Protein mobility and diffusive barriers in Escherichia coli: consequences of osmotic stress

Geert van den Bogaart, Nicolaas Hermans, Viktor Krasnikov, Bert Poolman*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

77 Citations (Scopus)
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Abstract

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.

Original languageEnglish
Pages (from-to)858-871
Number of pages14
JournalMolecular Microbiology
Volume64
Issue number3
DOIs
Publication statusPublished - May-2007

Keywords

  • GREEN FLUORESCENT PROTEIN
  • IN-VIVO
  • MITOCHONDRIAL MATRIX
  • DNA INTERACTIONS
  • GLYCINE BETAINE
  • LIVING CELLS
  • CYTOPLASM
  • MEMBRANE
  • VESICLES
  • OSMOLYTES

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