Slit Diaphragm and Actin Dynamics

狭缝隔膜和肌动蛋白动力学

• 批准号: 8230613
• 负责人: LAWRENCE B. HOLZMAN
• 金额: $ 33.32万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-11-03 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8230613
• 关键词: Accounting; Actins; Adaptor Signaling Protein; Address; Appearance; Architecture; Biology; Cells; Complex; Development; Diabetes Mellitus; Dialysis procedure; Disease; Economic Burden; End stage renal failure; Event; Extracellular Domain; Filtration; Foot Process; Gene Mutation; Gene Targeting; Glomerular Capillary; Goals; In Situ; Inherited; Injury; Intercellular Junctions; Kidney; Kidney Glomerulus; Kidney Transplantation; Medical Research; Microfilaments; Molecular; Molecular and Cellular Biology; Morbidity - disease rate; Morphology; Mus; NPHS2 protein; Nephrotic Syndrome; Pathogenesis; Patients; Phosphorylation; Play; Prevention; Process; Protein Kinase; Proteinuria; Recruitment Activity; Regulation; Renal function; Role; Signal Transduction; Testing; Transducers; Tyrosine; United States; Work; effective therapy; expectation; foot; in vivo; mortality; mouse model; nephrin; neuronal cell body; podocyte; polymerization; protein complex; receptor; response; slit diaphragm; therapeutic target; unpublished works

DESCRIPTION (provided by applicant): Glomerular visceral epithelial cells play a central role in maintaining the filtration barrier of the renal glomerulus. These cells are also called podocytes to describe the foot-like appearance of numerous interdigitating processes that arise from their cell bodies and surround glomerular capillary walls. In response to glomerular injury, podocytes undergo a dramatic change in morphology termed "foot process effacement", a process which is invariably associated with proteinuria, and which results from retraction and spreading of foot processes due to an alteration in podocyte cytoskeletal and intercellular junctional architecture. The goal of the project proposed herein is to contribute to understanding the cellular and molecular mechanisms that govern the dynamics of podocyte morphology and filter integrity with the expectation that providing a mechanistic understanding of these processes will expose potential disease mechanisms and therapeutic targets. Several unique protein complexes have been identified that are targeted to the foot process intercellular junction: among these is the Nephrin-Neph1-Podocin receptor complex, which appears to function as a transmembrane receptor. Loss of any one of the Nephrin-Neph1-Podocin receptor components, either in patients carrying an inherited genetic mutation or in experimental gene-targeted mice, causes proteinuria and effacement of podocyte foot processes. For this reason, we propose that the Nephrin-Neph1-Podocin complex functions to integrate podocyte cytoskeletal dynamics and intercellular junction dynamics. Our preliminary work suggests that the Nephrin complex transducers outside-in signals that participate in regulating actin dynamics. In work that first demonstrated this function of Nephrin, we showed that upon engagement of Nephrin's extracellular domain, the protein kinase Fyn catalyzes Nephrin phosphorylation during foot process formation and following induction of podocyte effacement. Nephrin phosphorylation results in recruitment of the Nck to the junction where it assembles an actin polymerization complex capable of nucleating and elongating actin filaments. In unpublished work described herein, we find that Neph1 behaves in a similar fashion, becoming tyrosine phosphorylated by Fyn and recruiting the adaptor protein Grb2, an event that is necessary for Neph1-induced actin polymerization. Moreover, Nephrin and Neph1 function cooperatively in regulating actin polymerization. While these results support our hypothesis, direct evidence that the Nephrin-Neph1 complex regulates actin dynamics in the podocyte in situ is lacking and the mechanisms by which the Nephrin-Neph1 complex regulates actin dynamics remain obscure. Therefore, this application represents a new proposal aimed at addressing these deficiencies by (1) Investigating cellular mechanisms by which Nephrin-Nck and Neph1-Grb2 cooperate to induce actin nucleation, (2) testing in vivo the relevance of the interaction between Neph1 and Grb2 by creating and characterizing a new mouse model in which the Neph1-Grb2 interaction is interrupted; (3) examining the relationship between the Nephrin and Crk-dependent regulation of actin dynamics. Project Narrative: Relevance. Diseases resulting in the nephrotic syndrome such as diabetes mellitus and other diseases of the glomerulus frequently cause progressive kidney damage resulting in irreversible loss of renal function and account for nearly 60% of end-stage renal disease (ESRD) in the United States (X). While dialysis and kidney transplantation are effective treatments for ESRD, they are expensive and imperfect. Indeed, ESRD is associated with a mortality rate of 60% at five years. Moreover, the economic burden of ESRD treatment in the United States is approaching $30 billion dollars per year. Because little is understood about the biology of diseases of the glomerulus effective therapies are lacking. Therefore, prevention of ESRD by understanding the molecular pathogenesis of these diseases is a critical problem requiring medical research. Diseases of the renal glomerulus that result in the nephrotic syndrome are important causes of morbidity and mortality. Unfortunately, the molecular mechanisms governing development of the nephrotic syndrome remain poorly understood. By continuing to study in detail the cellular and molecular biology of the junctional protein complex of the podocyte, it is anticipated that this project will contribute to a better understanding of the biology of the response of the podocyte to injury or disease.

