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监管机制。运动驱动的微管运输在细胞中扮演着许多重要的角色，然而细胞控制其特异性、时机和目的地的机制仍然是个谜。该提案的重点是双向运输的调节规则，双向运输是一种细胞内运输，在这种运输中，运动合作导致新的紧急现象，而不是通过孤立地研究单个电机来理解的。一个独特的模型系统，果蝇胚胎中的脂滴运动，提供了一个结合遗传学、生物化学、分子生物学和生物化学的实验技术的跨学科方法来解决细胞生物学中的这个长期存在的问题的机会。该提案侧重于液滴运输的第二阶段，在此阶段，Halo蛋白质充当方向性决定因素：在没有Halo的情况下，运输是负端定向的；在Halo存在的情况下，它是正端定向的。提出这些问题的目的是为了揭示Halo信号最终是如何传递到正端运动蛋白-1和负端运动细胞质动力蛋白的变化活动中的。提出了两种互补的方法，一方面侧重于信号Halo，另一方面侧重于SFO，这是电机机械的一个物理连接到电机的部件。1)为了了解Halo作为信号的作用，将确定Halo的蛋白质伙伴并进行Halo的结构-功能分析。对于选定的光晕突变体，物理运动参数将被量化，使用纳米尺度的跟踪和光学镊子的失速力测量。2)SFO被认为是一种“导体”，一种可以微调货物在一次不间断运动中行驶的距离的分子。实验被用来表征SFO的发育表达和定位，确定是否需要SFO来转导Halo信号的任何方面，并确定在体内重要的SFO结构域。这些实验将特别严格地检验SFO是否真的起到了导体的作用。如果成功，这些研究将提供一个框架，说明运输机械如何传递处理信号，以影响特定货物上的电机特性。因为Halo是果蝇整个蛋白质家族的创始成员，因为SFO从真菌到哺乳动物都是保守的，而且这两个分子在果蝇发育过程中都有表达，这些研究有望揭示许多不同的运输过程，无论是在果蝇还是其他真核生物中。 公共卫生相关性：这项拟议的工作对人类健康具有广泛的意义，因为运动功能异常与从阿尔茨海默氏症到癌症等各种疾病有关。这些研究还将阐明脂滴的基本生物学，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.