Cheek cell membrane fluidity measured by fluorescence recovery after photobleaching and steady-state fluorescence anisotropy

Membrane fluidity of human cheek cells was determined using fluorescence recovery after photobleaching (FRAP) and steady-state fluorescence anisotropy. The FRAP data showed that the lateral diffusion coefficient (D) and mobile fraction (%R) of lipid in the plasma membrane of control cells were 2.01×10^–9 cm²/ sec and 54.25%, respectively. Trypsin treatment increased D and %R to 6.4×10^–9 cm²/sec and 72.15%. In contrast, the anisotropy (r) for control cells was 0.270 which remained unchanged by trypsin treatment. The results show that diffusion of lipids in the plane of the membrane is restricted by trypsin-sensitive barriers.     

Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata

Abstract The lipid composition of a strain of each of two yeasts, Saccharomyces csrevisiae and Kloeckera apiculata , with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol: phospholipid and sterol: protein ratios and an increase in unsaturation index.     

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

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.     

Plasma membrane fluidity measurements on whole living cells by fluorescence anisotropy of trimethylammoniumdiphenylhexatriene

Trimethylammoniumdiphenylhexatriene (TMA-DPH) is a hydrophobic fluorescent probe with a high quantum yield, which was shown earlier to have specific localization properties in the plasma membranes of whole living cells. This probe was used in aqueous suspensions of L929 mouse fibroblasts, rat mast cells and ReH6 leukemic lymphocytes for determining plasma membrane fluidity from fluorescence stationary anisotropy measurements. TMA-DPH was only partially incorporated into the membranes, most of it remained as a stable form in the buffer solution; the distribution was governed by an equilibrium. The measurements were influenced by unavoidable parasitic scattered light and an appropriate correction is described. A set of precautions for the proper use of the probe is proposed. The results indicated that the fluidity was considerably lower in whole cells than in isolated membranes from the same system.     

Alterations in membrane fluidity during keratinocyte differentiation measured by fluorescence polarization

Summary The epidermis shows a distinctive pattern of differentiation wherein keratinocytes proliferate in the basal cell layer and mature into spinous and granular cells. Using a discontinuous density-gradient centrifugation method, guinea-pig keratinocytes were separated into high (HDF), intermediate (IDF), and low (LDF) density fractions. Morphological and flow cytometrical observations demonstrated that HDF, IDF, and LDF were basal, spinous, and granular cell-rich fractions, respectively. Membrane fluidity of the fractionated keratinocytes was measured by diphenylhexatriene fluorescence polarization. Polarization (p)-value of keratinocytes was negatively correlated with temperature. At each temperature, HDF cells showed a lower p-value than IDF or HDF cells except at 40° C. Since a low p-value indicates a high degree of Brownian motion, membrane fluidity is higher in basal cells and lower in spinous and granular cells. Our results indicate that membrane fluidity of guinea-pig keratinocytes decreases during their maturation.     

Effect of fatty acids on the membrane fluidity of cultured chick dorsal root ganglion measured by fluorescence photobleaching recovery

The effect of fatty acids on the membrane fluidity in tissue cultured chick embryo dorsal root ganglion was studied by fluorescence recovery method. Lateral motion of the lipid was measured by observing the fluorescent probe, 5-(octadecylthiocarbamoylamino) fluorescence, F18. The effective lateral diffusion coefficient of the membrane was around 0.30 X 10(-8) cm2/sec in control cells, 0.42 X 10(-8) cm2/sec in 2-decenoic acid treated cells, and 0.35 X 10(-8) cm2/sec in valeric acid treated cells. From these results it is concluded that effective mobilities of the membrane complex increased about 40% by the external application of 2-decenoic acid, while valeric acid increased it only 12%. From the physiological results that 2-decenoic acid inhibits the Na-channel, it is suggested that this increase in the membrane fluidity might affect the Na-channel.     

