In vivo regulation of bi-directional transport

描述（由申请人提供）：最近的生物物理研究在阐明体外单分子马达的功能方面取得了很大进展。然而，体内的分子功能远比这些体外研究预测的复杂：与体外观察到的单向运动相反，许多囊泡或其他货物沿沿着微管双向运动，经常逆转其行进方向。尽管如此，运输还是可以被控制的，这样货物就能平均地运到正确的地方。要了解这种运输是如何被调节的，需要开发新的工具，能够在体内以高的时间和空间分辨率量化单个货物的运动。该研究开发了这样的生物物理工具，并将其与遗传学和生物化学相结合，以研究体内马达或马达复合体的功能。 具体而言，双向运动的脂滴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。第一个是突变效应的生物物理表征，使用光镊和粒子跟踪和分析来确定蛋白质在运输途径中的具体物理作用。一个目标是测试假设存在具有某些功能的复合物的理论框架，并将每个蛋白质置于该框架内。第二种方法采用生物化学技术来鉴定参与转运调节的其他蛋白质，并确定不同蛋白质之间的相关相互作用。这些信息将在分子水平上阐明生物药理学决定的功能是如何产生的，并将直接研究假设的复合物。将使用生物物理测定直接研究在生物化学方法中鉴定的蛋白质的功能。 双向运输与公共卫生直接相关：疱疹等病毒以双向方式在细胞中传播;许多重要的货物如线粒体和内体双向移动。此外，在这里研究的一些基因突变，如Lisi，导致人类出生缺陷。最后，更好地了解运输可能允许设计新的药物输送系统。

DESCRIPTION (provided by applicant): Recent biophysical studies have made great progress in clarifying the function of single molecular motors in vitro. However, in vivo molecular function is far more complicated than these in vitro studies predict: contrary to the uni-directional motion observed in vitro, many vesicles or other cargos move bidirectionally along microtubules, frequently reversing their direction of travel. Nonetheless, transport can be regulated, so that the cargo moves-on average-to the right place. To understand how such transport is regulated requires the development of new tools capable of quantifying-in vivo-the motion of individual cargos with high temporal and spatial resolution. The research develops such biophysical tools, and combines them with genetics and biochemistry to investigate the function of motors or motor complexes in vivo. Specifically, bi-directional motion of lipid droplets in early Drosophila embryos is investigated. The work can be conceptually divided into two complimentary approaches. The first, the biophysical characterization of the effects of mutations, uses optical tweezers and particle tracking and analysis to determine the specific physical role of proteins in the transport pathway. One goal is to test a theoretical framework hypothesizing the existence of a complex with certain functions, and place each protein within this framework. The second approach employs biochemical techniques to identify additional proteins involved in the regulation of transport, and determine relevant interactions between the different proteins. This information will clarify at the molecular level how the biophysically determined functions come about, and will directly investigate the hypothesized complex. The function of the proteins identified in the biochemical approaches will be directly investigated using the biophysical assays. 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. Further, mutation in some of the genes investigated here, such as Lisi, lead to human birth defects. Finally, a better understanding of transport might allow the design of new drug delivery systems.