In vivo regulation of bi-directional transport

描述（由申请人提供）：过去的工作已经确定，许多重要的货物沿着微管双向移动，并且这些货物的分配和净运输可以通过调节正端与负端电机的相对贡献来控制。并建立了多个正端和多个负端电机一起工作。然而，调节这种运输的基本机制尚不清楚，参与电机数量的重要性（和调节）也不清楚。这里提出的工作将导致更深入地了解运输的调节，也控制和重要性的电机数量在这一调节。具体来说，脂滴的双向运动在早期果蝇胚胎进行了研究。这项工作在概念上可以分为两种互补的方法。首先，我们开发并严格测试了一个理论模型，该模型是如何通过调整单分子性质来控制这种运输的。理论方法是我们过去由多个电机驱动的传输建模的扩展，并依赖于蒙特卡罗模拟。然后用遗传学来改变马达的蛋白质剂量，以及特定的点突变体来测试理论预测。该模型的一些测试依赖于对个体货物在体内的运动进行高时间和空间分辨率的量化，并确定运动是否与模型的预测一致。在第二部分中，我们将重点放在由klar蛋白控制的参与马达数量及其力量产生的调节上。我们使用各种生物物理特征来确定klar突变的影响。特别是，我们使用光学镊子和粒子跟踪和分析来确定klar蛋白不同结构域的具体物理作用，然后使用互补的生化和细胞生物学技术来确定支撑这些物理作用的分子相互作用。这些信息将在分子水平上阐明生物物理决定的功能是如何产生的，因为我们已经证明了klar调节力的产生（以及可能的参与马达数量），这项研究将直接探讨参与马达的数量是如何被控制的，以及这种控制的功能含义。公共卫生相关性：双向运输与公共卫生直接相关：疱疹等病毒以双向方式通过细胞传播；许多重要的物质，如线粒体和核内体，是双向运动的。受损的囊泡运输与神经元变性有关。最后，更好地了解转运可能有助于设计新的药物输送系统。

DESCRIPTION (provided by applicant): Past work has established that many important cargos move bi-directionally along microtubules, and that the distribution and net transport of such cargos can be controlled by regulating the relative contributions of the plus-end versus minus-end motors. It has also established that multiple plus-end and multiple minus-end motors function together. However, both the fundamental mechanism of regulation of such transport is unclear, as is the importance (and regulation) of the number of engaged motors. The work proposed here will lead to a much deeper understanding both of the regulation of transport, and also of the control and importance of motor number in this regulation. Specifically, bi-directional motion of lipid droplets in early Drosophila embryos is investigated. The work can be conceptually divided into two complimentary approaches. In the first, we develop and critically test a theoretical model for how this transport is controlled by tuning single-molecule properties. The theoretical approach is an extension of our past modeling of transport driven by multiple kinesin motors, and rely on Monte Carlo simulations. The theoretical predictions are then tested using genetics to alter protein dosage of the motors, and also specific point mutants. Some of the testing of the model relies on quantifying-in vivo-the motion of individual cargos with high temporal and spatial resolution, and determining if the motion is consistent with the model's prediction. In the second, we focus on the regulation of the number of engaged motors and their force production, as controlled by the klar protein. We use a variety of biophysical characterizations to determine the effects of klar mutations. In particular, we use optical tweezers and particle tracking and analysis to determine the specific physical role of different domains of the klar protein, and then use complementary biochemical and cell biological techniques to determine the molecular interactions underpinning these physical roles. This information will clarify at the molecular level how the biophysically determined functions come about, and because we have already shown that klar regulates force production (and likely number of engaged motors), the study will directly probe how the number of engaged motors is controlled, and the functional implications of this control. PUBLIC HEALTH RELEVANCE: Bi-directional transport is directly related to public health: viruses such as herpes spread through cells in a bi-directional manner; many important cargos like mitochondria and endosomes move bi-directionally. Impaired vesicular transport is implicated in Neuronal degeneration. Finally, a better understanding of transport might allow the design of new drug delivery systems.