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.

总结 微管细胞骨架对细胞内成分的空间和时间组织是必需的 细胞分裂，发育的许多方面，以及神经元功能。微管细胞骨架缺陷 导致人类神经系统疾病。这项研究计划的长期目标是确定 该运输系统的多个部件-发动机及其轨道、货物、货物适配器，以及 监管机构一起工作。微管是动态的极性结构，其“正”端通常位于微管附近。 细胞外围和“负”端嵌入内部微管组织中心。动力蛋白向 微管的负端，而大多数驱动蛋白则以相反的方向移动。在人类中， 动力蛋白（细胞质动力蛋白-1）和~15种驱动蛋白负责细胞器的相间运输， 蛋白质和mRNA。病毒劫持了这些相同的马达。一小部分发动机是如何运输这样一个 大量不同种类的货物理解这一点是运输领域的主要前沿之一。目标 这项建议的目的是确定如何实现货物特异性细胞质动力蛋白-1，主要负- 真核细胞中的末端导向微管马达。传统的生物化学方法 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，我们正在使用基因和 蛋白质组学发现方法，以确定马达-货物相互作用，并确定它们是如何调节的。 遗传方法：在丝状真菌构巢曲霉中使用正向遗传筛选，我们共同研究了 发现过氧化物酶体“搭便车”早期内涵体实现运动。在此之前， 每一批货物都直接吸收了运输机械。我们将研究搭便车的机制 并确定它是否广泛用于真核生物的细胞器运动。蛋白质组学方法：哺乳动物 动力蛋白需要动力蛋白复合物和含有“激活剂”的卷曲螺旋以实现进行性运动。 利用邻近依赖的生物素化，我们鉴定了人肌细胞中人动力蛋白蛋白质组的组成部分， 间期胚胎肾细胞，包括新的动力蛋白激活剂。我们将使用这些蛋白质组学方法 以鉴定不同细胞类型和细胞周期不同阶段的其他新型动力蛋白激活剂。我们 将使用这些新的动力蛋白激活剂作为“垫脚石”，以确定它们的蛋白质组，并确定 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