Molecular Mechanisms Governing Cooperating Motors

描述（由申请人提供）：许多重要的亚细胞货物通过动力蛋白和/或动力蛋白运动蛋白群沿着微管细胞骨架运输。已知与疾病有关的特定蛋白质和物质，特别是神经退行性疾病，与多种运动分子相互作用，因此，集体运动功能与人类疾病之间可能存在密切联系。电动机组被认为是重要的特定运输挑战，可能需要高力生产。此外，许多货物结合动力蛋白和动力蛋白，并沿着微管双向移动。已知这种行为会影响细胞中物质的时空演化和最终分布。然而，控制集体机动运输的机制还没有得到很好的理解，总的来说，调查这些问题的现有方法由于无法精确描述或控制货物上的机动数量而受到限制。该项目通过建立我们新开发的生物合成策略来解决这些问题，以创建相互作用运动分子的结构定义系统，以及允许在单分子水平上监测集体运动动力学的生物物理分析。通过结合这些能力，我们已经确定了多个kinesin-1分子之间的相互作用对集体运输行为（例如，货物运行长度和力产生）有重大贡献，并且尽管它们有能力产生大的力，但kinesin马达组倾向于消极合作。拟议的工作将采用我们的合成技术以及精确的粒子跟踪和光学捕获方法来进一步评估细胞中多种驱动蛋白的负协同性对货物运输的影响程度（目的1）。我们还将研究由多个动力蛋白分子组成的运动系统中的类似合作效应（目的2）。这两项研究都将在单个运动分子的特性、运动组件内运动间相互作用的性质和集体运输参数之间建立联系。在每种情况下，将使用理论模型评估多个电机功能，这些模型可以解释单个电机的所有相关生化状态以及电机组件的微管结合配置。最后，我们将进行检测，监测由kinesin和dynein分子组成的结构定义的马达组件的运动（Aim 3）。在这里，了解每一类电机之间的集体行为，再加上系统调查电机数量和比例对双向货物流动的影响的能力，应该可以解决双向运输机制。总的来说，这项研究将有助于澄清对细胞中运动蛋白和动力蛋白明显的功能相互依赖的观察，并为检查和解释运动功能缺陷如何影响细胞内运输过程提供新的能力。

DESCRIPTION (provided by applicant): Many important subcellular cargos are transported along the microtubule cytoskeleton by groups of kinesin and/or dynein motor proteins. Specific proteins and cargos that are implicated in diseases, particularly neurodegeneration, are known to interact with multiple motor molecules, and hence, there are likely strong links between collective motor function and human diseases. The grouping of motors is believed to be important for specific transport challenges that may require high force production. Furthermore, many cargos bind to kinesin and dynein and move bidirectionally along microtubules. This behavior is known to influence the spatio-temporal evolution and final distributions of cargos in cells. Yet, mechanisms governing collective motor transport are not well understood, and overall, existing methods to investigate these problems are limited by inabilities to precisely characterize or control the number of motors on cargos. This project addresses these issues by building upon our newly developed biosynthetic strategies to create structurally- defined systems of interacting motor molecules, and biophysical assays that allow collective motor dynamics to be monitored at the single-molecule level. By combining these capabilities, we have established that interactions among multiple kinesin-1 molecules contribute significantly to collective transport behaviors (e.g., cargo run lengths, and force production), and that despite their abilities to produce large forces, groups of kinesin motors tend to cooperate negatively. The proposed work will employ our synthetic technologies along with precision particle tracking and optical trapping methods to further evaluate the extent to which negative cooperativity influences cargo transport by multiple kinesins in cells (Aim 1). We will also examine analogous cooperative effects in motor systems composed of multiple dynein molecules (Aim 2). Both of these studies will draw connections between the properties of individual motor molecules, the nature of inter-motor interactions within motor assemblies, and collective transport parameters. In each case, multiple-motor functions will be evaluated using theoretical models that can account for all relevant biochemical states of individual motors as well as microtubule-bound configurations of motor assemblies. Finally, we will perform assays that monitor the motions of structurally-defined motor assemblies composed of kinesin and dynein molecules (Aim 3). Here, knowledge of collective behaviors among each class of motors, coupled with the ability to systematically investigate the influence of motor number and ratio on bidirectional cargo motility, should allow bidirectional transport mechanisms to be resolved. Overall, the proposed study will help to clarify observations of kinesin and dynein's apparent functional interdependence in cells, and provide new abilities to examine and interpret how defects in motor function influence intracellular transport processes. PUBLIC HEALTH RELEVANCE: Resolving mechanisms of collective motor transport will provide a foundation to interpret how transport defects, genetic and/or environmental, influence the function of motor molecules in cells. Outcomes from this work may also suggest new methods to develop and evaluate therapeutic agents directed at molecular motors.