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。哺乳动物有三种亚型(Va、Vb和Vc)，它们具有不同的组织特异性和功能。其中两个亚型(Va和Vb)是过程的，这意味着它们可以在解离之前对肌动蛋白采取多个步骤。MyoVc是非过程性的，但都在货物运输中起作用。在单个肌动蛋白细丝上的单个运动的体外研究已经引起了很大的关注，主要是使用一个结构性的活性截断的myoVa版本。这项建议的首要目标是使用三种互补的方法，系统地将复杂性构建到V类肌球蛋白马达的研究中。首先是研究全长电机的特性，以及货物对其功能的影响。第二个是深入了解myoV发动机是如何协同工作的，因为蜂窝货物是由多个发动机运输的。第三种是将myoV马达引入细胞并跟踪它们的运动，即由具有已知特性的马达驱动的原生货物的运动。在此背景下，我们将评估三种myoV亚型之间的差异。在目标1中，我们将确定在没有或存在货物的情况下，受调控的全长myoVa的单分子性质与结构活性结构的单分子性质有何不同。在没有货物或有货物存在的情况下，将确定单个全长myoVa电机的运行长度、速度和步进模式。我们将测试货物接头蛋白是否会影响最初激活之外的运动功能。在目标2中，我们研究了两个myoV马达在空载和加载条件下如何协调它们的运动和力的产生。结合两个马达的DNA支架将被用来确定在空载和加载条件下，myoV马达对彼此性能的影响。要测量的参数包括速度、行程长度、步长和失速力。将测试各种马达组合。这些结果对通过绑定到相同的细胞内货物而机械耦合的多个马达的行为有一定的影响。在目标3中，我们比较和对比了myoVa、myoVb和myoVc在活细胞中的运动。通过胞饮作用将量子点(QDot)标记的myoV构建物导入哺乳动物细胞，将通过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。一种互补的可诱导货物贩运分析将被用于针对本地货物的发动机结构。我们认为，这里提出的综合办法提供了推动这一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.