The Molecular Basis for Myosin Regulation-Equipment Supplement

摘要 所有生物体的一个关键功能是在需要时移动的能力。这些细胞内的运动 运输、细胞分裂、肌肉收缩和细胞运动--都是由分子机器驱动的， 考虑到它们只有几纳米宽，这是一个惊人的力量。由于电机的种类繁多， 细胞中的蛋白质，一个关键问题是如何马达合作和竞争，同时移动货物和应用 力.一个新兴的范例是专门的马达的概念，或者说是经过微调以执行特定功能的马达。 具体功能。尽管这些马达蛋白的重要性，相对较少的是知道他们的个人 适应以及这些适应与细胞中发现的运动模式的关系。这项工作的重点是肌球蛋白-6和 肌球蛋白-10及其独特的细胞调节形式。肌球蛋白-6在细胞器中发挥重要作用 形态，细胞形态，胞质分裂，自噬和内吞，而肌球蛋白-10提供必要的 货物，如整联蛋白、钙粘蛋白和神经诱向蛋白受体的丝状伪足在细胞的前缘。两者都是 由于它们在膜运输、细胞迁移和转移中的作用，在肿瘤中过表达。的 这项工作将开发新的方法来控制肌球蛋白-6和分离其在细胞中的活性。的一个主要 方法将是用光隔离肌球蛋白-6，使细胞具有光学可转换的斯内尔氏细胞， Waltzer（肌球蛋白-6无效）表型。实验的目的是确定肌球蛋白-6何时充当乘客， 一个锚，或运输车在细胞，如果直接移交从一个货物适配器到另一个是必要的， 功能拟议的工作还将研究货物结合和肌球蛋白四级结构如何调节 肌球蛋白10的运动性。一个综合的方法，结合结构研究与功能 重建和单分子运动性测定。这一建议将检验货物和 细胞外配体结合都是肌球蛋白10激活所必需的，并且货物可以引导肌球蛋白10从 一种肌动蛋白网络到另一种完成这项研究将产生一个全面的看法， 细胞骨架马达蛋白被激活并被调节用于细胞中的不同任务。运动蛋白调节是 这是一个具有重要生物学意义的过程，但人们对此知之甚少。这项工作将指导今后的努力， 理解多种情境下的激活。

Abstract A critical function for all living organisms is the ability to move when needed. These movements--intracellular trafficking, cell division, muscle contraction, and cell motility-- are driven by molecular machines that exert an amazing amount of force considering that they are only a few nanometers across. Given the variety of motor proteins in the cell, a key question is how motors cooperate and compete while moving cargoes and applying forces. An emerging paradigm is the notion of specialized motors, or motors that are fine-tuned to perform a specific function. Despite the importance of these motor proteins, relatively little is known about their individual adaptations and how these relate to the motility patterns found in the cell. This work focuses on myosin-6 and myosin-10 and their unique forms of cellular regulation. Myosin-6 plays essential roles in organelle morphology, cell morphology, cytokinesis, autophagy, and endocytosis, while myosin-10 delivers essential cargoes such as integrins, cadherins and netrin receptors to filopodia at the leading edge of the cell. Both are overexpressed in tumors, owing to their roles in membrane trafficking, cell migration, and metastasis. The work will develop new approaches to control myosin-6 and isolate its activity in cells. One of the main approaches will be to sequester myosin-6 with light, making cells that have an optically switchable Snell's waltzer (myosin-6 null) phenotype. Experiments are designed to identify when myosin-6 acts as a passenger, an anchor, or a transporter in the cells, and if direct handoff from one cargo adaptor to another is required for function. The proposed work will also investigate how cargo binding and myosin quaternary structure tune myosin-10's motility. An integrated approach is developed, combining structural studies with functional reconstitution and single molecule motility assays. This proposal will test the hypotheses that cargo and extracellular ligand binding are both required for myosin-10 activation, and that cargo can steer myosin-10 from one type of actin network to another. Completion of this study will yield a comprehensive view of how cytoskeletal motor proteins are activated and regulated for distinct tasks in the cell. Motor protein regulation is a process of fundamental biological importance, but is poorly understood. This work will direct future efforts to understand activation in multiple contexts.