Mutational Studies of Processive Myosin Motors

 描述（由申请人提供）：V类肌球蛋白（myoV）在基于肌动蛋白的细胞器运输和膜运输中起关键作用，并且是从酵母到哺乳动物生存所必需的。在哺乳动物中有三种异构体，具有不同的组织特异性和功能。MyoVa是进行性的，这意味着运动可以在解离之前对肌动蛋白采取多个步骤。它与黑素体、胰岛素颗粒和内质网的转运有关。相反，据报道myoVc是非进行性的，并在腺组织中表达，在腺组织中它与大的外分泌颗粒相关。myoV中的突变导致人类疾病（例如Griscelli综合征、微绒毛包涵体病），证明它们在正常细胞功能中的重要作用。驱动蛋白和动力蛋白在微管通道上起着相似的转运作用。它们的功能障碍导致无数的神经退行性疾病。这项建议的一个广泛的主题是系统地建立复杂的货物运输马达的体外研究，以探测机制。每个目标都是由细胞水平的生物学观察激发的。在Aim 1中，通过使用全长myoVa，天然衔接子 蛋白质，和轨道组成的细胞质肌动蛋白与绑定原肌球蛋白，我们研究的机制参与货物本地化的V类肌球蛋白马达。将使用单分子方法和全内反射荧光（TIRF）显微镜。在目的2中，我们研究了myoVc的性质，myoVc是在外分泌组织中转运大分泌颗粒的同种型。我们希望了解肌动蛋白轨道形态如何可以是一个“持续合成因子”myoVc，以及myoVc的整体属性如何不同于myoVa，使用DNA折纸耦合多个电机。在目标3中，我们将重建一个最小的mRNP包含全长驱动蛋白和动力蛋白，衔接蛋白，和mRNA，以测试运动协调，激活和方向性的想法。作为一个整体，拟议的研究提供了一个重要的桥梁之间的定义明确的单电机研究与截断的结构，和纯粹的在体内细胞生物学研究的肌球蛋白，驱动蛋白，动力蛋白电机。

 DESCRIPTION (provided by applicant): Class V myosins (myoV) play crucial roles in actin-based organelle transport and membrane trafficking, and are needed for survival from yeast to mammals. There are three isoforms in mammals, with different tissue specificities and functions. MyoVa is processive, meaning that the motor can take multiple steps on actin before dissociating. It has been implicated in transport of melanosomes, insulin granules, and endoplasmic reticulum. In contrast, myoVc has been reported to be non-processive and expressed in glandular tissue where it associates with large exocrine granules. Mutations in myoV lead to human disease (e.g. Griscelli syndrome, microvillus inclusion disease), attesting to their important role in normal cellular function. Kinesin and dynein play similar transport role on microtubule tracks. Their dysfunction causes a myriad number of neurodegenerative diseases. A broad theme of this proposal is to systematically build complexity into the in vitro study of cargo-transport motors to probe mechanisms. Each aim is motivated by biological observations at the cellular level. In Aim 1, through the use of full- length myoVa, native adaptor proteins, and tracks composed of cytoplasmic actin with bound tropomyosin, we investigate mechanisms involved in cargo localization by class V myosin motors. Single- molecule approaches and total internal reflection fluorescence (TIRF) microscopy will be used. In Aim 2, we investigate the properties of myoVc, the isoform which transports large secretory granules in exocrine tissues. We wish to understand how actin track morphology can be a "processivity factor" for myoVc, and how the ensemble properties of myoVc differ from those of myoVa, using DNA origami to couple multiple motors. In Aim 3 we will reconstitute a minimal mRNP containing full-length kinesin and dynein, adaptor proteins, and mRNA, to test ideas of motor co-ordination, activation, and directionality. As a whole, the proposed studies provide an essential bridge between well-defined single motor studies with truncated constructs, and purely in vivo cell biological studies of myosin, kinesin, and dynein motors.