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoplasmic membrane fluidity measurements on intact living cells ofBacillus subtilis by fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene

A method for determination of membrane fluidity (microviscosity) in Bacillus subtilis cytoplasmic membrane under in vivo conditions is described. The membranes were labelled with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene during the exponential phase of growth. Fluorescence anisotropy measurements were carried out in an intact cell suspension having absorbance A as high as 0.2-0.3 (corresponding to a cell concentration of 100-300/nL).     

Influence of plasmalogen deficiency on membrane fluidity of human skin fibroblasts: a fluorescence anisotropy study

The influence of plasmalogen deficiency on membrane lipid mobility was determined by measuring fluorescence anisotropy of trimethylammoniumdiphenylhexatriene (TMA-DPH) and diphenylhexatrienylpropanoylhydrazylstachyose (glyco-DPH) inserted in the plasma membranes of human skin fibroblasts deficient in plasmalogens. The cells used were from patients affected with cerebrohepatorenal (Zellweger) syndrome (CHRS) or rhizomelic chondrodysplasia punctata. Their plasmalogen content (0-5% of total phospholipid) is significantly reduced compared with that of control cells from healthy donors (13-15% of total phospholipid) or of CHRS fibroblasts supplemented with the plasmalogen precursor, hexadecylglycerol. Plasmalogen-deficient cells consistently showed lower fluorescence anisotropies of membrane-bound DPH fluorophores corresponding to higher membrane lipid mobilities as compared to controls. However, very similar lipid mobilities were found for sonicated aqueous dispersions of phospholipids extracted either from CHRS or control cells. Therefore, the differences observed with living cells are not due to differences in the overall physical properties of the membrane lipid constituents. Other phenomena such as lipid asymmetry and/or plasmalogen-protein interactions may be responsible for the effects observed in the biomembranes.     

Effect of ethanol intake on human erythrocyte membrane fluidity and lipid composition

Erythrocyte membrane fluidity was evaluated in chronic alcoholic patients without any liver alteration, assuming different daily ethanol amounts, and in normal subjects and related to ghost fatty acid and total lipid composition obtained by high resolution gas chromatography. Erythrocyte membrane fluidity was significantly increased in a dose dependent manner in chronic alcoholic patients respect to normal subjects. This real fluidizing effect of ethanol "in vivo" was attributed mainly to a significant increase in the polyunsaturated fatty acids amount in patient ghosts in comparison with control subjects. On the other hand the cholesterol/phospholipid ratio was not significantly affected by chronic ethanol assumption.     

Order and fluidity of lipid membranes as determined by fluorescence anisotropy decay

The fluorescence anisotropy decay of four different probes in bilayers of dimyristoylphosphatidylcholine was measured. The probes are diphenylhexatriene, diphenyloctatetraene, trimethylaminodiphenylhexatriene, and trans-parinaric acid. The data for each probe were analyzed in terms of two orientational order parameters, the ordinary order parameter and a higher one, and two rotational diffusion coefficients. The order parameters are largely independent of probe size, but depend on the position of the probes along the membrane normal, thus reflecting the profile of lipid order. If a probe is located in the plateau region of lipid order, its order parameters are interpreted as representing the rigid-body order of lipids. According to this interpretation, the total lipid order in the plateau region originates about equally from rigid-body order and conformational order. The two order parameters obtained for each probe are used to derive approximate angular distributions of the probe molecules. The diffusion coefficient for rotation about the long molecular axis is found to be infinitely large, indicating unhindered rotation about this axis. The diffusion coefficient for rotation about the short molecular axes is evaluated for a viscosity which results as 0.2 poise. This viscosity for rotational diffusion is an order of magnitude smaller than the viscosity for lateral diffusion indicating that at least two viscosities are required to characterize the fluidity of a lipid membrane.Abbreviations FAD fluorescence anisotropy decay - DMR deuterium magnetic resonance - ESR electron spin resonance - DMPC dimyristoylphosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DPO 1,6-diphenyl-1,3,5,7-octatetraene - TMA-DPH 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene - tPnA trans-parinaric acid - NPN N-phenyl-1-naphthylamine - BBO 2,5-bis(4-biphenylyl)oxazole     

