Mechanisms of Mitochondrial Distribution

描述（由申请人提供）：在真核细胞中，线粒体运动或“运输”是通过将细胞器附着在分别沿着微管或肌动蛋白细丝行走的动蛋白或肌球蛋白运动蛋白上而发生的。这些线粒体运输事件是细胞质内线粒体分布所必需的，也是子细胞在分裂过程中线粒体遗传所必需的。线粒体运输机制的分子理解对人类健康至关重要，因为损害线粒体分布的遗传病变与越来越多的人类疾病有关，包括某些形式的肌肉萎缩症、心肌病、截瘫和神经变性。调节线粒体运输的基本机制从酵母到人类是保守的。这一建议的重点是分子和机制，介导在芽殖酵母，酿酒酵母分裂过程中基于肌动蛋白的线粒体运输。酵母线粒体运输缺陷干扰子细胞（芽）的线粒体遗传。先前的研究确定了调节酵母线粒体遗传的两个独立途径。第一种途径利用外周线粒体接头蛋白Mmr1。Mmr1与Myo2形成复合体，Myo2是V类肌球蛋白运动蛋白。本应用中的实验将测试Mmr1丰度、活性和/或定位的变化与细胞周期协调Mmr1介导的线粒体遗传的模型。第二种线粒体遗传途径需要内质网（ER）定位的Rab GTPase，称为Ypt11。Ypt11也与Myo2形成复合体。er锚定的Rab如何控制线粒体膜的遗传尚不清楚。在本应用中，提出并测试了两种替代模型来解释er定位的Ypt11如何影响线粒体遗传。首席研究员的实验室最近描述了线粒体遗传所需的第三条途径。该途径利用Gem1/Miro，这是一种尾锚定的线粒体外膜蛋白，含有两个GTPase结构域和两个Ca2+结合EF-hand基元。Gem1/Miro从酵母到人类都是保守的。在培养细胞中表达突变Miro会导致细胞凋亡，Gem1/Miro缺陷的果蝇会出现神经元缺陷。我们提出了实验来确定Gem1/Miro是否通过Myo2起作用，并确定其GTPase结构域的特异性效应物。最后，主要研究者的实验室将研究在基因筛选中发现的线粒体遗传途径的新成分。这些研究可能会确定哺乳动物的同源物，这将允许未来研究人类细胞和组织中的线粒体分布机制。公共卫生相关性：概述的研究将促进对基于肌动蛋白的线粒体运输机制的理解。这些结果将与人类发育和健康相关，因为一些感兴趣的分子具有人类同源物，包括Myo2（人类肌球蛋白Va）和Gem1（人类Miro1和Miro2）。人肌球蛋白Va缺陷导致胚胎死亡，肌球蛋白Va功能降低的患者患有格里塞利综合征，这是一种以免疫缺陷、神经系统和色素沉着缺陷为特征的致命疾病。哺乳动物Miro突变体的表达会改变线粒体形态并诱导培养细胞死亡，而果蝇Miro的缺陷会破坏轴突线粒体运输，这一功能可能在人类中是保守的。一些提出的实验可能导致鉴定同源控制肌动蛋白为基础的线粒体分布过程在人类细胞和组织。在这个过程中，科学家和临床医生可能会操纵这些分子的活动，以造福人类健康。

DESCRIPTION (provided by applicant): In eukaryotic cells, mitochondrial movement or `transport' occurs by attaching the organelle to kinesin or myosin motor proteins that walk along microtubules or actin filaments, respectively. These mitochondrial transport events are required to distribute mitochondria within the cytoplasm and are also essential for inheritance of mitochondria by daughter cells during division. A molecular understanding of mitochondrial transport mechanisms is critical for human health, as genetic lesions that compromise mitochondrial distribution are linked to a growing list of human disorders, including some forms of muscular dystrophy, cardiomyopathy, paraplegia, and neurodegeneration. The basic mechanisms regulating mitochondrial transport are conserved from yeast to man. This proposal focuses on the molecules and machineries that mediate actin-based mitochondrial transport during division in the budding yeast, S. cerevisiae. Defects in yeast mitochondrial transport interfere with mitochondrial inheritance by daughter cells (buds). Previous studies identified two independent pathways that regulate yeast mitochondrial inheritance. The first pathway utilizes a peripheral mitochondrial adaptor protein called Mmr1. Mmr1 forms a complex with Myo2, a class V myosin motor protein. Experiments in this application will test the model that changes in Mmr1 abundance, activity and/or localization coordinate Mmr1-mediated mitochondrial inheritance with the cell cycle. The second mitochondrial inheritance pathway requires an endoplasmic reticulum (ER)- localized Rab GTPase called Ypt11. Ypt11 also forms a complex with Myo2. How an ER-anchored Rab controls inheritance of mitochondrial membranes is not understood. In this application, two alternative models to explain how ER-localized Ypt11 influences mitochondrial inheritance are proposed and tested. The principle investigator's laboratory recently described a third pathway required for mitochondrial inheritance. This pathway utilizes Gem1/Miro, a tail-anchored outer mitochondrial membrane protein containing two GTPase domains and two Ca2+binding EF-hand motifs. Gem1/Miro is conserved from yeast to man. Expression of mutant Miro in cultured cells leads to apoptosis, and flies with defective Gem1/Miro display neuronal defects. Experiments are proposed to determine whether Gem1/Miro acts via Myo2 and to identify specific effectors of its GTPase domains. Finally, the principle investigator's laboratory will study novel components of mitochondrial inheritance pathways identified in genetic screens. These studies will likely identify mammalian homologs that will allow future investigation of mitochondrial distribution mechanisms in human cells and tissues. Public Health Relevance: The studies outlined will advance the understanding of actin-based mitochondrial transport mechanisms. The results will be relevant to human development and health, as several of the molecules of interest have human homologs, including Myo2 (human myosin Va) and Gem1 (human Miro1 and Miro2). Defects in human myosin Va cause embryonic lethality, and patients with reduced myosin Va function suffer from Griscelli's syndrome, an often fatal disorder characterized by immunodeficiency, and neurological and pigmentation defects. Expression of mutant mammalian Miro alters mitochondrial morphology and induce cell death in cultured cells, and defects in fly Miro disrupt axonal mitochondrial transport, a function that is likely conserved in humans. Some of the proposed experiments may lead to the identification of homologs that control actin-based mitochondrial distribution processes in human cells and tissues. What is learned in the process may allow scientists and clinicians to manipulate the activities of these molecules for the benefit of human health.