Cytoplasmic viscosity near the cell plasma membrane: translational diffusion of a small fluorescent solute measured by total internal reflection-fluorescence photobleaching recovery

Abstract

Total internal reflection-fluorescence recovery after photobleaching (TIR-FRAP) was applied to measure solute translational diffusion in the aqueous phase of membrane-adjacent cytoplasm. TIR fluorescence excitation in aqueous solutions and fluorescently labeled cells was produced by laser illumination at a subcritical angle utilizing a quartz prism; microsecond-resolution FRAP was accomplished by acousto-optic modulators and electronic photomultiplier gating. A mathematical model was developed to determine solute diffusion coefficient from the time course of photobleaching recovery, bleach time, bleach intensity, and evanescent field penetration depth; the model included irreversible and reversible photobleaching processes, with triplet state diffusion. The validity and accuracy of TIR-FRAP measurements were first examined in aqueous fluorophore solutions. Diffusion coefficients for fluorescein isothiocyanate-dextrans (10-2000 kDa) determined by TIR-FRAP (recovery t1/2 0.5-2.2 ms) agreed with values measured by conventional spot photobleaching. Model predictions for the dependence of recovery curve shape on solution viscosity, bleach time, and bleach depth were validated experimentally using aqueous fluorescein solutions. To study solute diffusion in cytosol, MDCK epithelial cells were fluorescently labeled with the small solute 2',7'-bis-2-carboxyethyl-5-carboxyfluorescein-acetoxymethyl-ester (BCECF). A reversible photobleaching process (t1/2 approximately 0.5 ms) was identified that involved triplet-state relaxation and could be eliminated by triplet-state quenching with 100% oxygen. TIR-FRAP t1/2 values for irreversible BCECF bleaching, representing BCECF translational diffusion in the evanescent field, were in the range 2.2-4.8 ms (0.2-1 ms bleach times), yielding a BCECF diffusion coefficient 6-10-fold less than that in water. These results establish the theory and the first experimental application of TIR-FRAP to measure aqueous-phase solute diffusion, and indicate slowed translational diffusion of a small solute in membrane-adjacent cytosol.

Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

“…The variation in the steady-state deprotonated-to-protonated SNARF-1 ratio may be due to actual pH cyt differences, variations in regional cytoplasmic microviscosity (28, 43,55,66), or even a different proportion of dye bound to cytoplasmic proteins (4). To properly interpret the differences in SNARF-1 protonated-to-deprotonated ratios, we have taken into account the behavior of the pH fluoroprobe in the cytoplasm, because it is heterogeneous in terms of composition and organization.…”

“…However, differences in the cytoplasmic microenvironment in terms of protein composition and viscosity could cause distinct diffusion mobility of fluorescent probes and distinct spectral properties. Indeed, studies by fluorescence recovery after photobleaching have shown that the translational diffusion of intracellular 2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxyfluorescein near the membrane and in the bulk cytoplasm is 6 -10 times and ϳ4 times lower than in water, respectively (55). However, the fluid-phase cytoplasmic viscosity in the absence of collisions or binding to cytoplasmic macromolecules is similar to the viscosity of water (26, 55).…”

Mobility of Human Immunodeficiency Virus Type 1 Pr55 Gag in Living Cells

“…For example, glucocorticoid receptor mobility significantly decreases upon ligand binding (42). Interaction with cytoskeletal components can also limit a protein's ability to diffuse (19,46). Analysis of protein mobility provides important insights into biochemical processes in the context of live cells.…”

Photoacoustic recovery after photothermal bleaching in living cells

“…Additionally, the intracellular components form a filamentous meshwork, which restricts diffusion as a cytosolic sieve. 22,23 Moreover, the GNPs tend to aggregate upon cellular uptake. 24 The aggregation of GNPs increases particles' effective diameters and thereby slows down their diffusion.…”