Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA.
Anal Biochem. 2013 Sep 1;440(1):40-8. doi: 10.1016/j.ab.2013.04.033. Epub 2013 Jun 7.
Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility.
荧光漂白后恢复（FRAP）和荧光相关光谱（FCS）是测量完整活细胞中分子扩散的两种最直接的方法。理想情况下，对于相同的系统，这两种方法应该产生相似的结果。我们使用这些方法监测两种 G 蛋白偶联受体及其相关蛋白在没有或包含称为 caveolae 的凹陷蛋白域的细胞膜中的扩散。FRAP 研究表明，caveolae 域增加了受体的不可动分数，而对其流动性没有显著影响。另一方面，FCS 研究表明，caveolae 相关蛋白的流动性出人意料地增加。我们的数据表明，caveolae 域的几何形状导致其附着蛋白的受限扩散，从而导致表观流动性增加。
