Mechanisms of Mitochondrial Distribution

描述(申请人提供)：在真核细胞中，线粒体运动或‘运输’是通过将细胞器分别连接到沿着微管或肌动蛋白细丝行走的动蛋白或肌球蛋白马达蛋白来实现的。这些线粒体转运事件是在细胞质内分布线粒体所必需的，也是子细胞在分裂过程中遗传线粒体所必需的。对线粒体运输机制的分子理解对人类健康至关重要，因为危及线粒体分布的遗传损伤与越来越多的人类疾病有关，包括某些形式的肌营养不良、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。这项建议侧重于在萌芽酵母酿酒酵母的分裂过程中，调节基于肌动蛋白的线粒体运输的分子和机制。酵母线粒体转运缺陷会干扰子代细胞(芽)的线粒体遗传。以前的研究发现了两条独立的调控酵母线粒体遗传的途径。第一种途径利用一种名为MMR1的外周线粒体适配器蛋白。MMR1与V类肌球蛋白运动蛋白Myo2形成复合体。这一应用中的实验将测试MMR1丰度、活性和/或定位的变化协调MMR1介导的线粒体遗传与细胞周期的模型。第二条线粒体遗传途径需要内质网(ER)定位的Rab GTP酶Ypt11。Ypt11还与Myo2形成复合体。内质网锚定的Rab如何控制线粒体膜的遗传尚不清楚。在这个应用中，提出了两个解释内质网定位Ypt11如何影响线粒体遗传的替代模型并进行了测试。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。这一途径利用了Gem1/Miro，这是一种尾部锚定的线粒体膜外膜蛋白，含有两个GTP酶结构域和两个钙结合的EF-Hand基序。Gem1/Miro在酵母和人类之间是保守的。突变的MIRO在培养细胞中的表达导致细胞凋亡，Gem1/MIRO缺陷的果蝇表现出神经元缺陷。实验是为了确定Gem1/Miro是否通过Myo2起作用，并确定其GTPase结构域的特定效应子。最后，首席研究员的实验室将研究基因筛查中确定的线粒体遗传途径的新组成部分。这些研究可能会识别哺乳动物的同源物，这将使未来能够研究线粒体在人类细胞和组织中的分布机制。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，因为几个感兴趣的分子具有人类同源基因，包括Myo2(人类肌球蛋白Va)和Gem1(人类Miro1和Miro2)。人类肌球蛋白Va的缺陷会导致胚胎死亡，肌球蛋白Va功能降低的患者会患上格里斯切利综合征，这是一种以免疫缺陷为特征的通常是致命的疾病，以及神经和色素缺陷。突变哺乳动物MIRO的表达改变了培养细胞中线粒体的形态并导致细胞死亡，Fly MIRO的缺陷扰乱了轴突线粒体的运输，这一功能在人类中可能是保守的。一些拟议的实验可能导致识别控制基于肌动蛋白的线粒体在人类细胞和组织中的分布过程的同系物。在这个过程中学到的东西可能会让科学家和临床医生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.