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运动蛋白为对分化至关重要的运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电机的功能受到影响，生存就会受到威胁因突变或衰老而受到损害，更好地了解运动正常光感受器中的蛋白质功能将有助于深入了解光感受器变性和视力丧失的机制。在这个我们检查细胞骨架马达驱动蛋白 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营业额，调查纤毛轴丝依赖的贡献运输到外段形成和维护，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，它是果蝇 ninaC 的脊椎动物同源物。我们的功能性战略对这些电机的分析需要评估妥协的影响它们在体外和体内的小鸡光感受器中的功能使用两种补充策略：(a) 瞬时敲除运动蛋白使用反义技术表达； (b) 逆转录病毒介导的过度表达显性负尾结构以使其失活内源性运动蛋白和饱和货物结合位点。驱动蛋白II 集中于脊椎动物的基体和轴丝光感受器。该电机已被证明需要运动鞭毛和感觉鞭毛的形态发生和维持，介导鞭毛内运输（IFT），即筏的运动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 颗粒肽并研究驱动蛋白 II 的可能作用和外节形态发生中轴丝依赖性运输使用小鸡光感受器测定系统进行维护。突变在人类肌球蛋白VIIA基因产生亚瑟氏综合症1B型，一种色素性视网膜炎伴有耳聋。肌球蛋白VIIA已被证明集中在连接纤毛的光感受器上，但它功能未知。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驱动与远端连接纤毛的轴丝相关的细丝。我们最近发现脊椎动物的视网膜选择性地表达果蝇肌球蛋白 (ninaC) 的同源物。由于飞行为空 ninaC 突变表现出光转导和光诱导改变光感受器变性，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雏鸡体外运动测定和功能分析感光系统。