In vivo regulation of bi-directional transport

双向运输的体内调节

• 批准号: 8000075
• 负责人: STEVEN P GROSS
• 金额: $ 10.6万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-07 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000075
• 关键词: Binding; Biochemical; Biological; Biological Models; Cell physiology; Cells; Development; Drops; Drosophila genus; Drug Delivery Systems; Dynein ATPase; Embryo; Endocytosis; Endosomes; Equilibrium; Family member; Genetic; Grant; Health; In Vitro; Individual; Intracellular Transport; Kinesin; Lead; Length; Light; Link; Lipids; Measurement; Measures; Methods; Microtubules; Mitochondria; Mitosis; Modeling; Molecular; Molecular Motors; Monitor; Motion; Motor; Motor Activity; Mutation; Nerve Degeneration; Neurons; Optics; Play; Plus End of the Microtubule; Post-Translational Protein Processing; Production; Property; Proteins; Public Health; Publishing; Regulation; Relative (related person); Resolution; Role; Running; System; Techniques; Technology; Testing; Theoretical model; Travel; Variant; Virus; War; Work; base; design; dosage; in vivo; insight; laser tweezer; mutant; particle; protein function; public health relevance; receptor; simulation; single molecule; trafficking; ward

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.

描述（由申请人提供）：过去的工作已经证实，许多重要的货物沿着微管双向移动，并且可以通过调节正端马达与负端马达的相对贡献来控制这些货物的分布和净运输。它还确定了多个正端和多个负端电机一起运行。然而，这种运输调节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人们对运输监管以及该监管中汽车数量的控制和重要性有更深入的了解。具体来说，研究了早期果蝇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，我们开发并严格测试了如何通过调整单分子特性来控制这种传输的理论模型。该理论方法是我们过去由多个驱动蛋白电机驱动的运输模型的延伸，并依赖于蒙特卡罗模拟。然后使用遗传学来改变电机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以高时间和空间分辨率量化单个货物的体内运动，并确定运动是否与模型的预测一致。在第二部分中，我们重点关注由 klar 蛋白控制的参与电机的数量及其产生的力的调节。我们使用各种生物物理特征来确定 klar 突变的影响。特别是，我们使用光镊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确定的功能是如何产生的，并且因为我们已经证明 klar 调节力的产生（以及可能的参与电机的数量），所以该研究将直接探讨如何控制参与电机的数量，以及这种控制的功能含义。公共卫生相关性：双向传播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；许多重要的货物，如线粒体和内体，都是双向移动的。囊泡运输受损与神经元变性有关。最后，更好地了解运输可能有助于设计新的药物输送系统。