Regulation of Microtubule-Dependent Motility

9513388 Gelfand这项研究的最终目标是了解细胞器沿微管的运输是如何被调节的。为了回答这个问题，将使用克隆的非洲爪哇黑素载体永生化细胞系。这些细胞的独特性质使它们对拟议的研究特别有吸引力。首先，与大多数其他系统不同的是，人们对触发细胞器运动的生理刺激知之甚少，而被称为黑素小体的数千个色素颗粒的运动可以很容易地通过在培养基中添加适当的激素来操纵。其次，黑素小体蛋白约占细胞总蛋白的10%。大量培养细胞的可获得性和一种简单的黑素小体纯化方案的开发使其成为纯化和表征参与运动的成分以及体外重建微管调节运动的首选系统。第三，在色素聚集和在黑素载体中分散的过程中，特定蛋白质组的磷酸化已被记录在案。第四，非洲爪哇黑素载体可以有效地导入外源DNA。因此，使用编码作为磷酸化目标的蛋白质的克隆基因，可以分析磷酸化在转基因细胞中的功能作用。这项拟议的研究将确定驱动黑素细胞和无细胞系统中细胞器顺行和逆行运动的马达蛋白。与这些运输过程的极性转换相关的马达或其他细胞器蛋白质的修饰将被识别，正在进行修饰的蛋白质的基因将被克隆和测序。修饰的功能作用将通过定点突变和对修饰蛋白的磷酸化状态和活性的分析来确定。这个项目的潜在意义远远超出了黑素小体运动的具体问题，因为类似的控制机制可能在有丝分裂、分泌、轴浆运输以及纤毛和鞭毛的跳动中发挥作用。因此，了解黑素小体运动的调节将显著增强我们对微管工作的了解，并将与细胞功能和功能障碍的基本方面的广泛研究相关。***

9513388 Gelfand The ultimate goal of this study is to understand how organelle transport along microtubules is regulated. To answer this question, a cloned immortalized cell line of Xenopus melanophores will be used. The unique properties of these cells make them especially attractive for the proposed study. First, unlike most other systems, where physiological stimuli triggering the movement of organelles are poorly understood, movement of thousands of pigment granules called melanosomes can easily be manipulated by the addition of the appropriate hormones to the culture medium. Second, melanosome proteins represent about 10% of the total cellular protein. Availability of a large number of cells grown in culture and the development of a simple protocol for melanosome purification makes it the system of choice for purification and characterization of the components involved in the motility and for the reconstitution of regulated motility along microtubules in vitro. Third, phosphorylation of specific sets of proteins during pigment aggregation and dispersion in melanophores has been documented. Fourth, Xenopus melanophores can be effectively transfected with exogenous DNA. Thus, using cloned genes that encode proteins that are targets for phosphorylation, the function role of phosphorylation can be analyzed in transgenic cells. The proposed studies will identify motor proteins that drive anterograde and retrograde movement of organelles in melanophores and in a cell-free system. Modifications of the motors or other organelle proteins that are associated with the switch in the polarity of these transport processes will be identified and the genes for proteins undergoing modification will be cloned and sequenced. The functional role of the modifications will be determined by site-directed mutagenesis and the analysis of the phosphorylation state and activities of the modified proteins in vitro and in vivo. The potential significance of this project extends well beyond the speci fic question of melanosome movement, because similar control mechanisms are likely to operate during mitosis, secretion, axoplasmic transport and the beating of cilia and flagella. Thus, understanding the regulation of melanosome movement will significantly enhance our knowledge of microtubules work, and will be relevant to a broad range of studies of fundamental aspects of cellular function and malfunction. ***