyrosine phosphorylation and suppression of signaling proteins Src and p130Cas, both of which regulate adhesion signaling pathways. Flii is upregulated in response to wounding, and overexpression of Flii inhibits paxillin activity and reduces adhesion signaling by modulating the activity of the Rho family GTPases. Overexpression of constitutively active Rac1 GTPase restores the spreading ability of Flii(Tg/Tg) fibroblasts and may explain the reduced adhesion, migration, and proliferation observed in Flii(Tg/Tg) mice and their impaired wound healing, a process dependent on effective cellular motility and adhesion.

Engagement of α(5)β(1)-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (Ca(L)) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing Ca(L). Using coimmunoprecipitation strategies, we show that coassociation of Ca(L) with α(5)- or β(1)-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between Ca(L) and α(5)β(1)-integrin and between Ca(L) and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of Ca(L) heterologously expressed in HEK293-T cells, we identified two regions of Ca(L) required for these interactions: 1) COOH-terminal residues Ser(1901) and Tyr(2122), known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type Ca(L) colocalization with β(1)-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, Ca(L) associates with α(5)β(1)-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of Ca(L) at Y(2122) and/or S(1901) is required for association of Ca(L) with α(5)β(1)-integrin; and 3) c-Src, via binding to PRDs that reside in the II-III linker region and/or the COOH terminus of Ca(L), mediates current potentiation following α(5)β(1)-integrin engagement. These findings provide new evidence for how interactions between α(5)β(1)-integrin and FN can modulate Ca(L) entry and consequently alter the physiological function of multiple types of excitable cells.

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.

The influence of mechanical forces on cell function has been well documented for many different cell types. Endothelial cells native to the aortic valve may play an important role in mediating tissue responses to the complex fluid environment, and may therefore respond to fluid flow in a different manner than more characterized vascular endothelial cells.Porcine endothelial cells of aortic and aortic valvular origin were subjected to 20 dynes/cm2 steady laminar shear stress for up to 48 hours, with static cultures serving as controls. The aortic valve endothelial cells were observed to align perpendicular to flow, in direct contrast to the aortic endothelial cells, which aligned parallel to flow. Focal adhesion complexes reorganized prominently at the ends of the long axis of aligned cells. Valvular endothelial cell alignment was dependent on Rho-kinase signaling, whereas vascular endothelial cell alignment was dependent on both Rho-kinase and phosphatidylinositol 3-kinase signal pathways.These differences in response to mechanical forces suggest a unique phenotype of valvular endothelial cells not mimicked by vascular endothelial cells, and could have implications for cardiovascular cell biology and cell-source considerations for tissue-engineered valvular substitutes.

The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in both the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p less than 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion.