Developmental control of organelle transport

描述（由申请人提供）：本提案的长期目标是了解允许细胞器运输与发育相结合的调节机制。马达驱动的运输沿着微管发挥许多重要的细胞作用，但细胞控制其特异性，时间和目的地的机制仍然是一个谜。该提案的重点是双向运输的调节规则，这是一种细胞内运输，其中电机合作导致新的紧急现象，通过孤立地研究单个电机不容易理解。一个独特的模型系统，果蝇胚胎中的脂滴运动，提供了一个机会，攻击这个长期存在的问题，在细胞生物学与跨学科的方法，结合遗传学，生物化学，分子生物学和生物化学的实验技术。该提案的重点是液滴运输的第二阶段，其中Halo蛋白作为方向性决定因素：在没有Halo的情况下，运输是负端定向的;在Halo的存在下，它是正端定向的。所问的问题旨在揭示Halo信号如何最终传递到正端运动驱动蛋白-1和负端运动细胞质动力蛋白的活性变化中。提出了两种互补的方法，一方面集中在信号光环，另一方面在SFO，一个组件的电机机械物理连接到电机。1)为了了解Halo如何作为信号，将鉴定Halo的蛋白质伴侣，并对Halo进行结构-功能分析。对于选定的Halo突变体，物理运动参数将被量化，使用纳米级跟踪和失速力测量与光学镊子。2)Sfo被认为是一种“导体”，一种可以精确调节货物在一次不间断运动中的运动距离的分子。实验提出了表征的发育表达和本地化的SFO，以确定是否需要SFO的Halo信号的任何方面，并确定SFO结构域在体内的重要。这些实验将特别严格地检验Sfo是否确实起导体的作用。如果成功的话，这些研究将为运输机械如何转导交易信号以影响特定货物上的电机特性提供一个框架。由于Halo是果蝇中整个蛋白质家族的创始成员，由于Sfo从真菌到哺乳动物都是保守的，并且由于这两种分子在整个果蝇发育过程中都有表达，因此这些研究有望阐明苍蝇和其他真核生物中许多不同的运输过程。 公共卫生相关性：这项工作对人类健康具有广泛的意义，因为异常的运动功能与从阿尔茨海默氏症到癌症的疾病有关。这些研究还将阐明脂滴的基础生物学，脂滴是肥胖和心血管疾病中重要的细胞器。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to understand the regulatory mechanisms that allow organelle transport to be integrated with development. Motor-driven transport along microtubules plays many essential cellular roles, yet the mechanisms with which cells control its specificity, timing and destination remain a mystery. The proposal focuses on the rules of regulation for bidirectional transport, a kind of intracellular transport in which motor cooperation results in novel emergent phenomena not easily understood by studying the individual motors in isolation. A unique model system, lipid-droplet motion in Drosophila embryos, provides the opportunity to attack this long-standing problem in cell biology with an interdisciplinary approach that combines experimental techniques from genetics, biochemistry, molecular biology, and biochemistry. The proposal focuses on Phase II of droplet transport in which the Halo protein acts as a directionality determinant: in the absence of Halo, transport is minus-end directed; in the presence of Halo, it is plus-end directed. The questions asked are designed to unravel how the Halo signal is ultimately transmitted into changed activity of the plus-end motor kinesin-1 and the minus-end motor cytoplasmic dynein. Two complementary approaches are proposed, focusing on the one hand on the signal Halo and on the other hand on Sfo, a component of the motor machinery physically connected to the motors. 1) To understand how Halo acts as a signal, protein partners of Halo will be identified and a structure-function analysis of Halo will be performed. For selected Halo mutants, physical motion parameters will be quantified, using nanometer-scale tracking and stall-force measurements with optical tweezers. 2) Sfo has been proposed to be a "conductor", a molecule that fine-tunes how far cargoes travel in a single uninterrupted motion. Experiments are proposed to characterize the developmental expression and localization of Sfo, to determine if Sfo is required to transduce any aspects of the Halo signal, and to identify Sfo domains important in vivo. These experiments will in particular critically test whether Sfo indeed acts as a conductor. If successful, these studies will provide a framework for how a transacting signal is transduced by the transport machinery to affect properties of motors on a specific cargo. Because Halo is the founding member of a whole protein family in Drosophila, because Sfo is conserved from fungi to mammals and because both molecules are expressed throughout Drosophila development, these studies are expected to shed light on many different transport processes, both in flies and other eukaryotes. PUBLIC HEALTH RELEVANCE: The proposed work has broad significance for human health since aberrant motor function is linked to diseases ranging from Alzheimer's to cancer. These studies will also illuminate the basic biology of lipid droplets, organelles important in obesity and cardiovascular disease.