Mechanosensitive determinants of podocyte physiology

项目摘要 肾足细胞在大量的生物力学应力下工作，作为其生理功能的一部分，以过滤 等离子体他们有一个专门的动态肌动蛋白细胞骨架结构，以加强脆弱的足突 这是其功能所必需的。利用体内蛋白质组学筛选和对NeuphroSeq数据的挖掘， 我们已经确定nebulette是一种新的支架蛋白，可能在组织中发挥作用， 维持足细胞的细胞骨架和粘附。Nebulette是一种肌动蛋白结合蛋白， 在心脏中高度;其星云蛋白重复结构域的突变与心脏异常有关。 生物力学和扩张性心脏肥大。我们假设，雾化器在稳定性中起着关键作用， 肌动蛋白丝在肾脏足细胞，增加其生物力学弹性对损伤。我们将测试这个 在体外和体内使用培养的原代足细胞和足细胞特异性nebulette敲除小鼠的假设， 分别我们将使用高内涵成像来表征与以下相关的形态学变化： 雾化消融和活细胞显微镜检查，以确定其对细胞运动性和钙动力学的影响。我们 还将使用原子力显微镜弹性成像定量表征细胞骨架生物力学。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- 基于动力学模型的细胞骨架中不同的双物理成分的贡献的量化 稳定性和焦点粘附分布和功能。这种计算机模拟过程也将确定可能的药物靶点 其可以以细胞特异性方式调节足细胞机械生物学。我们将最终测试 在足细胞特异性雾化中使用两种不同的疾病模型将雾化与体内足细胞生理学联系起来 敲除小鼠通过这些实验和计算建模，我们将彻底描述 nebulette在肾小球生理学中的作用，并可能确定新的途径来改变生物力学 足细胞在健康和疾病条件下的恢复力。

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.