Genetic Analysis of Retinal Cone Photoreceptor Function

描述(申请人提供)：光感受器是高度极化的、分隔的细胞。蛋白质合成始于内段，然后沿顶端方向向外段或基本向突触进行运输。近年来，几位研究人员在转运到外节的机制(S)方面取得了开创性的发现。相比之下，人们对去往突触的蛋白质的运输或去往不同细胞隔间的蛋白质内段的分类知之甚少。磷脂酰肌醇是膜运输的关键调节因子，在多种细胞类型中参与信号传递、规范和蛋白质募集。细胞磷脂酰肌醇的关键调节因子是脂质磷酸酶突触素I(Synaptojanin I，SynJ1)，它的主要细胞内靶标是PI(4，5)P2。我们已经建立了斑马鱼作为一个模型系统，在其中评估磷脂酰肌醇信号和SynJ1在锥体光感受器蛋白质分选过程中的功能。我们目前的工作发现SynJ1在内部细分市场中扮演了一个角色。我们发现，SynJ1集中在锥体内段，在缺乏该蛋白的NRC突变体中，异常积累大小泡和/或高尔基结构被破坏。我们假设，内段表型反映了去往突触的蛋白质的运输和分类的中断。我们提出了一系列实验来验证这一假设，并准确定义了我们在NRC内节中检测到的异常泡状结构、它们的内容和派生。我们建议的特定预期结果是详细了解当关键的PI(4，5)P2磷酸盐SynJ1的丢失导致光感受器内多磷肌醇平衡被破坏时，在NRC中检测到的内段缺陷。此外，我们的研究将提供关于多磷肌醇在野生型和NRC视锥感光器中的细胞分布的基本信息。最后，我们的研究将确定SynJ1不同结构域的重要性。我们从本提案中概述的研究中发现的关键的、基本的信息将有助于打开这一领域的大门，进行许多其他重要的调查。 公共卫生相关性：适当的磷脂酰肌醇稳态对于正常的光感受器功能是必不可少的。视锥感光细胞中关键的PI(4，5)P2磷酸酶突触素1的缺失导致视锥感光细胞内节段和突触小泡运输缺陷。我们提出的研究结果将产生新的和基本的信息，将加深我们对磷脂酰肌醇在视锥细胞中的信号的理解。

DESCRIPTION (provided by applicant): 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 zebra fish 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 phosphates, 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. PUBLIC HEALTH RELEVANCE: Proper phosphoinositide homeostasis is essential for normal photoreceptor function. Loss of the critical PI(4,5)P2 phosphatase synaptojanin 1 causes dramatic inner segment and synaptic vesicle transporting defects in cone photoreceptors. The findings from our proposed studies will yield novel and fundamental information that will deepen our understanding of phosphoinositide signaling in cones.