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发挥关键作用。存在三种具有不同组织特异性和功能的哺乳动物同种型（Va、Vb和Vc）。其中两种亚型（Va和Vb）是进行性的，这意味着它们在解离之前可以在肌动蛋白上进行多个步骤。MyoVc是非加工性的，但所有功能都在货物运输中。已经给予了很大的关注，在单个肌动蛋白丝上的单个电机运动的体外研究，主要是使用组成型活性截断版本的myoVa。这个建议的一个总体目标是系统地建立复杂的研究V类肌球蛋白马达，使用三种互补的方法。第一个是研究全长马达的特性，以及货物对其功能的影响。其次是深入了解myoV马达如何协同工作，因为细胞货物是由多个马达运输的。第三种是将myoV马达引入细胞并跟踪它们的运动，或者由具有已知特性的马达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，我们将确定在不存在或存在货物的情况下，受调节的全长myoVa的单分子性质如何不同于组成型活性构建体的单分子性质。将确定单个全长myoVa电机在没有或存在货物的情况下的运行长度、速度和步进模式。我们将测试货物衔接蛋白是否影响初始激活以外的运动功能。在目标2中，我们研究了两个myoV电机如何在空载和负载条件下协调它们的运动和力的产生。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。要测量的参数是速度、运行长度、步长和失速力。将测试各种电机组合。这些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影响。在目标3中，我们比较和对比活细胞内myoVa、myoVb和myoVc的运动。量子点（Qdot）标记的myoV构建体的轨迹，通过胞饮引入哺乳动物细胞，将遵循高分辨率全内反射荧光（TIRF）显微镜和单粒子跟踪。将两种进行性myoV同种型（myoVa和myoVb）的运动与非进行性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。互补的诱导型货物运输测定将用于将马达构建体靶向天然货物。我们认为，这里提出的综合办法提供了推动该领域向前发展的最大潜力。

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.