Mechanosensitive determinants of podocyte physiology

项目摘要 肾足细胞在相当大的生物力学压力下工作，这是其生理功能的一部分，以过滤 血浆。他们有一个专门的动态肌动蛋白细胞骨架结构，以加强脆弱的脚部过程 它们的功能所需的形态。使用体内蛋白质组筛选和NephroSeq数据挖掘， 我们已经确定星云是一种新的支架蛋白，它可能在组织和 维持足细胞的细胞骨架和粘附性。星云是一种肌动蛋白结合蛋白，表达 在心脏中高度存在；其星云蛋白重复区域的突变与心脏异常有关 生物力学和扩张性心肌肥厚。我们假设星云在恒星的稳定性中起着关键作用。 肾脏足细胞中的肌动蛋白细丝，增加其对损伤的生物力学弹性。我们将对此进行测试 在体外和体内使用培养的原代足细胞和足细胞特异性星云剔除小鼠的假说， 分别进行了分析。我们将使用高含量成像来表征与以下相关的形态变化 星云消融和活细胞显微镜以确定其对细胞运动和钙动力学的影响。我们 还将使用原子力显微镜弹性成像来定量表征细胞骨架的生物力学。我们 然后将使用蛋白质组学和网络分析来构建空间特定的偏微分方程式- 基于动力学模型的星云不同生物物理成分在细胞骨架中的贡献 稳定性和灶性粘连的分布和功能。这一计算机处理过程还将识别可能的药物靶点。 这可能会以细胞特有的方式调节足细胞的机械生物学。我们将最终测试一下 足细胞特异性星云中两种不同疾病模型对体内足细胞生理学的影响 基因敲除老鼠。通过这些实验和计算模型，我们将彻底表征 星云在肾小球生理学中的作用，并有可能确定修改生物力学的新途径 足细胞在健康和疾病条件下的弹性。

Project Summary Kidney podocytes operate under substantial biomechanical stress as part of their physiological function to filter plasma. They have a specialized dynamic actin cytoskeleton structure to reinforce the fragile foot process morphology that is required for their function. Using in vivo proteomic screening and mining of NephroSeq data, we have identified nebulette as a novel scaffolding protein that may play a role in organization and maintenance of the podocyte cytoskeleton and adhesion. Nebulette is an actin-binding protein expressed highly in the heart; mutations on its nebulin repeat domains have been linked to abnormal cardiac biomechanics and dilated cardiac hypertrophy. We hypothesize that nebulette plays a critical role in stability of actin filaments in kidney podocytes, increasing their biomechanical resilience against injury. We will test this hypothesis in vitro and in vivo using cultured primary podocytes and podocyte-specific nebulette knockout mice, respectively. We will use high-content imaging to characterize morphological changes associated with nebulette ablation and live-cell microscopy to determine its effects on cell motility and calcium dynamics. We will also quantitatively characterize cytoskeletal biomechanics using atomic force microscope elastography. We will then use proteomics and network analyses to construct a spatially specific partial differential equations- based dynamical model to quantify contribution of different biphysical components of nebulette in cytoskeletal stability and focal adhesion distribution and function. This in silico process will also identify likely drug targets that may modulate podocyte mechanobiology in a cell-specific manner. We will finally test the contribution of nebulette to in vivo podocyte physiology using two different disease models in podocyte-specific nebulette knockout mice. Through these experiments and computational modeling, we will thoroughly characterize the role of nebulette in glomerular physiology, and potentially identify novel pathways to modify biomechanical resilience of podocytes under healthy and disease conditions.