Change of intracellular fluidity during keratinocyte differentiation measured by fluorescence polarization

Summary The fluorescence polarization method was applied to measure the intracellular fluidity of fractionated guinea pig keratinocytes. Guinea pig epidermal cell suspension was obtained by treatment with EDTA and trypsin, and was separated into high, intermediate, and low density fractions using Percoll density gradient centrifugation. Morphological observation and cytofluorometric analysis of DNA content in the fractionated epidermal cells showed that the high, intermediate, and low density fractions were basal, spinous, and granular cell-rich fractions, respectively. Intracellular fluorescence polarization of each fraction was determined by a polarization spectrofluorometer (Hitachi MPF-4, prototype) with fluorescein diacetate. The P-values were calculated for high, intermediate, and low density fractions as 0.192 ± 0.021, 0.172 ± 0.019, and 0.147 ± 0.012, respectively. Since low P-values indicate a high degree of fluidity, the results indicate that intracellular fluidity of keratinocytes is lower in basal cells and higher in granular cells. Dye-binding experiments showed that fluorescein-binding proteins were not detected in the soluble fraction of the epidermal cells. The present findings suggest that intracellular fluidity of the guinea pig keratinocyte increases during the process of its differentiation.     

Modulation of membrane fluidity in living protoplasts of Nicotiana plumbaginifolia by catalytic hydrogenation

A homogeneous water-soluble Ru catalyst, has been incorporated into mesophyll protoplasts isolated from Nicotiana plumbaginifolia leaves. In the presence of hydrogen gas this complex causes an extensive loss of unsaturated fatty acid bonds and a concomitant increase in microviscosity of the cellular membranes. Although the gradual reduction of the level of unsaturation, per se, is accompanied by considerable cell damage, there is an optimum reaction time where approximately 50% of the protoplasts are still living and about 20% of the double bounds initially present in fatty acyl residues have undergone hydrogenation. The possible mechanism of the self regulatory process competing with the hydrogenation in the early stages of the reaction is also discussed.     

Effect of chromium on the rate of carbon dioxide evolution fromSaccharomyces cerevisiae

The rate of carbon dioxide evolution by whole yeast cells was measured under conditions of both chromium starvation and availability. Preincubation experiments show that there is no evidence for the view thatSaccharomyces cerevisiae can synthesize a biologically important form of chromium from simple chromium(III) salts. Chromium enters the yeast cell, possibly by way of the glucose transport system, and exerts a slight inhibitory effect on the fermentation rate but does not affect the rate of cell growth. Glucose tolerance factor fractions show a stimulatory effect in the yeast fermentation assay irrespective of whether the yeast is chromium depleted. The evidence shows that chromium is not an essential trace element for normal yeast metabolism and hence the activity of glucose tolerance factor preparations is unlikely to be due to a chromium complex as is commonly assumed. Some other as yet unidentified compound or compounds must be responsible for the observed increase in the rate of carbon dioxide evolution.     

Transbilayer effects of ethanol on fluidity of brain membrane leaflets

Previous work on membrane effects of ethanol focused on fluidization of the bulk membrane lipid bilayer. That work was extended in the present study to an examination of ethanol's effect on lipid domains. Two independent methods were developed to examine the effects of ethanol on the inner and outer leaflets of synaptic plasma membranes (SPM). First, differential polarized phase and modulation fluorometry and selective quenching of diphenyl-1,3,5-hexatriene (DPH) were used to examine individual leaflets. Both limiting anisotropy and rotational relaxation time of DPH in SPM indicated that the outer leaflet was more fluid than the inner leaflet. Second, plasma membrane sidedness selective fluorescent DPH derivatives, cationic 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and anionic 3-[p-6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PRO-DPH), confirmed this transmembrane fluidity difference. TMA-DPH and PRO-DPH preferentially localized in the inner and outer leaflets of SPM, respectively. Ethanol in vitro had a greater fluidizing effect in the outer leaflet as compared to the inner leaflet. Thus, ethanol exhibits a specific rather than nonspecific fluidizing action within transbilayer SPM domains. This preferential fluidization of the SPM outer leaflet may have a role in ethanol affecting transmembrane signaling in the nervous system.     

