Genetic Analysis of Retinal Cone Photoreceptor Function

光感受器是高度极化的、区室化的细胞。蛋白质合成起始于内节 然后转运沿顶端方向朝向外节或沿基部方向朝向突触进行。在 近年来，一些研究人员已经取得了关于运输机制的开创性发现， 外段相比之下，很少有人知道蛋白质的运输注定突触或 在蛋白质的内部片段中进行分类，分配到不同的细胞区室。磷酸肌醇是 已知是膜运输中的关键调节因子，并参与信号传导、特化和蛋白质 在多种细胞类型中招募。细胞磷酸肌醇的一个关键调节因子是脂质 磷酸酶，synaptojanin I（SynJ 1），其主要细胞内靶点是PI（4，5）P2。我们建立 斑马鱼作为模型系统，在其中评估磷酸肌醇信号传导和SynJ 1功能， 视锥细胞感光器中蛋白质分类的过程。我们目前的工作发现了SynJ 1在内段中的作用。 我们发现SynJ 1集中在锥体内段，并且大囊泡异常累积和/或 在缺乏该蛋白质的nrc突变体中，高尔基体结构被破坏。我们假设内节 表型反映了预定用于突触的蛋白质的运输和分选的破坏。我们提出了一个 一系列的实验来验证这一假设，并精确定义我们检测到的异常囊泡结构 在NRC内部段中，它们的内容和派生。我们的建议的具体预期成果是一个详细的 当多聚磷酸肌醇平衡时， 由于关键的PI（4，5）P2磷酸酶SynJ 1的丧失，光感受器被破坏。另外我们 这些研究将提供关于多磷酸肌醇在两种细胞中的细胞分布的基本信息。 野生型和NRC锥体光感受器。最后，我们的研究将定义不同结构的重要性 SynJ 1的结构域。我们从本提案中概述的研究中发现的关键、基本信息 将有助于打开这一领域的许多其他重要的调查。

Photoreceptors are highly polarized, compartmentalized cells. Protein synthesis initiates in the inner segment and then transport proceeds in the apical direction toward the outer segment or basally toward the synapse. In recent years, several investigators have made seminal discoveries about the mechanism(s) of transport to the outer segment. In contrast, very little is known about the transport of proteins destined for the synapse or the sorting within the inner segment of proteins destined for different cellular compartments. Phosphoinositides are known to be key regulators in membrane trafficking and are involved in signaling, specification and protein recruitment in a wide variety of cell types. A key regulator of cellular phosphoinositides is the lipid phosphatase, synaptojanin I (SynJ1), whose primary intracellular target is PI(4,5)P2. We have established zebrafish as a model system in which to evaluate both phosphoinositide signaling and SynJ1 function in the process of protein sorting in cone photoreceptors. Our current work finds a role for SynJ1 in the inner segment. We find that SynJ1 concentrates in the cone inner segment and that large vesicles abnormally accrue and/or the Golgi architecture is disrupted in nrc mutants lacking this protein. We hypothesize that the inner segment phenotype reflects a disruption of transport and sorting of proteins destined for the synapse. We propose a series of experiments that test this hypothesis and define precisely the abnormal vesicular structures we detect in nrc inner segments, their content and derivation. The specific expected outcome of our proposal is a detailed understanding of the inner segment defect detected in nrc when the balance of polyphosphoinositides within the photoreceptor is disrupted due to the loss of the critical PI(4,5)P2 phosphatase, SynJ1. In addition, our studies will provide fundamental information about the cellular distribution of polyphosphoinositides in both wild-type and nrc cone photoreceptors. Finally, our studies will define the importance of different structural domains of SynJ1. The critical, fundamental information we discover from the studies outlined in this proposal will help open this field to many additional important investigations.