Mechanisms of signal-dependent photoreceptor protein localization and transport

描述(申请人提供)：视网膜视杆感光细胞中涉及光信号的三种高表达蛋白质，转导蛋白、恢复素和arrestin，根据环境照明条件定位于不同的光感受器区域。这种运输对于调节光感受器的光敏感度很重要。此外，它还起到保护视杆感光细胞免受我们每天所经历的光线水平的持续刺激所造成的损害的作用，以及保护可能导致许多先天性视网膜退行性疾病的异常光感受器活动的作用。然而，这些蛋白质被定位到光感受器隔间的机制，它们在隔间之间运输的机制，以及环境光线水平的变化启动这种重新定位的机制尚不清楚。 我们已经开发了使用多光子显微镜的新方法来直接检查蛋白质的局部、隔室特有的行为以及活的、功能的光感受器内信号分子的局部变化。使用这些新方法，我们将检查： 目的1：蛋白质定位于杆状感光细胞隔间的机制。 目的2：信号依赖的蛋白质在杆状室之间的运输方式。 目标3：启动蛋白质运输的信号。 AIMS 1和AIMS 2中的实验将定量检测与绿色荧光蛋白的变体--可光激活GFP融合的蛋白质的局部和远程迁移率，以确定定位和运输方式的机制。然后，我们将利用我们从这些研究中学到的东西，构建一个定量模型来测试迁移率参数是否足以解释蛋白质定位和光驱动运输的模式。Aim 3的实验旨在使用新开发的、可表达的信号转导传感器来识别告诉蛋白质移动的信号。 了解光诱导蛋白质运输的机制可以揭示直接测试运输的适应性或保护性作用的实验策略，并可能导致治疗干预策略以减缓或逆转先天性疾病的光感受器退化。这项拟议的工作符合视网膜疾病计划下的国家眼睛和视力研究计划。

DESCRIPTION (provided by applicant): Three highly expressed proteins involved in light signaling in retinal rod photoreceptors, transducin, recoverin and arrestin, localize to different photoreceptor compartments depending on the conditions of ambient illumination. This transport is important for adjusting photoreceptor light sensitivity. Moreover, it plays a role in protecting rod photoreceptors from damage caused by continual stimulation from light levels we experience every day, and from aberrant photoreceptor activity that may underlie many congenital retinal degenerative diseases. Yet the mechanisms by which these proteins are localized to photoreceptor compartments, by which they are transported among compartments and by which changes in ambient light levels initiate this re-localization are not known. We have developed new methods using multiphoton microscopy to directly examine local, compartment-specific behavior of the proteins and local changes in signaling molecules within living, functioning photoreceptors. Using these new methods we will examine the: Aim 1: Mechanisms underlying protein localization to rod photoreceptor compartments. Aim 2: Mode of signal-dependant protein transport between rod compartments. Aim 3: Signals that initiate protein transport. Experiments in aims 1 and 2 will quantitatively examine the local and long distance mobilities of the proteins fused with a variant of the green fluorescent protein, photoactivatable GFP, to identify the mechanisms of localization and modes of transport. We will then take what we have learned from these studies and construct a quantitative model to test if the mobility parameters are sufficient to explain the patterns of protein localization and light-driven transport. Experiments in aim 3 are designed to identify the signals that tell the proteins to move using newly developed, expressible signal transduction sensors. Understanding the mechanisms of light-induced protein transport could reveal experimental strategies for testing adaptive or protective roles of transport directly and may lead to strategies for therapeutic intervention to slow or reverse photoreceptor degeneration in congenital disease. The proposed work fits into the National Plan for Eye and Vision Research under the Retinal Disease Program.