描述（由申请人提供）： 肾小球内脏上皮细胞在维持肾小球滤过屏障中起着重要作用。这些细胞也被称为足细胞，以描述从其细胞体产生并围绕肾小球毛细血管壁的许多交错突起的足样外观。在肾小球损伤的反应中，足细胞经历了称为“足突消失”的形态学的急剧变化，这是一个总是与蛋白尿相关的过程，并且由于足细胞细胞骨架和细胞间连接结构的改变而导致足突的收缩和扩展。本文提出的项目的目标是有助于理解控制足细胞形态和过滤器完整性的细胞和分子机制，期望提供对这些过程的机械理解将揭示潜在的疾病机制和治疗靶点。已经鉴定了几种独特的蛋白质复合物，其靶向足突细胞间连接：其中包括Nephrin-Neph 1-Podocin受体复合物，其似乎起跨膜受体的作用。在携带遗传性基因突变的患者或实验性基因靶向小鼠中，任何一种Nephrin-Neph 1-Podocin受体成分的丢失都会导致蛋白尿和足细胞足突消失。出于这个原因，我们建议，Nephrin-Neph 1-Podocin复杂的功能，整合足细胞骨架动力学和细胞间连接动力学。我们的初步工作表明，Nephrin复杂的传感器外，在信号参与调节肌动蛋白动力学。在首次证明了Nephrin的这种功能的工作中，我们表明，在参与Nephrin的细胞外结构域时，蛋白激酶Fyn在足突形成期间和足细胞消失诱导之后催化Nephrin磷酸化。Nephrin磷酸化导致Nck募集到连接处，在那里它组装能够成核和伸长肌动蛋白丝的肌动蛋白聚合复合物。在本文所述的未发表的工作中，我们发现Neph 1以类似的方式表现，通过Fyn使酪氨酸磷酸化并募集衔接蛋白Grb 2，这是Neph 1诱导的肌动蛋白聚合所必需的事件。此外，Nephrin和Neph 1在调节肌动蛋白聚合中起协同作用。虽然这些结果支持我们的假设，直接的证据表明，Nephrin-Neph 1复合物调节肌动蛋白在足细胞原位动力学是缺乏和机制的Nephrin-Neph 1复合物调节肌动蛋白动力学仍然模糊。因此，本申请提出了一种新的建议，旨在通过以下方式解决这些缺陷：（1）研究Nephrin-Nck和Neph 1-Grb 2协同诱导肌动蛋白成核的细胞机制，（2）通过创建和表征其中Neph 1-Grb 2相互作用被中断的新小鼠模型，在体内测试Neph 1和Grb 2之间相互作用的相关性;（3）研究Nephrin与Crk依赖的肌动蛋白动力学调节之间的关系。 项目叙述：相关性。导致肾病综合征的疾病，如糖尿病和其他肾小球疾病，经常引起进行性肾损伤，导致不可逆的肾功能丧失，占美国终末期肾病（ESRD）的近60%（X）。虽然透析和肾移植是ESRD的有效治疗方法，但它们昂贵且不完善。事实上，ESRD与五年内60%的死亡率相关。此外，美国ESRD治疗的经济负担每年接近300亿美元。由于对肾小球疾病的生物学知之甚少，因此缺乏有效的治疗方法。因此，通过了解这些疾病的分子发病机制来预防ESRD是一个需要医学研究的关键问题。导致肾病综合征的肾小球疾病是发病率和死亡率的重要原因。不幸的是，肾病综合征的发展的分子机制仍然知之甚少。通过继续详细研究足细胞连接蛋白复合物的细胞和分子生物学，预计该项目将有助于更好地了解足细胞对损伤或疾病的反应的生物学。