Cellular control of microtubule-based transport.

基于微管的运输的细胞控制。

• 批准号: 9923705
• 负责人: SAMARA L RECK-PETERSON
• 金额: $ 35.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9923705
• 关键词: 3-Dimensional; Algorithms; Aspergillus; Aspergillus nidulans; Behavior; Biochemical; Biological Assay; Biotinylation; Cell Cycle Stage; Cell division; Cells; Complex; Cytoskeleton; Defect; Development; Dynein ATPase; Early Endosome; Embryo; Endoplasmic Reticulum; Endosomes; Eukaryota; Eukaryotic Cell; Focal Adhesions; Genetic; Genetic Screening; Goals; Grant; Human; In Vitro; Interphase; Interphase Cell; Kinesin; Label; Light; Lipids; Mammalian Cell; Maps; Mass Spectrum Analysis; Messenger RNA; Methods; Microscopy; Microtubule-Organizing Center; Microtubules; Minus End of the Microtubule; Mitotic; Molds; Molecular Machines; Motor; Movement; Neurodegenerative Disorders; Neurons; Organelles; Problem Solving; Protein Family; Proteins; Proteome; Proteomics; Research; Role; Site; Specificity; Structure; System; Virus; Work; base; cell motility; cell type; dynactin; experimental study; follow-up; frontier; genetic approach; insight; kidney cell; live cell imaging; migration; nervous system disorder; ninein; novel; peroxisome; programs; reconstitution; recruit; single molecule

SUMMARY The spatial and temporal organization of intracellular components by the microtubule cytoskeleton is required for cell division, many aspects of development, and neuronal function. Defects in the microtubule cytoskeleton cause neurological diseases in humans. The long-term goal of this research program is to determine how the multiple components of this transport system—the motors and their tracks, cargos, cargo adaptors, and regulators–work together. Microtubules are dynamic polar structures, with “plus” ends usually located near the cell periphery and “minus” ends embedded in internal microtubule organizing centers. Dyneins move towards the minus ends of microtubules, whereas most kinesins move in the opposite direction. In humans, a single dynein (cytoplasmic dynein-1) and ~15 kinesins are responsible for the interphase transport of organelles, proteins, and mRNAs. Viruses hijack these same motors. How does a small subset of motors transport such a large and diverse set of cargos? Understanding this is one of the major frontiers in the transport field. The goal of this proposal is to determine how cargo specificity is achieved for cytoplasmic dynein-1, the major minus- end-directed microtubule-based motor in eukaryotic cells. Traditional biochemical approaches have yielded surprisingly little information about these mechanisms. To solve this problem, we are using both genetic and proteomic discovery approaches to identify motor-cargo interactions and to determine how they are regulated. Genetic approach: Using a forward genetic screen in the filamentous fungus Aspergillus nidulans we co- discovered that peroxisomes “hitchhike” on early endosomes to achieve motility. Previously, the paradigm was that each cargo directly recruited the transport machinery. We will investigate the mechanism of hitchhiking and determine if it is widely used for organelle motility across eukaryotes. Proteomic approach: Mammalian dynein requires the dynactin complex and a coiled coil containing “activator” to achieve processive motility. Using proximity-dependent biotinylation we identified the components of the human dynein proteome in human interphase embryonic kidney cells, including novel dynein activators. We will use these proteomic approaches to identify additional novel dynein activators in different cell types and at different stages of the cell cycle. We will use these novel dynein activators as “stepping stones” to identify their proteomes and determine which cargos they transport.

概括 需要微管细胞骨架的细胞内组件的空间和临时组织 对于细胞分裂，发育的许多方面和神经元功能。微管细胞骨架中的缺陷 引起人类神经疾病。该研究计划的长期目标是确定 该运输系统的多个组件 - 电动机及其轨道，货物，货物适配器和 监管机构 - 一起工作。微管是动态的极性结构，其“加号”末端通常位于 细胞周围和“负”末端嵌入内部微管组织中心。动力蛋白朝着 微管的减去末端，而大多数驱动蛋白朝相反的方向移动。在人类中，一个 动力蛋白（细胞质动力蛋白1）和〜15驱动蛋白负责细胞器的相间转运， 蛋白质和mRNA。病毒劫持了这些相同的电机。一小部分电动机如何运输这样的电动机 大型和潜水员的货物集？了解这是运输领域的主要边界之一。目标 该建议的是确定如何实现细胞质动力蛋白-1的货物特异性，这是主要负量 - 真核细胞中基于微管的终导向。传统的生化方法已经产生 令人惊讶的是，关于这些机制的信息很少。为了解决这个问题，我们同时使用遗传和 蛋白质组学的发现方法以识别运动型货车相互作用并确定如何调节它们。 遗传方法：在丝状曲霉中使用正向遗传筛选，我们共同 发现早期内体上的过氧化物体“搭便车”以达到运动性。以前，范式是 每辆货物都直接招募了运输机械。我们将研究搭便车的机制 并确定它是否广泛用于整个真核生物的细胞器运动。蛋白质组学方法：哺乳动物 Dynein需要Dynactin复合物和包含“激活剂”的盘绕线圈才能实现加工运动。 使用接近度依赖性生物素化我们确定了人类动力蛋白蛋白质组的成分 相间胚胎肾细胞，包括新型动力蛋白激活剂。我们将使用这些蛋白质组学方法 在不同的细胞类型和细胞周期的不同阶段中鉴定其他新型的动力蛋白激活剂。我们 将使用这些新型的动力蛋白激活剂作为“步进石材”来识别其蛋白质组并确定哪个 他们运输的货物。

项目成果

Hitching a Ride: Mechanics of Transport Initiation through Linker-Mediated Hitchhiking

搭便车：通过链接器介导的搭便车启动传输机制

• DOI: 10.1016/j.bpj.2020.01.024
• 发表时间: 2020
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Mogre, Saurabh S;Christensen, Jenna R.;Niman, Cassandra S.;Reck-Peterson, Samara L;Koslover, Elena F
• 通讯作者: Koslover, Elena F