Protein mobility and diffusive barriers in Escherichia coli:: consequences of osmotic stress
Protein mobility and diffusive barriers in Escherichia coli:: consequences of osmotic stress
复制标题
DOI：
10.1111/j.1365-2958.2007.05705.x
复制
发表时间：
2007-05-01
影响因子：
3.6
通讯作者：
Poolman, Bert
中科院分区：
文献类型：
--
作者：
van den Bogaart, Geert;Hermans, Nicolaas;Poolman, Bert
使用光漂白后脉冲 FRAP 荧光恢复的脉冲版本，研究了渗透应激对大肠杆菌中蛋白质细胞内扩散的影响。该方法采用激光脉冲序列，仅部分漂白细胞中的荧光团。由于考虑了细胞尺寸和几何形状，脉冲 FRAP 能够测量不同形状的非常小的细胞中的扩散。我们发现，在氯化钠或山梨醇施加的渗透压从0.15 Osm上升到0.6 Osm后，移动绿色荧光蛋白（GFP）的表观细胞内扩散（D）从3.2下降到0.4 μ m(2) s(-1)，而膜渗透性甘油则没有影响。将大肠杆菌细胞暴露于较高的渗透压（> 0.6 Osm）会导致 GFP 分隔成离散的池，而 GFP 无法从其中逃逸。尽管通过细胞的自由扩散受到阻碍，但这些池中 GFP 的迁移率仍然相对较高（D 类似于 0.4 μ m(2) s(-1)）。渗透保护剂的存在恢复了渗透压对蛋白质迁移率和表观区室化的影响。此外，将渗透压从 0.6 Osm 降低至 0.15 Osm 恢复了 GFP 的流动性。讨论了这些发现对细胞内异质性和扩散屏障的影响。
The effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo-bleaching, pulsed-FRAP. This method employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed-FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion (D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 mu m(2) s(-1), whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high (D similar to 0.4 mu m(2) s(-1)). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed.