Department of Medicine, Renal Electrolyte and Hypertension Division, University of Pennsylvania, Philadelphia, PA 19104, USA.
Mol Cell Biol. 2011 May;31(10):2134-50. doi: 10.1128/MCB.05051-11. Epub 2011 Mar 14.
The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.
足细胞蛋白 Nephrin 和 Neph1 在足细胞细胞膜上形成信号复合物，为功能性肾小球滤过屏障提供结构框架。目前尚不清楚调节这些蛋白在膜内外运动的机制。本研究发现 Nephrin 与肌球蛋白 1c（Myo1c）之间存在一种新的相互作用，Myo1c 在将 Nephrin 靶向足细胞细胞膜中发挥积极作用。通过体内和体外实验，我们提供的数据支持 Nephrin 与肌球蛋白 1c 之间存在直接相互作用，该相互作用具有动态性和肌动蛋白依赖性。与野生型 Myo1c 不同，在表达显性失活 Myo1c 的足细胞中，Neph1 的膜定位显著减少。此外，在 Myo1c 耗尽的足细胞中，Neph1 未能定位在足细胞细胞膜和细胞连接处。我们进一步证明，类似于 Nephrin，Myo1c 还与 nephrin 结合，并减少其在足细胞细胞膜上的定位。Myo1c 敲低细胞的功能分析表明，细胞迁移缺陷，如划痕试验所示。此外，Myo1c 敲低细胞形成紧密连接的能力受损，如跨上皮电阻（TER）和牛血清白蛋白（BSA）通透性测定所示。这些结果确定了一种新的肌球蛋白 1c 依赖性分子机制，介导了 Nephrin 和 nephrin 在裂隙隔膜处的动态组织，这对于足细胞功能至关重要。
