Motor protein Myo1c participates in Nephrin and Neph1 signaling

 描述(由申请人提供)：我们鉴定了肌动蛋白Myo1c是缝隙隔膜的一种新成分，它与Nephin和Neph1相互作用，并调节它们的运动。为了研究Myo1c在体内的功能，我们产生了Myo1c小鼠，用于构建纯合子缺失和足细胞特异性Myo1c缺失小鼠。虽然Myo1c纯合子缺失小鼠被报道会在产前死亡，但12周龄小鼠的Myo1c完全缺失增加了它们对阿霉素诱导的蛋白尿的敏感性(在C57BL/6J背景下)，这与我们的斑马鱼研究一致。有趣的是，对足细胞特异性Myo1c基因敲除小鼠的分析显示，当小鼠出生到8个月时，没有出现蛋白尿或功能异常。然而，当饲养在阿霉素敏感的背景下时，这些小鼠对阿霉素诱导的肾小球病变具有抵抗力；与对照组小鼠相比，它们没有出现蛋白尿，这表明Myo1c具有多种功能。尽管这些结果令人惊讶，但这些结果与足细胞中参与信号和运输的蛋白质的丢失是一致的，如rac1和Crk。足细胞对损伤的反应通常是通过增加NePhin和Neph1的磷酸化来评估的，这会启动它们的重新分布，并组装细胞内的信号级联，导致足细胞的消失。这些事件的损伤可能会削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Neph1信号转导可以保护足细胞免受损伤。由于Myo1c具有将其货物蛋白与膜和肌动蛋白联系在一起的拴系功能，我们假设Myo1c参与了一种机制，该机制调节这些蛋白在膜上的运动，这对于指导NePhin和Neph1组装信号复合体以及启动细胞内信号和运输事件至关重要。事实上，Myo1c结合的丧失减弱了Neph1在膜上的动态运动，这一点通过实时FRAP分析得到了证明。此外，我们用Myo1c特异性抑制剂五氯伪林(PCIP)处理培养的足细胞，这种抑制剂可以阻止膜和细胞内小泡的运动，这表明Myo1c参与了肌动蛋白依赖的细胞事件，这是产生细胞损伤反应的关键。这进一步表明，这些细胞将损害损伤反应。事实上，根据肌动蛋白细胞骨架重组的测量，这些细胞抵抗了鱼精蛋白硫酸盐(PS)的损伤。在特定的目标1中，我们将研究这样的假设，即Myo1c由于其膜和肌动蛋白结合功能，参与通过足细胞产生适当的损伤反应。这涉及到调控损伤诱导的NePhin和Neph1蛋白到细胞内隔室的重新分配，以及驱动它们在细胞内的信号和运输的信号复合体的组装。在第二个目标中，我们将研究小鼠发育不同阶段的Myo1c缺失对肾小球功能的影响。此外，我们将通过确定足细胞特异性Myo1c缺失是否减轻各种急性和慢性肾小球损伤模型中的疾病表型，来研究Myo1c是否是一个治疗靶点。

 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.