Mechanism by Which the Bicaudal D2-Nuclear Pore Protein 358 Interaction Activates Microtubule-based Cargo Transport

双尾 D2-核孔蛋白 358 相互作用激活基于微管的货物运输的机制

• 批准号: 10809832
• 负责人: M Yusuf Ali
• 金额: $ 15.6万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10809832
• 关键词: Adaptor Signaling Protein; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Autophagosome; Axon; Binding; Binding Proteins; Biological; Biological Assay; Cell physiology; Collaborations; Complex; Coupling; Cytoplasm; Data; Dynein ATPase; Exhibits; Fluorescence Microscopy; Frequencies; Huntington Disease; Impairment; In Vitro; Intracellular Transport; Kinesin; LIS1 protein; Length; Link; Liquid substance; Membrane; Messenger RNA; Microtubule-Associated Proteins; Microtubules; Minus End of the Microtubule; Mitochondria; Molecular Conformation; Monitor; Motion; Motor; Movement; Neurodegenerative Disorders; Neurons; Nuclear Pore; Organelles; Parkinson Disease; Play; Plus End of the Microtubule; Pore Proteins; Positioning Attribute; Proteins; Registries; Regulation; Role; Running; Secretory Vesicles; Signaling Protein; Spastic Paraplegia; Speed; Tail; Testing; Time; Vesicle; Work; cell motility; cofactor; dynactin; experimental study; in vivo; insight; intracellular protein transport; knock-down; mutant; novel; protein complex; reconstitution; recruit; scaffold; sensor; single molecule

Project Summary/Abstract There are many cargo such as secretory vesicles, autophagosomes, and liquid droplets that are bi-directionally transported along microtubules by the opposing motors cytoplasmic dynein and kinesin. However, it is still unclear how these two biological motors collaborate to position the cargo and achieve cellular functions. Impaired bidirectional transport leads to a variety of neurodegenerative diseases, including Alzheimer's disease. Microtubule-associated protein MAP7 and dynein cofactor Lis1 are two important proteins that play critical roles in cargo transport. While MAP7 enhances the processive motion of kinesin, Lis1 relieves the auto- inhibition of dynein. In this proposal, we aim to reconstitute the DDBE-K (dynein, dynactin, BicD, Egl, kinesin) complex and observe its motion on microtubule tracks in the presence of both MAP7 and Lis1 using total internal reflection fluorescence (TIRF) microscopy. Given that MAP7 has the ability to prolong the attachment of kinesin to microtubules and Lis1 prefers binding two dyneins, we predict that MAP7 and Lis1 will play key roles in determining the direction of motion of the complexes on microtubules. Using single-molecule assays, we will investigate whether the adapter protein BicD not only functions as a linker between cargo and motor but also plays an essential role in sensing cargo and motor binding. These proposed studies will provide novel insights into the roles of dynein and cargo adapter proteins and contribute to our understanding of how cytoplasmic dynein and kinesin work together to transport cargo bidirectionally on microtubules.

项目概要/摘要 分泌囊泡、自噬体、液滴等多种货物 通过相反的马达细胞质动力蛋白沿着微管双向运输 驱动蛋白。然而，目前尚不清楚这两种生物马达如何协作来定位 货物并实现细胞功能。双向运输受损会导致多种 神经退行性疾病，包括阿尔茨海默病。微管相关蛋白 MAP7 和动力蛋白辅因子 Lis1 是两种重要的蛋白质，在货物中发挥着关键作用 运输。 MAP7 增强了驱动蛋白的进行性运动，而 Lis1 则缓解了自动运动 动力蛋白的抑制。在本提案中，我们的目标是重建 DDBE-K（dynein、dynactin、BicD、 Egl，驱动蛋白）复合物并在 MAP7 存在的情况下观察其在微管轨道上的运动 和 Lis1 使用全内反射荧光 (TIRF) 显微镜。鉴于 MAP7 有 能够延长驱动蛋白与微管的附着，并且 Lis1 更喜欢结合两个动力蛋白， 我们预测 MAP7 和 Lis1 将在确定运动方向方面发挥关键作用 微管上的复合物。使用单分子测定，我们将研究是否 衔接蛋白 BicD 不仅充当货物和马达之间的连接物，而且还发挥 在感知货物和电机结合方面发挥着重要作用。这些拟议的研究将提供新颖的 深入了解动力蛋白和货物衔接蛋白的作用并有助于我们的理解 细胞质动力蛋白和驱动蛋白如何协同工作以双向运输货物 微管。