REGULATION OF MYOSIN DRIVEN ORGANELLE TRANSPORT

细胞器和大分子复合体通过细胞质的运输对每个真核细胞的生存都是必不可少的。这一过程是由马达蛋白执行的，马达蛋白沿着细胞骨架细丝、微管和肌动蛋白细丝移动货物。细胞器运输的时空调控对细胞分裂、分泌和胚胎发育等过程至关重要。我们的长期目标是了解细胞质中细胞器运输的分子机制。本实验室以前的工作建立了非洲爪蛙色素细胞(黑素细胞)的永久细胞系，作为在分子水平上研究细胞器运动调节的模型系统。当cAMP浓度较高时，黑素载体将色素细胞器从细胞中心移动到外围，而在cAMP水平较低时，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/肌球蛋白。我们鉴定了与这些细胞中的色素细胞器结合的肌球蛋白为肌球蛋白V，这是一类已知参与其他系统的细胞器运输的肌球蛋白。这项建议的目的是分析肌球蛋白V在非洲爪哇黑素细胞细胞器运动中的作用，并研究其活性的调节机制。我们将从非洲爪哇获得一个编码肌球蛋白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在黑素载体中色素沿肌动蛋白细丝移动中的作用。为了研究肌球蛋白V对细胞器运输的调控，我们将分析该马达在色素聚集和分散过程中的磷酸化。磷酸化位点将通过定点突变来定位和改变。编码这些突变蛋白的DNA将被导入黑素载体，并将检测突变对细胞聚集和分散色素能力的影响。使用类似的技术，我们还将研究肌球蛋白V的活性在细胞分裂过程中是如何调节的。此外，我们还将鉴定和鉴定细胞器表面与肌球蛋白V结合相关的蛋白质。我们将把我们的发现扩展到其他类型的细胞，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.