Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging

Abstract

Membrane fluidity was measured in the isolated perfused proximal tubule from rabbit kidney. The apical and basolateral plasma membranes of tubule cells were stained separately with the fluidity-sensitive fluorophore trimethylammonium-diphenyl-hexatriene (TMA-DPH) by luminal or bath perfusion. Fluorescence anisotropy (r) of TMA-DPH was mapped with spatial resolution using an epifluorescence microscope (excitation 380 nm, emission greater than 410 nm) equipped with rotatable polarizers and a quantitative imaging system. To measure r without the confounding effects of fluorophore orientation, images were recorded with emission polarizer parallel and perpendicular to a continuum of orientations of the excitation polarizer. The theoretical basis of this approach was developed and its limitations were evaluated by mathematical modeling. The tubule inner surface (brush border) was brightly stained when the lumen was perfused with 1 microM TMA-DPH for 5 min; apical membrane r was 0.281 +/- 0.006 (23 degrees C). Staining of the tubule basolateral membrane by addition of TMA-DPH to the bath gave a significantly lower r of 0.242 +/- 0.010 (P less than 0.005); there was no staining of the brush border membrane. To interpret anisotropy images quantitatively, effects of tubule geometry, TMA-DPH lifetime, fluorescence anisotropy decay, and objective-depolarization were evaluated. Steady-state and time-resolved r and lifetimes in the intact tubule, measured by a nanosecond pulsed microscopy method, were compared with results in isolated apical and basolateral membrane vesicles from rabbit proximal tubule measured by cuvette fluorometry; r was 0.281 (apical membrane) and 0.276 (basolateral membrane) (23 degrees C). These results establish a methodology to quantitate membrane fluidity in the intact proximal tubule, and demonstrate a significantly higher fluidity in the basolateral membrane than in the apical membrane.

Solvent drag measurement of transcellular and basolateral membrane NaCl reflection coefficient in kidney proximal tubule.

“…Kidneys from female New Zealand white rabbits (1.5-2 kg) were cut in coronal slices. Individual segments of PST (1.5-2 ram) were dissected in solution 1 (see Table I) at 4°C as described previously (Burg et al, 1966;Fushimi et al, 1990). Tubules were transferred to a laminar flow bath (Strange and Spring, 1986) in which solutions were exchanged in under 0.5 s. Tubules were mounted at the perfusion site by a tightly fitting holding pipette of 40 ~m inner diameter pulled from glass capillary tubes (Drummond Scientific Co., Broomall, PA) on a vertical pipette puller.…”

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

“…Because of spatial heterogeneity in the composition and organization of the cytosol, separate measurements are probably required for "bulk" cytosol, "peripheral" cytosol, and "membrane-adjacent" cytosol. The rheology of cytosol has been the subject of a series of biophysical investigations based on the analysis of viscosity-sensitive probes by electron spin resonance (Lepock et al, 1983;Mastro and Keith, 1984) and fluorescence spectroscopy (Luby-Phelps et al, 1986;Dix and Verkman, 1990;Fushimi et al, 1990). Two independent approaches have established recently that the "fluid-phase" cytoplasmic viscosity, de-fined as the viscosity sensed by a small molecule in the absence of collisions with or binding to cytosolic macromolecules Kao et al, 1993), is not much different from the viscosity of water.…”

Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry.

“…Furthermore, the linear extrapolation to a probe size of zero may not give the correct value of fluid-phase cytoplasmic viscosity because of the possible nonlinear relationship between the probe size and viscosity. Lastly, steadystate fluorescence anisotropy was used to estimate the fluidphase cytoplasmic viscosity (Lindmo and Steen, 1977;Hashimoto and Shinozaki, 1988;Dix and Verkman, 1990).…”