REGULATION AND FUNCTION OF RETINAL PHOSPHOINOSITIDES

目标是了解视网膜中磷酸肌醇合成、降解和定位的调节 和视网膜色素上皮 (RPE)，并了解磷酸肌醇在视网膜信号传导中的作用， 发展、健康和疾病。通过阐明膜运输等过程的分子细节， 自噬、吞噬作用、内吞作用和胞吐作用，这种理解可以帮助我们更好地理解如何 这些过程在视网膜疾病中被破坏，以及治疗干预如何利用它们 保护视力。具体目标是： 1. 确定流程中各个步骤之间的关系 光转导级联和 PI(3)P 的调节 PI(4)P 和杆中 PI(4,5)P2 的调节。光， 与一天中的时间无关，驱动内节磷酸肌醇水平大幅增加。 初步结果表明驱动这种增加的信号位于光转导级联的下游， 我们发表的结果证明了 III 类 PI-3 激酶 Vps34 的关键作用。目前尚不清楚 光如何导致 Vps34 活性上调或驱动 PI(4,5)P2 增加的因素。以下 将测试假设： A. 光激发视紫红质的转导蛋白激活对于光驱动的 增加。 B. PDE6 活性对于光驱动的增加至关重要。 C. 环核苷酸门控通道是 对于增加至关重要。 2.确定PI(4)P-5激酶活性在PIP2（磷脂酰肌醇- (4,5)二磷酸)在外视网膜中的调节以及PI(4,5)P2的功能作用。我们会 产生可诱导杆细胞或 RPE 细胞特异性敲除主要酶的小鼠 在神经元中合成 PI(4,5)P2、PIP-5-激酶γ，并确定表型 磷酸肌醇水平、细胞形态和存活、蛋白质运输、内吞作用、吞噬作用和 自噬。这些实验将检验以下假设： A. PI(4,5)P2 主要在棒中合成 和 RPE 通过 PIP-5-激酶γ 同种型的作用，使用 ATP 和 PI(4)P 作为底物； B、PI(4,5)P2 合成对于一系列膜运输功能和细胞活力至关重要。如果需要的话我们也会测试 α 和 β 亚型的全局敲除，这是可行的，以及双重和三重。 3. 确定 LC3 募集和溶酶体融合的独特磷酸肌醇调节机制 自噬和吞噬作用。我们发现 LC3 招募标准模型存在缺陷 自噬体和吞噬体，所以我们将体内实验和RPE细胞实验结合起来 细胞系以确定哪些蛋白质对于 RPE 细胞中 LC3 的募集至关重要以及事件的顺序 在两条途径中都会导致这一关键事件。我们还将鉴定对 PI(3)P 至关重要的结合蛋白 LC3 募集至吞噬体以及自噬体和溶酶体融合所必需的区域 吞噬体。

The goal is to understand regulation of phosphoinositide synthesis, degradation and localization in the retina and retinal pigmented epithelium (RPE), and to understand the role of phosphoinositides in retinal signaling, development, health and disease. By illuminating molecular details of such processes as membrane trafficking, autophagy, phagocytosis, endocytosis and exocytosis, this understanding can help us better understand how these processes are disrupted in retinal disease and how therapeutic interventions could make use of them to preserve vision. The specific aims are: 1. Determine the relationship between each step in the phototransduction cascade and regulation of PI(3)P PI(4)P, and PI(4,5)P2 regulation in rods. Light, independently of time-of-day, drives massive increases in the levels of inner segment phosphoinositides. Preliminary results suggest the signal driving this increase is downstream of the phototransduction cascade, and our published results demonstrate a critical role for the Class III PI-3-kinase, Vps34. It remains unclear how light leads to upregulation of the activity of Vps34 or what drives PI(4,5)P2 increases. The following hypotheses will be tested: A. Transducin activation by photoexcited rhodopsin is essential for the light-driven increases. B. PDE6 activity is essential for light-driven increases. C. The cyclic nucleotide-gated channel is essential for the increases. 2. Determine the role of PI(4)P-5 kinase activity in PIP2 (phosphatidylinositol- (4,5)bisphosphate) regulation in the outer retina and the functional role there of PI(4,5)P2. We will generate mice with inducible rod-cell- or RPE-cell-specific knockouts of the principal enzyme responsible for synthesizing PI(4,5)P2 in neurons, PIP-5-kinaseγ, and determine the phenotype with respect to phosphoinositide levels, cell morphology and survival, protein trafficking, endocytosis, phagocytosis and autophagy. These experiments will test the following hypotheses: A. PI(4,5)P2 is primarily synthesized in rods and RPE by the action of the PIP-5-kinaseγ isoform using ATP and PI(4)P as substrates; B. PI(4,5)P2 synthesis is essential for a range of membrane trafficking functions and cell viability. If needed, we will also test the global knockouts of the α and β isoforms, which are viable, as well as double and triple. 3. Determine the distinct phosphoinositide-regulated mechanisms of LC3 recruitment and lysosome fusion in autophagy and phagocytosis. We have found deficiencies in the standard models of LC3 recruitment to autophagosomes and phagosomes, so we will combine in vivo experiments with experiments with RPE cell lines to determine which proteins are critical for LC3 recruitment in RPE cells and what sequence of events leads to this key event in both pathways. We will also identify the PI(3)P binding proteins that are essential for LC3 recruitment to phagosomes and those that are essential for lysosome fusion of both autophagosomes and phagosomes.