Mechanisms of signal-dependant photoreceptor protein localization transport

描述（由申请人提供）：本项目的目的是确定视网膜光感受器内可溶性和外周膜蛋白转运和定位的生物物理基础。光感受器是高度极化的神经元，其细胞功能被分离成离散的隔室。光信号发生在睫状体外节，一个专门的隔室，包含光转导机制。光转导级联的某些组分的水平通过尚不清楚的机制以信号依赖性方式进行调节。竞争的生物物理机制似乎在发挥作用，包括蛋白质寡聚化，膜协会通过翻译后脂质化和空间体积排阻（SVE）。将使用活细胞多光子/共聚焦荧光成像系统地检查各种生物物理成分。荧光蛋白探针或视网膜光感受器中内源蛋白的融合物将在转基因非洲爪蟾或小鼠光感受器中表达。蛋白质动力学和扩散的检查将使用光转换后多光子荧光恢复（FRAP）和荧光相关光谱（FCS）来实现。活细胞中的蛋白质-蛋白质相互作用将通过福斯特共振能量转移（FRET）和荧光互相关光谱（FCCS）来检查。此外，将通过小角X射线散射（SAXS）和沉降速度（SV）或沉降平衡（SE）评估体外蛋白质寡聚化和蛋白质-蛋白质缔合。将通过质谱法分析来自光感受器提取物的蛋白质脂化状态。具体目标：目标1。确定蛋白质多聚化和DSVE如何控制视杆细胞中arrestin-1的转运和定位。目标2.确定转导蛋白膜联合如何控制其在视杆细胞中的分布和迁移。目标3：确定脂质屏蔽伴侣蛋白如何促进主要视杆细胞间转导素亚基的溶解性和光依赖性转运。

DESCRIPTION (provided by applicant): The objectives of this project are to determine the biophysical underpinnings of soluble and peripheral membrane protein transport and localization within retinal photoreceptors. Photoreceptors are highly polarized neurons with cellular functions segregated into discrete compartments. Light signaling takes place in the ciliary outer segments, a specialized compartment that contains the phototransduction machinery. The levels of some components of the phototransduction cascade are modulated in a signal-dependent manner through mechanisms that are not understood. Competing biophysical mechanisms appear to be at play, including protein oligomerization, membrane association via post-translational lipidation and steric volume exclusion (SVE). The various biophysical components will be systematically examined using live cell multiphoton/confocal fluorescence imaging. Fluorescent protein probes or fusions of endogenous proteins in retinal photoreceptors will be expressed in transgenic Xenopus laevis or mouse photoreceptors. Examination of protein dynamics and diffusion will be achieved using multiphoton fluorescence recovery after photoconversion (FRAP) using photo-switchable fluorescent proteins and fluorescence correlation spectroscopy (FCS). Protein-protein interactions in live cells will be examined by Forster resonance energy transfer (FRET) and fluorescence cross correlation spectroscopy (FCCS). Additionally protein oligomerization and protein-protein associations, in vitro, will be assessed by small angle X-ray scattering (SAXS) and sedimentation velocity (SV) or sedimentation equilibrium (SE). Protein lipidation states from photoreceptor extracts will be analyzed by mass spectrometry. Specific aims: Aim 1. Determine how protein multimerization and DSVE control transport and localization of arrestin-1 in rods. Aim 2. Determine how transducin membrane association governs its distribution and mobility in rods. Aim 3. Determine how lipid-shielding chaperone proteins promote solubility and the light-dependent transport of transducin subunits between the major rod compartments.