Michaud Jean-Louis R, Hosseini-Abardeh Mona, Farah Kevin, Kennedy Chris R J
Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ontario, Canada.
Cell Motil Cytoskeleton. 2009 Mar;66(3):166-78. doi: 10.1002/cm.20339.
Podocytes are epithelial cells that line the outer aspect of renal blood vessels and provide a platform for the kidney's filtering apparatus, the slit diaphragm. Mutations in alpha-actinin-4, an actin bundling protein highly expressed in podocytes, result in increased affinity for actin and cause a familial form of focal segmental glomerulosclerosis. We hypothesized that such gain-of-affinity mutations would override alpha-actinin-4's sensitivity to regulatory factors such as calcium (acting via two EF-hand motifs), and phosphoinositides. We generated calcium- (mutEF) and phosphoinositide- (mutPI) insensitive variants of alpha-actinin-4, comparing their properties to a disease-associated mutant (K256E) and to the wildtype (wt) protein. alpha-Actinin-4(mutPI) displayed increased affinity for actin, while the affinity of alpha-actinin-4(mutEF) was unchanged. Addition of calcium to actin sedimentation assays caused a decrease in the association of alpha-actinin-4(wt) with filamentous actin, while phosphoinositides generally increased this association. Similar to alpha-actinin-4(K256E), alpha-actinin-4(mutPI) was mislocalized in cultured podocytes, being preferentially associated with filamentous actin and focal adhesions. Fluorescence recovery after photobleaching experiments revealed a rapid turnover of alpha-actinin-4(wt) and alpha-actinin-4(mutEF) along stress fibers and focal adhesions, while the turnover of alpha-actinin-4(K256E) and alpha-actinin-4(mutPI) was dramatically reduced at these subcellular locales. Equibiaxial mechanical stimulation of podocytes, a mimic of intraglomerular forces, reduced podocyte surface area by 50%; this decrease was more severe (70%) in the presence of high-affinity mutants of alpha-actinin-4. These data suggest that dynamic regulation of alpha-actinin-4/actin interactions may be necessary for maintaining podocyte structure in response to glomerular hydrostatic forces.
足细胞是位于肾血管外侧的上皮细胞，为肾脏的滤过装置——裂孔隔膜提供了一个平台。α-辅肌动蛋白-4是一种在足细胞中高度表达的肌动蛋白成束蛋白，其突变会导致对肌动蛋白的亲和力增加，并引发家族性局灶节段性肾小球硬化症。我们推测，这种亲和力增加的突变会使α-辅肌动蛋白-4对诸如钙（通过两个EF手基序起作用）和磷酸肌醇等调节因子的敏感性失效。我们生成了对钙（mutEF）和磷酸肌醇（mutPI）不敏感的α-辅肌动蛋白-4变体，并将它们的特性与一种疾病相关突变体（K256E）和野生型（wt）蛋白进行比较。α-辅肌动蛋白-4（mutPI）对肌动蛋白的亲和力增加，而α-辅肌动蛋白-4（mutEF）的亲和力未发生变化。在肌动蛋白沉降试验中添加钙会导致α-辅肌动蛋白-4（wt）与丝状肌动蛋白的结合减少，而磷酸肌醇通常会增加这种结合。与α-辅肌动蛋白-4（K256E）类似，α-辅肌动蛋白-4（mutPI）在培养的足细胞中定位错误，优先与丝状肌动蛋白和粘着斑相关联。光漂白后荧光恢复实验显示，α-辅肌动蛋白-4（wt）和α-辅肌动蛋白-4（mutEF）沿着应力纤维和粘着斑快速周转，而α-辅肌动蛋白-4（K256E）和α-辅肌动蛋白-4（mutPI）在这些亚细胞区域的周转显著减少。对足细胞进行双轴机械刺激（模拟肾小球内压力）会使足细胞表面积减少50%；在存在α-辅肌动蛋白-4高亲和力突变体的情况下，这种减少更为严重（70%）。这些数据表明，α-辅肌动蛋白-4/肌动蛋白相互作用的动态调节对于响应肾小球静水压维持足细胞结构可能是必要的。
