Arrio-Dupont M, Foucault G, Vacher M, Devaux P F, Cribier S
Gènes et Protéines Musculaires, EP CNRS 1088, F91405 Orsay, F75005 Paris, France.
Biophys J. 2000 Feb;78(2):901-7. doi: 10.1016/S0006-3495(00)76647-1.
Modulated fringe pattern photobleaching (MFPP) was used to measure the translational diffusion of microinjected fluorescein isothiocyanate (FITC)-labeled proteins of different sizes in the cytoplasm of cultured muscle cells. This technique, which is an extension of the classical fluorescence recovery after photobleaching (FRAP) technique, allows the measurement of the translational diffusion of macromolecules over several microns. Proteins used had molecular masses between 21 and 540 kDa. The results clearly indicated that the diffusivity of the various proteins is a decreasing function of their hydrodynamic radius. This decrease is more rapid with globular proteins than with FITC-labeled dextrans (, Biophys. J. 70:2327-2332), most likely because, unlike globular proteins, dextrans are randomly coiled macromolecules with a flexible structure. These data do not exclude the possibility of a rapid diffusion over a short distance, unobservable with our experimental set-up, which would take place within the first milliseconds after bleaching and would correspond to the diffusion in restricted domains followed by impeded diffusion provoked by the network of microtubules, microfilaments, and intermediate filaments. Thus our results may complement rather than contradict those of Verkman and collaborators (, J. Cell Biol. 138:1-12). The biological consequence of the size-dependent restriction of the mobility of proteins in the cell cytoplasm is that the formation of intracellular complexes with other proteins considerably reduces their mobility.
调制条纹图案光漂白（MFPP）被用于测量微注射的异硫氰酸荧光素（FITC）标记的不同大小蛋白质在培养肌肉细胞胞质中的平移扩散。该技术是经典光漂白后荧光恢复（FRAP）技术的扩展，可测量大分子在几微米范围内的平移扩散。所使用的蛋白质分子量在21至540 kDa之间。结果清楚地表明，各种蛋白质的扩散系数是其流体动力学半径的递减函数。球形蛋白质的这种下降比FITC标记的葡聚糖更快（，《生物物理学杂志》70:2327 - 2332），最有可能的原因是，与球形蛋白质不同，葡聚糖是具有柔性结构的无规卷曲大分子。这些数据并不排除在漂白后的最初几毫秒内发生短距离快速扩散的可能性，这种扩散在我们的实验装置中无法观察到，它将对应于在受限区域内的扩散，随后是由微管、微丝和中间丝网络引发的受阻扩散。因此，我们的结果可能是对Verkman及其合作者（，《细胞生物学杂志》138:1 - 12）结果的补充而非矛盾。蛋白质在细胞质中移动性受大小依赖性限制的生物学后果是，与其他蛋白质形成细胞内复合物会大大降低其移动性。
