MOLECULAR MOTORS IN RETINAL CELL MOTILITY

我们研究的最终目标是了解细胞骨架 马达蛋白为分化关键的能动过程提供动力 和脊椎动物感光器的功能。由于光感受器 如果特定的马达的功能被破坏， 受突变或老化的影响，更好地了解运动 正常光感受器中的蛋白质功能将提供对 光感受器退化和视力丧失的机制。 在这 应用程序，我们研究的作用，细胞骨架马达驱动蛋白II 和肌球蛋白VIIA在感光细胞形态发生和外节中的作用 周转，研究睫状体轴突依赖性的贡献 运输到外部段的形成和维护，以及进一步 描述了我们发现的一种新的肌球蛋白，肌球蛋白III， 果蝇的脊椎动物同源物 我们的功能性战略 对这些发动机的分析需要评估损害的影响， 它们在体外和体内鸡光感受器中的功能， 互补策略：（a）瞬时敲除马达蛋白 （B）逆转录病毒介导的 显性负尾构建体的过表达 内源性马达蛋白和饱和货物结合位点。 驱动蛋白II 主要集中在脊椎动物的基体和轴丝上 光感受器 该电动机已被证明是必要的， 运动和感觉鞭毛的形态发生和维持， 介导鞭毛内运输（IFT）， 轴丝和质膜之间的多肽颗粒。 我们 建议使用简并引物PCR来寻找脊椎动物的同源物， IFT颗粒肽和研究驱动蛋白II的可能作用 和外节形态发生中的轴丝依赖性运输， 使用小鸡光感受器测定系统进行维持。突变 人肌球蛋白VIIA基因产生Usher综合征1B型，一种 视网膜色素变性伴耳聋。肌球蛋白VIIA已被证明 集中在连接纤毛的感光细胞上， 功能未知。我们提出检验圆盘 形态发生是由肌球蛋白VIIA与肌动蛋白的相互作用提供动力的 在末端连接纤毛处与轴丝相连的细丝。 我们最近发现，脊椎动物视网膜选择性地表达一种 果蝇肌球蛋白（ninaC）的同系物。 因为带null的苍蝇 ninaC的突变表现出改变的光转导和光诱导 光感受器退化，我们感兴趣的是确定 脊椎动物肌球蛋白III的性质和功能。 为此我们 将使用异源表达进行生物化学表征， 鸡的体外运动试验和功能分析 感光系统

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.