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，对多种生物学现象（如分泌、神经元信号传导、内膜组织和有丝分裂）具有根本重要性。细胞内运输的驱动力是由结合在货物细胞器表面并沿沿着微管（MT）和肌动蛋白丝（AF）运动的分子马达提供的。这两种类型的运输之间的转换必须精确调节，以将细胞器运送到细胞质的特定区域。然而，这种调节的机制仍然是一个谜。在这里，我们建议回答的问题，使用非洲爪蟾黑色素细胞作为一个实验系统之间的转换调节MT和AF。这些细胞在细胞质中迅速重新分布数千个膜结合的色素颗粒，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的分子马达分散在整个细胞质中。色素颗粒在MT和AF之间的转换受第二信使cAMP水平的控制。主要研究人员的背景数据表明，两种类型的轨道之间的切换是基于运输系统之间的持续拔河。拔河的结果是由MT为基础的和AF为基础的分子马达同时绑定到同一色素颗粒的相对活动决定。初步数据还表明，除了分子马达，细胞器运输和MT和AF轨道之间的切换涉及额外的蛋白质，调节对接的细胞器的目的地轨道。该提案将使用分子，细胞和生物化学的方法来测试的假设，即聚集和分散信号调节细胞器对接和颗粒结合的分子马达的活动，通过相互关联的机制，产生不同的动力学转移色素颗粒之间的MT和AF在聚集和分散。为了研究这些调节机制，将确定对接分子，并阐明它们与运输轨道和色素颗粒结合的调节。还将确定分子马达亚基磷酸化在调节其活动中的作用。 公共卫生相关性：这个建议的目标是了解膜细胞器如何在两种类型的细胞骨架运输轨道，微管和肌动蛋白丝之间切换。这种转换是细胞内转运过程的关键部分，这对正常细胞功能至关重要。细胞内转运的缺陷是许多人类疾病的原因，并且我们对这些缺陷的分子基础的理解为诸如癌症、糖尿病/沃尔科特-雷利森综合征和包括阿尔茨海默病和亨廷顿病的神经变性疾病的未来有效治疗铺平了道路。

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. PUBLIC HEALTH RELEVANCE: The goal of this proposal is an understanding of how membrane organelles switch between the two types of cytoskeletal transport tracks, microtubules and actin filaments. Such switching is a critical part of the intracellular transport process, which is essential for normal cell function. Defects in intracellular transport are responsible for many human diseases and our understanding of the molecular basis of these defects is paving the way to future effective therapeutics of diseases such as cancer, diabetes/Wolcott- rallison syndrome, and neurodegenerative disorders including Alzheimer and Huntington diseases.