In vivo regulation of bi-directional transport

描述(申请人提供)：过去的工作已经证实，许多重要的货物沿着微管双向移动，这种货物的分布和净运输可以通过调节正端和负端电机的相对贡献来控制。它还确定了多个正端电机和多个负端电机一起工作。然而，这两种运输调节的基本机制都不清楚，参与的马达数量的重要性(和调节)也不清楚。本文提出的工作将使我们更深入地理解交通运输的规则，以及马达数量在这一规则中的控制和重要性。具体地说，研究了果蝇早期胚胎中脂滴的双向运动。这项工作在概念上可以分为两种互补的方法。首先，我们开发并批判性地测试了如何通过调节单分子的性质来控制这种传输的理论模型。理论方法是我们过去对多个运动蛋白马达驱动的运输建模的扩展，并依赖于蒙特卡罗模拟。然后，使用遗传学改变马达的蛋白质剂量以及特定的点突变来检验理论预测。该模型的一些测试依赖于以高时间和空间分辨率在体内量化单个货物的运动，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蛋白控制的参与运动的数量和它们的力的产生的调节上。我们使用各种生物物理特征来确定KLAR突变的影响。特别是，我们使用光钳和粒子跟踪和分析来确定KLAR蛋白不同结构域的特定物理作用，然后使用互补的生化和细胞生物学技术来确定支持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，而且因为我们已经证明KLAR调节力产生(以及可能的参与马达的数量)，这项研究将直接探索参与的马达数量是如何控制的，以及这种控制的功能含义。公共卫生相关性：双向运输直接关系到公共健康：疱疹等病毒以双向方式通过细胞传播；许多重要的货物，如线粒体和内体，是双向传播的。囊泡转运受损与神经元变性有关。最后，对运输的更好理解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.