Characterization of effectors and ER in Drp1-mediated mitochondrial division

描述（由申请人提供）：我们建议对线粒体分裂动力学的调节机制进行详细分析。Drp1是线粒体裂变的核心调节因子，是一种与动力蛋白相关的GTPase，在细胞凋亡中也起着重要的调节作用。Drp1聚集在线粒体膜上进行分裂；然而，调节这些事件的机制在很大程度上仍然未知。我们正在努力鉴定和表征Drp1效应物，以确定这些蛋白如何介导Drp1的募集和组装以执行线粒体分裂（目的1）。最近观察到内质网（ER）与线粒体接触，这些接触是线粒体收缩和分裂的部位，表明内质网强烈影响线粒体动力学。然而，协调这一过程的机制尚不清楚。我们将使用质谱法和蛋白质组学方法鉴定er -线粒体拴系复合体的成分，并确定这些位点如何影响线粒体分裂动力学（目的2）。我们将使用已建立的显微镜和基于细胞的方法，以及siRNA方法来研究效应物如何在细胞线粒体分裂途径中发挥作用，并在体外实验中专门研究效应物影响Drp1组装和GTP水解的机制，并使用负染色电镜确定组装的Drp1效应物复合物的结构。我们将使用定位于er -线粒体接触点的候选蛋白来识别新的系带成分，并表征它们对线粒体分裂动力学的影响。鉴于内质网影响线粒体分裂的事实，我们将研究Drp1分裂效应物与ER-线粒体系固复合物之间的关系，以了解调节线粒体分裂位点和执行的机制。内质网和线粒体在应激条件下均发生动态重塑；我们将运用我们所学到的关于健康条件下线粒体分裂的调节的知识来了解影响应激反应中发生的重塑的具体机制。效应蛋白的不同组合以及翻译后修饰可能会影响线粒体分裂动力学的速率和位点。从长远来看，我们希望了解内质网和线粒体如何在健康和病理条件下协调它们的行为，以调节内质网和线粒体的功能、分布和诱导凋亡，从而了解这些过程对细胞健康的影响。了解线粒体分裂及其调控的基本机制与理解与内质网应激和线粒体分裂失调相关的越来越多的疾病的基础直接相关，如阿尔茨海默氏症、帕金森病和糖尿病、心脏病和中风，并将潜在地允许开发针对线粒体分裂的治疗方法来治疗各种人类疾病。

DESCRIPTION (provided by applicant): We propose a detailed analysis of the mechanisms regulating mitochondrial division dynamics. The core mitochondrial fission regulator, Drp1 is a dynamin-related GTPase that also plays an important regulatory role in intrinsic apoptosis. Drp1 assembles on the mitochondrial membrane to execute division; however the mechanisms regulating these events remain largely unknown. We are working to identify and characterize Drp1 effectors to determine how these proteins mediate Drp1 recruitment and assembly for execution of mitochondrial division (Aim 1). The recent observation that the endoplasmic reticulum (ER) contacts mitochondria, and these contacts are sites of mitochondrial constriction and division, suggests that the ER strongly influences mitochondrial dynamics. However the mechanisms that coordinate this process are uncharacterized. We will identify components of the ER-mitochondrial tethering complexes using mass spectrometry and proteomics approaches, and identify how these sites influence mitochondrial division dynamics (Aim 2). We will use established microscopic and cell-based approaches, as well as siRNA methods to study how effectors function in a pathway of mitochondrial division in cells, and in vitro assays to specifically address the mechanism by which effectors influence Drp1 assembly and GTP hydrolysis, and determine the structure of assembled Drp1-effector complexes using negative-stain electron microscopy. We will use candidate proteins that localize to ER-mitochondrial contacts to identify new tethering components, and characterize their influence on mitochondrial division dynamics. Given the fact that the ER influences mitochondrial division, we will study the relationship between the Drp1 division effectors and ER-mitochondrial tethering complexes in order to understand the mechanisms that regulate the site and execution of mitochondrial division. Both ER and mitochondria undergo dynamic remodeling under stressed conditions; we will apply what we learn regarding the regulation of mitochondrial division under healthy conditions to learn about the specific mechanisms that influence the remodeling that occurs in response to stress. It is likely that different combinations of effector proteins, as well as post-translational modifications, influence the rates and sites of mitochondrial division dynamics. In the long term we wish to understand how ER and mitochondria coordinate their behavior in healthy and pathologic conditions to regulate ER and mitochondrial function, distribution, and the induction of apoptosis in order to understand the impact of these processes on cellular health. Understanding the fundamental mechanism of mitochondrial division and its regulation is directly relevant to understanding the basis of an increasing number of diseases associated with ER stress and dysregulated mitochondrial division, such as Alzheimer's, Parkinson's and diabetes, heart disease and stroke, and will potentially allow for the development of therapies targeting mitochondrial division to treat a wide variety of human diseases.