REGULATION OF MYOSIN DRIVEN ORGANELLE TRANSPORT

细胞器和大分子复合物通过细胞质的运输是每一个真核细胞生存所必需的。这个过程是由运动蛋白完成的，运动蛋白沿着细胞骨架丝、微管和肌动蛋白丝移动它们的货物。细胞器运输的时空控制对细胞分裂、分泌和胚胎发育等过程至关重要。我们的长期目标是了解调节细胞器运输在细胞质中的分子机制。本实验室在前期工作中建立了一种来自非洲爪蟾（Xenopus laevis）的永久性色素细胞（黑色素细胞）细胞系，作为在分子水平上研究细胞器运动调控的模型系统。在cAMP浓度高的情况下，黑色素细胞将色素细胞器从细胞中心移动到周围，而在cAMP浓度低的情况下，色素聚集到细胞中心。在这个系统中，细胞器的运动需要两个细胞骨架系统的协调活动：微管/微管马达和肌动蛋白丝/肌凝蛋白。我们在这些细胞中发现了一种与色素细胞器结合的肌凝蛋白，称为肌凝蛋白V，这是一类已知参与其他系统中细胞器运输的肌凝蛋白。本研究旨在分析myosin V在非洲爪蟾细胞器运动中的作用，并研究其活性的调控机制。我们将从非洲爪蟾获得一个编码肌凝蛋白V的cDNA克隆，并将使用显性负突变和抑制抗体来检测肌凝蛋白V在黑色素细胞中沿肌动蛋白丝运动中的作用。为了研究肌凝蛋白V是如何调控细胞器运输的，我们将分析该马达在色素聚集和分散过程中的磷酸化。磷酸化位点将被定位并通过位点定向诱变改变。编码这些突变蛋白的DNA将被转染到黑色素细胞中，突变对细胞聚集和分散色素能力的影响将被检验。使用类似的技术，我们还将研究在细胞分裂过程中肌凝蛋白V的活性是如何被调节的。此外，我们将识别和表征参与肌球蛋白V结合的细胞器表面的蛋白质。我们将把我们的发现扩展到其他细胞类型，如神经元，以了解控制肌动蛋白和肌球蛋白依赖的细胞器运输的一般机制以及肌球蛋白V在神经细胞中的作用。

Transport of organelles and macromolecular complexes through the cytoplasm is essential for survival of every eukaryotic cell. This process is performed by motor proteins that move their cargo along the cytoskeletal filaments, microtubules and actin filaments. Spatial and temporal control of organelle transport is critical for such processes as cell division, secretion and embryonic development. Our long term goal is to understand molecular mechanisms regulating organelle transport in the cytoplasm. Previous work by this laboratory established a permanent cell line of pigment cells (melanophores) from the frog Xenopus laevis as a model system to study the regulation of organelle movement at the molecular level. Melanophores move pigment organelles from the cell center to the periphery in response to high concentrations of cAMP, while at low cAMP levels pigment aggregates to the cell center. Movement of organelles in this system requires the coordinated activity of two cytoskeletal systems: microtubules/microtubule motors and actin filaments/myosin. We identified a myosin bound to pigment organelles in these cells as myosin V, a class of myosins known to be involved in organelle transport in other systems. The goal of this proposal is to analyze the role of myosin V in organelle movement in Xenopus melanophores and to study the mechanisms regulating its activity. We will obtain a cDNA clone encoding myosin V from Xenopus and will use dominant negative mutations and inhibitory antibodies to examine the role of myosin V in the movement of pigment along actin filaments in melanophores. In order to study how organelle transport by myosin V is regulated, we will analyze phosphorylation of this motor during pigment aggregation and dispersion. Phosphorylation sites will be mapped and changed by site-directed mutagenesis. DNA encoding these mutant proteins will be transfected into melanophores and the effects of the mutations on the ability of cells to aggregate and disperse pigment will be examined. Using similar techniques we will also study how activity of myosin V is regulated during cell division. In addition, we will identify and characterize proteins on the surface of organelles that are involved in myosin V binding. We will extend our findings to other cell types, such as neurons, in order to understand general mechanisms controlling actin and myosin-dependent organelle transport and the role of myosin V in nerve cells.