Regulation of cytoplasmic dynein in vivo

体内细胞质动力蛋白的调节

• 批准号: 9311086
• 负责人: XIN XIANG
• 金额: $ 29.51万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9311086
• 关键词: APOBEC2 gene; Actins; Affect; Amyotrophic Lateral Sclerosis; Aspergillus nidulans; Behavior; Binding; Binding Proteins; Binding Sites; Biological Assay; Brain; Collaborations; Complex; Cytoskeleton; Data; Dynein ATPase; Early Endosome; Eukaryotic Cell; Filament; Fluorescence Resonance Energy Transfer; Genetic; Golgi Apparatus; Health; Human; Image; In Vitro; Intracellular Transport; Label; Link; Mass Spectrum Analysis; Mediating; Methods; Microtubules; Molds; Molecular; Molecular Conformation; Motor; Movement; Mutation; Neurodegenerative Disorders; Pathway interactions; Plus End of the Microtubule; Positioning Attribute; Process; Proteins; Proteomics; Regulation; Role; Site; Testing; Transport Vesicles; Vesicle; Vesicle Transport Pathway; Work; base; cell motility; developmental disease; dynactin; experience; genetic approach; in vivo; interest; lissencephaly; mutant; novel

Abstract Microtubule-based intracellular transport is critical for the function of all eukaryotic cells. Cytoplasmic dynein is a minus-end-directed microtubule motor that transports a variety of cargoes, and dynein-cargo interactions require the dynactin complex and specific cargo adapters. How dynein-cargo interactions are regulated in vivo and how dynein activity is coordinated with cargo binding are major open questions in the field. Recently, structural studies and in vitro motility assays led to the hypothesis that the binding of cargo to the pointed end of dynactin's Arp1 filament may unlock dynactin's p150 subunit via a change in its conformation, leading to dynein activation. However, in vivo evidence supporting this hypothesis is missing and the identity of proteins involved in p150's conformational change remain to be defined. We previously found that dynactin's pointed-end protein p25 in Aspergillus nidulans is important for dynein- early endosome interaction. Recently, we discovered that p25 is required for the interaction between dynein-dynactin and HookA, the early endosome dynein adapter. Based on our recent data, we hypothesize that p25 is a key protein regulating dynactin conformation. Specific Aim 1 is to reveal the roles of dynactin p25 and the cargo adapter HookA in regulating dynactin. Sensitized emission-based FRET imaging, microtubule pelleting and in vitro motility assays will be used to achieve this aim. In addition, our recent genetic work has uncovered a vezatin-like protein, VezA, as a novel regulator for dynein-cargo interaction. VezA is not a cargo adapter like HookA but affects the accumulation of dynein at the cargo-loading site, the microtubule plus end. How VezA regulates dynein remains a mystery. Specific Aim 2 is to determine the mechanism of VezA action in dynein-mediated vesicle transport. We will use proteomic and genetic approaches to identify new players in the VezA-dynein pathway.

抽象的 基于微管的细胞内运输对于所有真核细胞的功能至关重要。细胞质 动力蛋白是一种负端定向的微管马达，可运输各种货物，动力蛋白货物 相互作用需要动力蛋白复合物和特定的货物适配器。动力蛋白与货物的相互作用如何 体内调节以及动力蛋白活性如何与货物结合协调是主要的悬而未决的问题 领域。最近，结构研究和体外运动测定得出了这样的假设： 将货物运送到 dynactin 的 Arp1 细丝的尖端可能会通过改变来解锁 dynactin 的 p150 亚基 其构象，导致动力蛋白激活。然而，支持这一假设的体内证据是 缺失以及参与 p150 构象变化的蛋白质的身份仍有待确定。我们 先前发现构巢曲霉中的 dynactin 尖端蛋白 p25 对于动力蛋白很重要 早期内体相互作用。最近，我们发现 p25 是两者之间相互作用所必需的。 动力蛋白-动力蛋白和 HookA，早期内体动力蛋白接头。根据我们最近的数据，我们 假设 p25 是调节 dynactin 构象的关键蛋白。具体目标 1 是揭示 dynactin p25 和货物接头 HookA 在调节 dynactin 中的作用。基于敏化发射 FRET 成像、微管沉淀和体外运动测定将用于实现这一目标。在 此外，我们最近的基因工作发现了一种类似vezatin的蛋白质，VezA，作为一种新的调节剂 动力蛋白-货物相互作用。 VezA 不像 HookA 那样是货物适配器，但会影响动力蛋白的积累 在货物装载处，微管正端。 VezA 如何调节动力蛋白仍然是个谜。 具体目标 2 是确定 VezA 在动力蛋白介导的囊泡运输中的作用机制。我们 将使用蛋白质组学和遗传学方法来识别 VezA-dynein 通路中的新参与者。