MOLECULAR MOTORS IN RETINAL CELL MOTILITY

我们研究的最终目标是了解细胞骨架是如何 马达蛋白能驱动对分化至关重要的运动过程 脊椎动物光感受器的功能。因为光感受器 如果特定运动的功能 因突变或衰老而受损，对运动有更好的理解 正常光感受器的蛋白质功能将为深入了解 光感受器退化和视力丧失的机制。在这 应用：我们研究细胞骨架马达激动素II的作用 和肌球蛋白VIIA在光感受器形态发生和外节中的作用 周转，调查睫状轴丝依赖的贡献 运输至外段形成和维护，并进一步 描述我们发现的一种新的肌球蛋白，肌球蛋白III，它是一种 果蝇的脊椎动物同源物。我们的职能部门战略 对这些发动机的分析需要评估危害的影响 它们在鸡体内外光感受器中的作用 互补策略：(A)瞬时敲除马达蛋白 使用反义技术表达；和(B)逆转录病毒介导 过度表达显性负尾结构以使其失活 内源性马达蛋白和饱和的货物结合部位。激动素II 主要集中在脊椎动物的基体部和轴丝 光感受器。这台马达已被证明是 运动和感觉鞭毛的形态发生和维持，其中它 介导鞭毛内转运(IFT)，即 轴丝和质膜之间的多肽颗粒。我们 建议用简并引物聚合酶链式反应寻找脊椎动物的同源物 IFT粒子肽和研究激动素II的可能作用 和依赖轴丝的运输在外段形态发生和 使用鸡光感受器检测系统进行维护。基因突变 人类肌球蛋白VIIA基因导致亚瑟综合征1B型，一种 视网膜色素变性伴有耳聋。肌球蛋白VIIA已被证明 集中在连接纤毛的光感受器，但其 功能未知。我们建议检验磁盘的假设 肌球蛋白VIIA与肌动蛋白相互作用推动形态发生 在远端连接的纤毛上与轴丝相关的花丝。 我们最近发现脊椎动物的视网膜选择性地表达一种 果蝇肌球蛋白的同源物(NINAC)。由于空值为空值 NINAC基因突变表现为光转导和光诱导的改变 光感受器退化，我们有兴趣确定 脊椎动物肌球蛋白的性质和功能。为此，我们 将使用异源表达进行生化鉴定和 雏鸡的体外动力测定和功能分析 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.