Mechanisms of signal-dependant photoreceptor protein localization transport

项目摘要 该项目的目标是确定可溶性和外周性的生物物理基础， 视网膜光感受器内的膜蛋白转运和定位。光感受器高度 具有细胞功能的极化神经元分离成离散的隔室。光信号发生 在睫状体外节中，一个包含光传导机制的专门隔室。 光转导级联的一些组分的水平以信号依赖性的方式被调节。 通过不被理解的机制。相互竞争的生物物理机制似乎在 作用，包括蛋白质寡聚化，通过翻译后脂质化和空间位阻的膜缔合 体积排阻（SVE）。各种生物物理成分将使用活细胞系统地检查， 多光子/共聚焦荧光成像。荧光蛋白探针或内源性蛋白质的融合物， 视网膜光感受器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实现使用多光子荧光恢复 使用光可转换荧光蛋白和荧光相关性的光转换后（FRAP） 光谱（FCS）。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进行检查 转移（FRET）和荧光交叉相关光谱（FCCS）。另外蛋白质 寡聚化和蛋白质-蛋白质缔合，在体外，将通过小角X射线散射进行评估 （SAXS）和沉降速度（SV）或沉降平衡（SE）。蛋白质脂化状态， 通过质谱分析感光体提取物。 具体目标： 目标1.确定蛋白质多聚化和DSVE如何控制arrestin-1的转运和定位， 棒。 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溶解性和光依赖性 转导素亚单位在主要视杆细胞间的转运。

Project summary 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.