REGULATION OF MYOSIN DRIVEN ORGANELLE TRANSPORT

细胞器和大分子复合物通过细胞质的运输对于每个真核细胞的生存是必不可少的。这一过程是由马达蛋白完成的，马达蛋白沿着细胞骨架丝、微管和肌动蛋白丝移动它们的货物。 细胞器运输的空间和时间控制对于细胞分裂、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在细胞质中运输的分子机制。 该实验室以前的工作建立了一个永久的色素细胞（黑色素细胞）的细胞系，从青蛙非洲爪蟾作为模型系统，研究细胞器运动的调节在分子水平上。 黑色素细胞响应于高浓度的cAMP将色素细胞器从细胞中心移动到外周，而在低cAMP水平下色素聚集到细胞中心。 细胞器在该系统中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/肌球蛋白。 我们将这些细胞中与色素细胞器结合的肌球蛋白鉴定为肌球蛋白V，这是一类已知参与其他系统中细胞器运输的肌球蛋白。 本研究的目的是分析肌球蛋白V在非洲爪蟾细胞器运动中的作用，并研究其活性调节机制。 我们将从非洲爪蟾获得编码肌球蛋白V的cDNA克隆，并将使用显性负突变和抑制性抗体来研究肌球蛋白V在黑色素细胞中色素沿着肌动蛋白丝运动中的作用。 为了研究肌球蛋白V如何调节细胞器运输，我们将分析在色素聚集和分散过程中该马达的磷酸化。 将通过定点诱变对磷酸化位点进行定位和改变。 将编码这些突变蛋白的DNA转染到黑色素细胞中，并检查突变对细胞聚集和分散色素的能力的影响。 使用类似的技术，我们也将研究肌球蛋白V的活性是如何在细胞分裂过程中进行调节。 此外，我们将确定和表征蛋白质的细胞器的表面参与肌球蛋白V的结合。 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.