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展相结合的调节机制。沿着微管的马达驱动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、时间和目的地的机制仍然是一个谜。双向运输是细胞内运输的一种，在这种运输中，运动合作导致了新的涌现现象，通过孤立地研究单个运动是不容易理解的。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，来解决这个长期存在的问题。该建议侧重于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；在光晕的存在下，它是正向的。所提出的问题旨在揭示Halo信号如何最终传递到正端运动动力蛋白-1和负端运动细胞质动力蛋白的活性变化。提出了两种互补的方法，一方面关注信号Halo，另一方面关注与电机物理连接的电机机械组件Sfo。1)为了了解Halo作为信号的作用机制，我们将鉴定Halo的蛋白伙伴，并对Halo进行结构-功能分析。对于选定的Halo突变体，将使用纳米级跟踪和光学镊子的失速力测量来量化物理运动参数。Sfo被认为是一种“导体”，一种微调货物在一次不间断运动中行进距离的分子。我们提出了实验来表征Sfo的发育表达和定位，以确定Sfo是否需要转导Halo信号的任何方面，并确定体内重要的Sfo结构域。这些实验将特别严格地测试Sfo是否确实起到导体的作用。如果成功，这些研究将提供一个框架，说明运输机械如何传导交易信号，从而影响特定货物上电机的性能。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，因为这两种分子在果蝇的发育过程中都有表达，这些研究有望揭示果蝇和其他真核生物中许多不同的运输过程。

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.