MOLECULAR MOTORS IN RETINAL CELL MOTILITY

视网膜细胞运动中的分子马达

基本信息

批准号：
6125186
负责人：
MARY BETH BURNSIDE
金额：
$ 31.53万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-12-01 至 2003-11-30
项目状态：
已结题

项目摘要

The ultimate goal of our research is to understand how cytoskeletal motor proteins power motile processes critical to the differentiation and function of the vertebrate photoreceptor. Since photoreceptor survival is jeopardized if the function of specific motors is compromised by mutation or aging, a better understanding of motor protein function in normal photoreceptors will provide insight into mechanisms of photoreceptor degeneration and vision loss. In this application we examine the roles of the cytoskeletal motors kinesin II and myosin VIIA in photoreceptor morphogenesis and outer segment turnover, investigate the contribution of ciliary axoneme-dependent transport to outer segment formation and maintenance, and further characterize a new myosin we have discovered, myosin III, which is a vertebrate homologue of Drosophila ninaC. Our strategy for functional analysis of these motors entails assessing the effects of compromising their function in chick photoreceptors in vitro and in vivo using two complementary strategies: (a) transient knockout of motor protein expression using antisense technology; and (b) retroviral-mediated overexpression of dominant negative tail constructs to inactivate endogenous motor proteins and saturate cargo binding sites. Kinesin II is concentrated at the basal body and axoneme of vertebrate photoreceptors. This motor has been shown to be required for morphogenesis and maintenance of motile and sensory flagella, where it mediates intraflagellar transport (IFT), the movement of rafts of multipeptide particles between the axoneme and plasma membrane. We propose to use degenerate primer PCR to seek vertebrate homologues of IFT particle peptides and investigate the possible roles of kinesin II and axoneme-dependent transport in outer segment morphogenesis and maintenance using the chick photoreceptor assay system. Mutations in the human myosin VIIA gene produce Usher's Syndrome type 1B, a form of retinitis pigmentosa accompanied by deafness. Myosin VIIA has been shown to be concentrated at the photoreceptor connecting cilium but its function is not known. We propose to test the hypothesis that disk morphogenesis is powered by the interaction of myosin VIIA with actin filaments associated with the axoneme at the distal connecting cilium. We have recently found that vertebrate retinas selectively express a homologue of the Drosophila myosin (ninaC). Since flies with null mutations for ninaC exhibit altered phototransduction and light-induced photoreceptor degeneration, we are interested in determining the properties and functions of the vertebrate myosin III. To this end we will use heterologous expression for biochemical characterization and in vitro motility assays, and functional analysis in the chick photoreceptor system.

我们研究的最终目标是了解细胞骨架如何电机蛋白动力运动过程对分化至关重要脊椎动物光感受器的功能。由于感光器如果特定电机的功能是因突变或衰老而妥协，对电机有更好的理解正常感光器中的蛋白质功能将提供有关光感受器变性和视力丧失的机制。在这个应用我们检查了细胞骨架电动机动力蛋白II的作用和肌球蛋白VIIA在光感受器形态发生和外部段营业额，研究纤毛轴突依赖性的贡献运输到外部细分市场的形成和维护，然后进一步描述了我们发现的新肌球蛋白，肌球蛋白III，这是一个果蝇Ninac的脊椎动物同源物。我们的功能策略对这些电动机的分析需要评估妥协的影响它们在体外和体内使用两个互补策略：（a）运动蛋白的短暂敲除使用反义技术表达；（b）逆转录病毒介导灭活的主要负尾巴结构的过表达内源性运动蛋白和饱和货物结合位点。动力素II 集中在脊椎动物的基体和轴突上感受器。已证明该电动机是必需的运动和感觉鞭毛的形态发生和维护，介导flagellar运输（IFT），筏的运动轴突和质膜之间的多肽颗粒。我们建议使用退化底漆PCR寻求脊椎动物的同源物 IFT颗粒肽并研究驱动蛋白II的可能作用以及外部段形态发生的轴突依赖性转运和使用雏鸡光感受器测定系统进行维护。突变人肌球蛋白VIIA基因产生usher综合征1B，一种形式色素性视网膜炎伴有耳聋。肌球蛋白VIIA已显示要集中在连接纤毛的感光器上，但功能尚不清楚。我们建议检验磁盘的假设形态发生由肌球蛋白VIIA与肌动蛋白的相互作用提供动力与远端连接纤毛的轴突相关的细丝。我们最近发现脊椎动物视网膜有选择地表达果蝇肌球蛋白（NINAC）的同源物。因为苍蝇与空飞 NINAC的突变表现出改变的光转导和光诱导的变化光感受器变性，我们有兴趣确定脊椎动物肌球蛋白III的特性和功能。为此，我们将使用异源表达进行生化表征和体外运动测定和雏鸡的功能分析感光器系统。