Departments of 1Physiology and Biophysics, MetroHealth System Campus, Case Western Reserve University, Cleveland, Ohio, USA.
Am J Physiol Renal Physiol. 2012 Jan 1;302(1):F103-15. doi: 10.1152/ajprenal.00419.2011. Epub 2011 Sep 7.
Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. Wt1-interacting protein (Wtip), an Ajuba family LIM domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in adherent but isolated podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Noncontacted shWtip podocytes did not assemble actin stress fibers, and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and β-catenin clustered in irregularly distributed spots that failed to laterally expand. Cell surface biotinylation showed diminished plasma membrane cadherin, β-catenin, and α-catenin in shWtip podocytes, although protein expression was similar in shWtip and control cells. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined whether Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor C3 toxin and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix contacts requires Wtip, a process that may be mediated by spatiotemporal regulation of RhoA activity through appropriate targeting of Arhgef12.
足细胞通过重塑其裂孔隔膜和细胞基质黏附连接来响应环境信号。WT1 相互作用蛋白（Wtip）是一种在足细胞中表达的 Ajuba 家族 LIM 结构域支架蛋白，它协调细胞黏附变化和转录反应，以调节足细胞表型可塑性。我们使用增益和缺失功能方法评估了 Wtip 对足细胞细胞-细胞和细胞-基质接触组织的影响。内源性 Wtip 靶向附着但分离的足细胞中的粘着斑，然后在细胞建立稳定的同质接触后转移到黏着连接。用 Wtip 敲低（shWtip）的足细胞可以附着，但不能正常扩散。未接触的 shWtip 足细胞不会组装肌动蛋白应力纤维，其粘着斑也不会成熟。随着 shWtip 足细胞建立细胞-细胞接触，稳定的黏着连接无法形成，F-肌动蛋白结构紊乱。在 shWtip 细胞中，钙粘蛋白和 β-连环蛋白聚集在不规则分布的斑点中，无法侧向扩展。细胞表面生物素化显示 shWtip 足细胞的质膜钙粘蛋白、β-连环蛋白和α-连环蛋白减少，尽管 shWtip 和对照细胞中的蛋白表达相似。由于正常的肌动蛋白动力学是黏着连接和粘着斑组织所必需的，我们确定了 Wtip 是否调节 F-肌动蛋白组装。未分化的足细胞不会形成 F-肌动蛋白应力纤维，但当被诱导过度表达 WTIP 时，会形成丰富的应力纤维，这一过程被 RhoA 抑制剂 C3 毒素和 RhoA 激酶抑制剂阻断。WTIP 直接与 Rho 鸟嘌呤核苷酸交换因子（GEF）12（Arhgef12）相互作用，Arhgef12 是一种富含肾小球的 RhoA 特异性 GEF。总之，足细胞黏着连接和细胞-基质接触的稳定组装需要 Wtip，这一过程可能通过适当靶向 RhoA 活性的时空调节来介导，从而通过适当靶向 RhoA 活性的时空调节来介导。
