Motor protein Myo1c participates in Nephrin and Neph1 signaling

 描述（由申请人提供）：我们鉴定了运动蛋白Myo 1c作为狭缝隔膜的一种新组分，其与Nephrin和Neph 1相互作用并调节其运动。为了研究Myo 1c的体内功能，我们产生了Myo 1c floxed小鼠，用于构建纯合子null和足细胞特异性Myo 1c null小鼠。虽然Myo 1c纯合子缺失小鼠在出生前死亡，但12周龄小鼠的Myo 1c完全缺失增加了其对阿霉素诱导的蛋白尿的敏感性（在C57 BL/6 J背景下），这与我们的斑马鱼研究一致。有趣的是，足细胞特异性Myo 1c敲除小鼠的分析显示，当年龄达到8个月时，没有蛋白尿或功能异常。然而，当在阿霉素敏感的背景下繁殖时，这些小鼠对阿霉素诱导的肾小球病具有抗性;与对照小鼠相比，它们没有发生蛋白尿，这表明Myo 1c具有多种功能。尽管令人惊讶，但这些结果与足细胞中参与信号传导和运输的蛋白质（如Rac 1和Crk）的丢失一致。足细胞对损伤的反应通常通过增加Nephrin和Neph 1的磷酸化来评估，所述磷酸化启动它们的再分布和导致足细胞消失的细胞内信号级联的组装。这些事件的损害可能会减弱足细胞对损伤的反应能力，从而诱导保护。事实上，我们最近的研究表明，抑制Neph 1信号保护足细胞免受损伤。由于Myo 1c具有将其货物蛋白与膜和肌动蛋白相关联的拴系功能，因此我们假设Myo 1c参与调节这些蛋白在膜上的运动的机制，该机制对于指导由Nephrin和Neph 1组装信号复合物并启动细胞内信号传导和贩运事件至关重要。事实上，Myo 1c结合的损失减弱了Neph 1在膜上的动态运动，如使用活FRAP分析所示。此外，我们用Myo 1c特异性抑制剂pentachloropropyldilin（PCIP）处理培养的足细胞，该抑制剂阻止了膜和细胞内囊泡的运动，这表明Myo 1c参与了肌动蛋白依赖的细胞事件，这些事件对于产生细胞对损伤的反应至关重要。这进一步表明这些细胞将具有受损的损伤反应。事实上，这些细胞抵抗硫酸鱼精蛋白（PS）的损伤，通过肌动蛋白细胞骨架重组。在具体目标1中，我们将研究Myo 1c由于其膜和肌动蛋白结合功能而参与足细胞产生适当损伤反应的假设。这涉及调节损伤诱导的Nephrin和Neph 1蛋白向细胞内区室的再分布以及驱动其细胞内信号传导和运输的信号传导复合物的组装。在第二个目标中，我们将研究Myo 1c在小鼠发育的各个阶段的缺失如何影响肾小球功能。此外，我们将通过确定足细胞特异性Myo 1c缺失是否减弱各种急性和慢性肾小球损伤模型中的疾病表型来研究Myo 1c是否是治疗靶点。

 DESCRIPTION (provided by applicant): We identified motor protein Myo1c as a novel component of slit diaphragm that interacts with Nephrin and Neph1 and regulates their movement. To study the in vivo function of Myo1c, we generated Myo1c floxed mice that were used to construct homozygous null and podocyte-specific Myo1c null mice. While Myo1c homozygous null mice were reported to die prenatally, complete Myo1c deletion in 12 week old mice increased their sensitivity to adriamycin-induced proteinuria (on a C57BL/6J background), which is in agreement with our zebrafish studies. Interestingly, the analysis of podocyte specific Myo1c knockout mouse showed no proteinuria or functional abnormality when aged to 8 months. However, when bred to an adriamycin-sensitive background, these mice were resistant to adriamycin-induced glomerulopathy; they did not develop proteinuria as compared to the control mice suggesting multiple functions for Myo1c. Although surprising, these results are consistent with loss of proteins in podocytes that are involved in signaling and trafficking such as Rac1 and Crk. Podocytes response to injury is commonly assessed through increased phosphorylation of Nephrin and Neph1 that initiates their redistribution and the assembly of an intracellular signaling cascade leading to podocyte effacement. Impairment of these events may attenuate podocytes ability to respond to injury thus inducing protection. Indeed, our recent study demonstrated that inhibiting Neph1 signaling protected podocytes from injury. Since Myo1c has a tethering function that associates its cargo proteins with membranes and actin, we hypothesized that Myo1c participates in a mechanism that regulates movement of these proteins at the membrane that is critical for directing the assembly of a signaling complex by Nephrin and Neph1 and initiating intracellular signaling and trafficking events. Indeed, loss of Myo1c binding attenuated the dynamic movement of Neph1 at the membrane as demonstrated using live FRAP analysis. Furthermore, we treated cultured podocytes with a Myo1c specific inhibitor pentachloropseudilin (PCIP) that arrested membrane and intracellular vesicles movements suggesting the involvement of Myo1c in actin dependent cellular events that are critical for generating cellular response to injury. This further suggested that these cells will hve impaired injury response. Indeed, these cells resisted injury by protamine sulphate (PS) as measured by actin cytoskeleton reorganization. In the Specific Aim 1, we will investigate the hypothesis that Myo1c due to its membrane and actin binding functions, participates in generating an appropriate injury response by podocytes. This involves regulating injury-induced redistribution of Nephrin and Neph1 proteins to intracellular compartments and the assembly of signaling complexes that drives their intracellular signaling and trafficking. In the second Aim, we will investigate how Myo1c depletion at various stages of mouse development affects glomerular function. In addition, we will investigate if Myo1c is a therapeutic target by determining whether podocyte-specific deletion of Myo1c attenuates the disease phenotype in various acute and chronic glomerular injury models.