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Protein mobility and diffusive barriers in Escherichia coli : consequences of osmotic stress
Author(s) -
Van Den Bogaart Geert,
Hermans Nicolaas,
Krasnikov Victor,
Poolman Bert
Publication year - 2007
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2007.05705.x
Subject(s) - green fluorescent protein , osmotic shock , intracellular , biophysics , compartmentalization (fire protection) , biology , escherichia coli , fluorescence recovery after photobleaching , cell membrane , osmotic concentration , microbiology and biotechnology , cell , biochemistry , membrane , gene , enzyme
Summary 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 μm 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 ∼0.4 μm 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.