Department of Ophthalmology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.
J Gen Physiol. 2012 Sep;140(3):249-66. doi: 10.1085/jgp.201210818. Epub 2012 Aug 13.
G protein-coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell-imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti-sapphire laser focused to the diffraction limit, which produced small sub-cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient D(lat) = 0.130 ± 0.012 µm(2)s(-1). Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.
G 蛋白偶联受体 (GPCR) 级联反应依赖于膜蛋白扩散进行信号传递，通常存在于空间受限的亚细胞微区室中。然而，这些微区室的几何形状如何影响级联活性，目前尚不清楚，主要是因为当前的活细胞成像技术无法解析这些小结构。在这里，我们使用一种新的高分辨率方法检查了活光感受器中离散信号微区室中 GPCR 视紫红质的动力学。绿色荧光蛋白变体（增强型绿色荧光蛋白（EGFP）或光激活型 PAGFP（Rho-E/PAGFP））融合的视紫红质通过转基因在非洲爪蟾杆状光感受器中表达，并通过共焦成像解析了由片状盘膜及其切迹裂隙形成的光信号微区室的几何形状。然后，使用聚焦到衍射极限的钛宝石激光进行多光子荧光弛豫后光转换实验，在离散微区室内产生了光转换分子的小亚立方微米体积。开发了一种分子扩散模型，允许指定正在检查的特定隔室的几何形状。这用于解释实验结果。使用这种独特的方法，我们表明视紫红质在盘表面上的迁移是高度异质的。在微区室内光激活的 Rho-PAGFP 荧光的整体弛豫是双相的，快速相持续数秒，而缓慢相的持续时间可变，需要数分钟才能达到平衡。然而，在定义的隔室内局部 Rho-EGFP 扩散是单调的，具有有效侧向扩散系数 D(lat) = 0.130 ± 0.012 µm(2)s(-1)。与模型预测的 Rho-PAGFP 弛豫时间曲线的比较表明，微区室几何形状本身可以解释快速局部视紫红质扩散及其在较大盘膜上的缓慢平衡。我们的方法首次允许在活细胞信号微区室内直接检查 GPCR 动力学，并对隔室几何形状对 GPCR 活性的影响进行定量评估。