The plasma membrane ATPase of Kloeckera apiculata: purification,characterization and effect of ethanol on activity

Partially (6-fold) purified plasma membrane ATPase from an ethanol-sensitive yeast, Kloeckera apiculata, had an optimum pH of 6.0, an optimum temperature of 35°C, a K _m of 3.6 mm ATP and a V _max of 11 mol P_i/min.mg protein. SDS-PAGE of the semi-purified plasma membrane showed a major band of 106 kDa. No in vivo activation of the ATPase by glucose was observed. Although 4% (v/v) ethanol decreased the growth rate by 50% it did not affect the ATPase. Concentrations of ethanol 2% (v/v) did, however, inhibit the enzyme in vitro. The characteristics of the enzyme did not change during growth in the presence of ethanol.     

Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy

Self-assembly properties of HIV-1 integrase were investigated by time-resolved fluorescence anisotropy using tryptophanyl residues as a probe. From simulation analyses, we show that suitable photon counting leads to an accurate determination of long rotational correlation times in the range of 20-80 ns, permitting the distinction of the monomer, dimer, and tetramer from higher oligomeric forms of integrase. The accuracy of correlation times higher than 100 ns is too low to distinguish the octamer from other larger species. The oligomeric states of the widely used detergent-solubilized integrase were then studied in solution under varying parameters known to influence the activity. In the micromolar range, integrase exists as high-order multimers such as an octamer and/or aggregates and a well-defined tetramer, at 25 and 35 degrees C, respectively. However, integrase is monomeric at catalytically active concentrations (in the sub-micromolar range). Detergents (NP-40 and CHAPS) and divalent cation cofactors (Mg(2+) and Mn(2+)) have a clear dissociative effect on the high multimeric forms of integrase. In addition, we observed that Mg(2+) and Mn(2+) have different effects on both the oligomeric state and the conformation of the monomer. This could explain in part why these two metal cations are not equivalent in terms of catalytic activity in vitro. In contrast, addition of Zn(2+) stimulates dimerization. Interestingly, this role of Zn(2+) in the multimerization process was evident only in the presence of Mg(2+) which by itself does not induce oligomerization. Finally, it is highly suggested that the presence of detergent during the purification procedure plays a negative role in the proper self-assembly of integrase. Accordingly, the accompanying paper [Leh, H., et al. (2000) Biochemistry 39, 9285-9294] shows that a detergent-free integrase preparation has self-assembly and catalytic properties different from those of the detergent-solubilized enzyme.     

DPH标记法研究杀虫剂对二化螟线粒体膜流动性的影响

以DPH为荧光探剂,采用荧光偏振法研究了几种常用农药对二化螟Chilo supperssalis(Walekr)线粒体膜流动性的影响。结果表明,DPH是一种有效的荧光探剂,可以用来研究线粒体膜脂的流动性。不同种类的农药对二化螟线粒体膜的流动性都有一定的影响,但是以三氟氯氰菊酯、高效氯氰菊酯和硫丹影响较大,甲胺磷、三唑磷和克百威影响较小。三氟氯氰菊酯和高效氯氰菊酯可使膜的流动性下降,而硫丹、甲胺磷、三唑磷和克百威则使膜的流动性增强。对膜影响较大的三氟氯氰菊酯和硫丹对膜流动性的影响,还存在一定的剂量-效应关系。另外,膜的流动性受温度的影响很大,在温度分别为17、27、37℃的条件下,在药剂浓度为1×10-4mol/L时,甲胺磷在3个温度下对膜的流动性影响都很小,在误差范围内几乎没有影响;硫丹不同温度下都使膜的流动性增强,而三氟氯氰菊酯则使膜的流动性降低。