Mechanisms of photoreceptor protein transport and compartmentalization

项目总结/摘要 本研究的目的是确定感光蛋白的作用机制， 划分视网膜光感受器是极化神经元，其主要功能是， 包括接收和发送信号，被划分为离散子蜂窝 域.区室化对于正常的感光细胞活动和视力下降或 失明是由于蛋白质分离不当造成的。尽管这些机制很重要， 光感受器或任何其他神经元中潜在的蛋白质区室化仍然很差， 明白理解区室化的关键是 光感受器细胞质，注定要成为的蛋白质的生物物理特性 蛋白质被分隔，驱动蛋白质积累的力量， 浓度梯度，进入特定的隔室。我们发现了一个基本的 生物物理机制，可能是核心的蛋白质运输和隔离，在所有的电 活性细胞：在光感受器产生的电场中运输带电蛋白质 神经元活动我们把这种新机制称为轴向动态电泳蛋白 运输（ADEPT）。我们将使用最先进的活细胞荧光成像工具， 我们的实验室，强大的转基因和基因编辑技术在非洲爪蟾，和复杂的 生物化学和细胞生物学方法，以实现以下目标： 目的1：绘制视杆细胞中的轴向胞质电场Eax。 目的2：确定ADEPT对光感受器蛋白转运的影响， 在活体光感受器中的区室化。 目的3：确定Arrestin相互作用的位置及其对分布的影响 和动力学。

PROJECT SUMMARY/ABSTRACT The objectives of this proposal are to determine the mechanisms of photoreceptor protein compartmentalization. Retinal photoreceptors are polarized neurons whose major functions, include receiving and transmitting signals, are compartmentalized into discrete subcellular domains. Compartmentalization is critical for normal photoreceptor activity and reduced vision or blindness result from improper segregation of proteins. Despite their importance, the mechanisms underlying protein compartmentalization in photoreceptors, or any other neuron, remain poorly understood. Essential for understanding compartmentalization are the biophysical properties of the photoreceptor cytoplasm, the biophysical properties of the proteins that are destined to be compartmentalized and the forces that drive accumulation of proteins, against significant concentration gradients, into the specific compartments. We have uncovered a fundamental biophysical mechanism that may be central to protein transport and segregation in all electrically active cells: transport of charged proteins within the electrical field generated by the photoreceptor neuronal activity. We call this novel mechanism axial dynamic electrophoretic protein transport (ADEPT). We will use state of the art live cell fluorescence imaging tools developed in our lab, powerful transgenic and gene editing techniques in Xenopus, and sophisticated biochemical and cell biological approaches to address the following aims: Aim 1: Map the axial cytoplasmic electric field, Eax, in rod photoreceptors. Aim 2: Determine the impact of ADEPT on photoreceptor protein transport and compartmentalization in living photoreceptors. Aim 3: Determine the locations and influence of Arrestin interactions on their distributions and dynamics in living rods and cones.