Mutational studies of processive myosin motors

进行性肌球蛋白运动的突变研究

• 批准号: 8499349
• 负责人: KATHLEEN M TRYBUS
• 金额: $ 33.73万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8499349
• 关键词: Actins; Affect; Architecture; Attention; Behavior; Binding; Biological; Biological Assay; Blood Vessels; Cell physiology; Cells; Collaborations; Coupled; Cytomegalovirus Infections; DNA; Disease; Exhibits; Face; Fluorescence Microscopy; Generations; Goals; Griscelli Syndrome; In Vitro; Individual; Label; Lead; Length; Life; Lower Organism; Mammalian Cell; Mammals; Measures; Membrane Protein Traffic; Microfilaments; Molecular; Motion; Motor; Movement; Mutation; Myosin ATPase; Myosin Type V; Organelles; Pattern; Pinocytosis; Play; Production; Property; Protein Isoforms; Quantum Dots; Resolution; Role; Running; Specificity; Speed; System; Testing; Therapeutic Intervention; Tissues; Work; adapter protein; base; cell type; cellular microvillus; design; human disease; in vivo; insight; particle; scaffold; single molecule; trafficking

DESCRIPTION (provided by applicant): Class V myosins (myoV) play crucial roles in actin-based organelle transport and membrane trafficking. There are three mammalian isoforms (Va, Vb, and Vc) with different tissue specificities and functions. Two of these isoforms (Va and Vb) are processive, meaning that they can take multiple steps on actin before dissociating. MyoVc is non-processive, yet all function in cargo transport. Much attention has been given to the in vitro study of single motor motion on individual actin filaments, primarily using a constitutively active truncated version of myoVa. An overarching goal of this proposal is to systematically build complexity into the study of class V myosin motors, using three complementary approaches. The first is to investigate the properties of the full-length motor, and how cargo impacts on its function. The second is to gain insight into how myoV motors work together, because cellular cargo is transported by multiple motors. The third is to introduce myoV motors into cells and track their motion, or the motion of native cargo driven by motors with known properties. In this context, we will assess differences between the three myoV isoforms. In Aim 1 we will determine how the single molecule properties of regulated full- length myoVa differ from those of constitutively active constructs, in the absence or presence of cargo. Run lengths, speed, and stepping patterns of single full-length myoVa motors, in the absence or presence of cargo, will be determined. We will test if cargo adapter proteins affect motor function beyond initial activation. In Aim 2 we investigate how two myoV motors co-ordinate their motion and force generation under unloaded and loaded conditions. DNA scaffolds that bind exactly two motors will be used to determine the effect that myoV motors have on each others' properties, under unloaded and loaded conditions. Parameters to be measured are speed, run length, step size, and stall force. Various combinations of motors will be tested. The results have implications for the behavior of multiple motors that are mechanically coupled by being bound to the same intracellular cargo. In Aim 3 we compare and contrast the motion of myoVa, myoVb, and myoVc within a living cell. The trajectories of Quantum-dot (Qdot) labeled myoV constructs, introduced into mammalian cells by pinocytosis, will be followed by high resolution total internal reflection fluorescence (TIRF) microscopy and single particle tracking. Movement of the two processive myoV isoforms (myoVa and myoVb) will be compared to non-processive myoVc. Different cell types with varying actin architectures will be used. A complementary inducible cargo trafficking assay will be used to target motor constructs to native cargo. We believe that a combined approach as presented here provides the greatest potential to move the field forward.

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。有三种哺乳动物同种异构体（Va， Vb和Vc）具有不同的组织特异性和功能。其中两种同工异构体（Va和Vb）是渐进的，这意味着它们在解离肌动蛋白之前可以在肌动蛋白上经过多个步骤。MyoVc是非进程性的，但在货物运输中都有功能。人们对单个肌动蛋白丝的单个运动的体外研究给予了很大的关注，主要是使用一种组成活性的截断版myoVa。本提案的首要目标是使用三种互补的方法系统地构建V类肌球蛋白马达的复杂性研究。首先是研究全长电机的特性，以及货物对其功能的影响。其次是深入了解myoV马达是如何协同工作的，因为细胞货物是由多个马达运输的。第三种方法是将myoV电机引入细胞并跟踪它们的运动，或者由具有已知特性的电机驱动的天然货物的运动。在这种情况下，我们将评估三种myoV亚型之间的差异。在目的1中，我们将确定在没有或存在货物的情况下，受调节的全长myoVa的单分子特性与构成活性结构的单分子特性有何不同。在没有或有货物的情况下，将确定单个全长myoVa电机的运行长度，速度和步进模式。我们将测试货运适配器蛋白是否会影响初始激活后的运动功能。在Aim 2中，我们研究了两个myoV电机在卸载和加载条件下如何协调它们的运动和力产生。精确结合两个马达的DNA支架将用于确定myoV马达在卸载和加载条件下对彼此特性的影响。要测量的参数有速度、运行长度、步长和失速力。将测试各种电机组合。该结果对通过与相同的细胞内货物结合而机械耦合的多个马达的行为具有启示意义。在目标3中，我们比较和对比myoVa， myoVb和myoVc在活细胞内的运动。通过胞吞作用将量子点（Qdot）标记的myoV构建物引入哺乳动物细胞后，将采用高分辨率全内反射荧光（TIRF）显微镜和单粒子跟踪来跟踪其轨迹。两种进展性myoV亚型（myoVa和myoVb）的运动将与非进展性myoVc进行比较。将使用具有不同肌动蛋白结构的不同细胞类型。一个互补的诱导货物运输试验将用于目标电机结构到本地货物。我们相信，这里提出的综合方法提供了推动该领域向前发展的最大潜力。