Increasing the Complexity of Microtubule-based transport: Cargo adaptors and Hitchhiking on Vesicles.

项目摘要 细胞内货物（细胞器、囊泡和大分子）的正确定位对细胞生长至关重要， 成熟和生存。真核细胞使用分子马达动力蛋白和驱动蛋白来运输货物， 微管轨迹基于微管的运输缺陷和马达本身的突变，是一个 许多神经发育和神经退行性疾病的潜在特征。由于每种货物都是 为了适应特定的细胞功能，它们沿沿着微管的装载、移动和卸载需要 不同的运输机制。微管上的货物特异性是如何实现的？该提案将解决 这与基于微管的运输的两种主要模式的深入分析。典型的观点， 作为货物衔接模式，是每个货物与能够募集 分子发动机。货物有选择地装卸这些货物的监管机制 适配器还没有被很好地理解。这项提议的一个目标是确定一个保守的磷酸化 囊泡结合Rab GTP酶上的位点影响与动力蛋白货物衔接子的相互作用，并最终影响这些衔接子如何与动力蛋白货物衔接子相互作用。 相互作用影响细胞功能。重要的是，该Rab磷酸化位点是免疫调节的主要靶点。 帕金森病相关激酶LRRK2。另一种运输方式被称为细胞器搭便车。在 搭便车，Rab-vesicle可以指导微管上细胞器的运动。为了实现这一点，细胞器 在膜接触位点附着（或"搭便车"）马达驱动的Rab-vesicle。细胞器搭便车是一种新的， 相对未开发的基于微管的运输范例，和潜在的分子机制是 没有被很好地理解。什么是细胞器搭便车的分子连接器、系链和调节器？这是 基于微管的运输模式这项提案将使用遗传学、显微镜和生物化学 在真菌和哺乳动物模型系统中解决这些问题。综合来看， 基于微管的运输的主要模式是极其重要的，考虑到Rabs，囊泡和 搭便车的货物在发育和与年龄有关的疾病中发挥作用。

PROJECT SUMMARY Proper positioning of intracellular cargos (organelles, vesicles, and macromolecules) is critical for cell growth, maturation, and survival. Eukaryotic cells use the molecular motors dynein and kinesin to transport cargos along microtubule tracks. Defects in microtubule-based transport and mutations in the motors themselves, are an underlying feature of many neurodevelopmental and neurodegenerative diseases. Since each type of cargo is tuned for specific cellular functions, their loading, movement and unloading along microtubules requires divergent transport mechanisms. How is cargo specificity on microtubules achieved? This proposal will address this with an in-depth analysis of the two major modes of microtubule-based transport. The canonical view, known as the cargo adaptor mode, is that each cargo interacts with specific adaptor proteins capable of recruiting molecular motors. The regulatory mechanisms by which cargos selectively load and unload from these cargo adaptors are not well understood. One goal of this proposal is to determine how a conserved phosphorylation site on vesicle-bound Rab GTPases affects interactions with dynein cargo adaptors and ultimately, how these interactions affect cellular function. Importantly, this Rab phosphorylation site is the predominant target of the Parkinson’s disease-linked kinase LRRK2. The other mode of transport is called organelle hitchhiking. In hitchhiking, Rab-vesicles can direct the movement of organelles on microtubules. To accomplish this, organelles attach to (or ‘hitchhike’ on) motor-driven Rab-vesicles at membrane contact sites. Organelle hitchhiking is a new, relatively unexplored paradigm of microtubule-based transport, and the underlying molecular mechanisms are not well understood. What are the molecular linkers, tethers, and regulators of organelle hitchhiking? Is this a prominent mode of microtubule-based transport? This proposal will use genetics, microscopy, and biochemistry in both fungal and mammalian model systems to address these questions. Taken together, studying the two main modes of microtubule-based transport is extremely important, given the critical roles Rabs, vesicles, and hitchhiking cargos play in development and age-related diseases.