Regulation of intracellular transport

描述（由申请人提供）：细胞内转运在动物细胞中普遍存在，对多种生物现象，如分泌、神经元信号、内膜组织和有丝分裂等具有重要意义。胞内运输的驱动力是由结合在货物细胞器表面并沿着微管和肌动蛋白丝运动的分子马达提供的。为了将细胞器运送到细胞质的特定区域，必须精确调节这两种运输方式之间的切换。然而，这种调控的机制仍然是个谜。在此，我们提出以非洲爪蟾为实验系统来回答关于mtts和AFs之间转换的调控问题。这些细胞在细胞质中迅速重新分配成千上万的膜结合色素颗粒，这些色素颗粒聚集在细胞中心或通过使用MT和AF轨道的色素颗粒结合分子马达分散在细胞质中。色素颗粒在MTs和AFs之间的切换是由第二信使cAMP的水平控制的。首席研究员的背景数据表明，在两种轨道之间的切换是基于运输系统之间的持续拉锯战。拔河的结果取决于同时结合在同一色素颗粒上的mt基分子马达和af基分子马达的相对活性。初步数据还表明，除了分子马达外，细胞器运输及其在MT和AF通道之间的切换还涉及调节细胞器与目的地通道对接的其他蛋白质。本提案将使用分子、细胞和生化方法来验证聚集和分散信号通过相互关联的机制调节细胞器对接和颗粒结合分子马达的活动的假设，这些机制在聚集和分散过程中在mtts和AFs之间产生不同的色素颗粒转移动力学。为了研究这些调节机制，将鉴定对接分子，并阐明它们与运输轨道和色素颗粒结合的调节。分子马达亚基的磷酸化在调节其活动中的作用也将被确定。

DESCRIPTION (provided by applicant): Intracellular transport is ubiquitous in animal cells and has fundamental importance for diverse biological phenomena, such as secretion, neuronal signaling, organization of endomembranes, and mitosis. The driving force for intracellular transport is provided by molecular motors bound to the surface of cargo organelles and moving along microtubules (MTs) and actin filaments (AFs). Switching between these two types of transport must be precisely regulated for the delivery of organelles to specific regions of the cytoplasm. However, the mechanisms of such regulation remain a mystery. Here, we propose to answer the question about the regulation of switching between MTs and AFs using Xenopus melanophores as an experimental system. These cells rapidly redistribute in the cytoplasm thousands of membrane-bounded pigment granules, which aggregate in the cell center or disperse throughout the cytoplasm by means of pigment granule-bound molecular motors that use both MT and AF tracks. Switching of pigment granules between MTs and AFs is controlled by the levels of the second messenger cAMP. Background data by the principal investigator suggest that switching between the two types of tracks is based on a continuous tug-of-war between transport systems. The outcome of the tug-of-war is decided by the relative activities of MT-based and AF-based molecular motors simultaneously bound to the same pigment granule. Preliminary data also indicate that besides molecular motors, organelle transport and its switching between MT and AF tracks involves additional proteins that regulate docking of organelles to the destination track. This proposal will use molecular, cellular, and biochemical approaches to test the hypothesis that aggregation and dispersion signals regulate organelle docking and activities of granule-bound molecular motors through interconnected mechanisms that generate distinct kinetics of transferring pigment granules between MTs and AFs during aggregation and dispersion. To examine these regulatory mechanisms, docking molecules will be identified, and the regulation of their binding to transport tracks and pigment granules will be elucidated. The role of phosphorylation of subunits of molecular motors in regulation of their activities will be also determined.