Regulation of Microtubule-Dependent Motility

小行星9513388 本研究的最终目的是了解细胞器运输沿着微管的调节。 为了回答这个问题，将使用克隆的黑色素爪蟾永生化细胞系。 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，在这些系统中，人们对触发细胞器运动的生理刺激知之甚少，通过向培养基中添加适当的激素，可以很容易地操纵数千个称为黑素体的色素颗粒的运动。 第二，黑素体蛋白占总细胞蛋白的约10%。 大量的细胞生长在培养基中的可用性和一个简单的协议黑素体纯化的发展，使其成为系统的选择纯化和参与的运动性和重建的组件在体外调控的运动性沿着微管。 第三，在黑色素细胞中色素聚集和分散过程中特定蛋白质组的磷酸化已被记录。 第四，外源DNA可以有效地转染非洲爪蟾黑色素细胞。 因此，使用编码磷酸化靶蛋白的克隆基因，可以在转基因细胞中分析磷酸化的功能作用。 拟议的研究将确定马达蛋白，驱动顺行和逆行运动的细胞器在黑色素细胞和无细胞系统。 将鉴定与这些运输过程极性转换相关的马达或其他细胞器蛋白的修饰，并克隆和测序进行修饰的蛋白质的基因。 修饰的功能作用将通过定点诱变和体外和体内修饰蛋白的磷酸化状态和活性的分析来确定。 该项目的潜在意义远远超出了黑素体运动的具体问题，因为类似的控制机制可能在有丝分裂，分泌，轴质运输以及纤毛和鞭毛的跳动中起作用。 因此，了解黑素体运动的调节将大大提高我们对微管工作的认识，并将与细胞功能和故障的基本方面的广泛研究有关。 ***

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. ***