Polarization of plasma membrane microviscosity during endothelial cell migration

Cell movement is characterized by anterior-posterior polarization of multiple cell structures. We show here that the plasma membrane is polarized in moving endothelial cells (EC); in particular, plasma membrane microviscosity (PMM) is increased at the cell leading edge. Our studies indicate that cholesterol has an important role in generation of this microviscosity gradient. In vitro studies using synthetic lipid vesicles show that membrane microviscosity has a substantial and biphasic influence on actin dynamics; a small amount of cholesterol increases actin-mediated vesicle deformation, whereas a large amount completely inhibits deformation. Experiments in migrating ECs confirm the important role of PMM on actin dynamics. Angiogenic growth factor-stimulated cells exhibit substantially increased membrane microviscosity at the cell front but, unexpectedly, show decreased rates of actin polymerization. Our results suggest that increased PMM in lamellipodia may permit more productive actin filament and meshwork formation, resulting in enhanced rates of cell movement.     

Changes in the microviscosity of lipid bilayer membranes of various malignant cells and tumor transplantability

It is shown that cholesterol incorporation into the membranes of Zajdel hepatoma cells, lymphoblast leukemia cells L1210 and into those of ovary tumour causes an increase in the membrane phospholipid bilayer microviscosity measured by pyrene as fluorescent probe. The increase in the membrane lipid microviscosity resulted in a decrease in the activity of Na,K-ATPase and 5-nucleotidase of the tumour cells. After the injection of tumour cells with an increase of cholesterol/phospholipid ratio we observed an increase of the life-span of experimental animals as compared to the control groups.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Quantification of lipid bilayer effective microviscosity and fluidity effect induced by propofol

Electron spin resonance (ESR) spectroscopy with nitroxide spin probes was used as a method to probe the liposome microenvironments. The effective microviscosities have been determined from the calibration of the ESR spectra of the probes in solvent mixtures of known viscosities. In the first time, by measuring ESR order parameter (S) and correlation time (tau(c)) of stearic spin probes, we have been able to quantify the value of effective microviscosity at different depths inside the liposome membrane. At room temperature, local microviscosities measured in dimyristoyl-l-alpha phosphatidylcholine (DMPC) liposome membrane at the different depths of 7.8, 16.95, and 27.7 A were 222.53, 64.09, and 62.56 cP, respectively. In the gel state (10 degrees C), those microviscosity values increased to 472.56, 370.61, and 243.37 cP. In a second time, we have applied this technique to determine the modifications in membrane microviscosity induced by 2,6-diisopropyl phenol (propofol; PPF), an anaesthetic agent extensively used in clinical practice. Propofol is characterized by a unique phenolic structure, absent in the other conventional anaesthetics. Indeed, given its lipophilic property, propofol is presumed to penetrate into and interact with membrane lipids and hence to induce changes in membrane fluidity. Incorporation of propofol into dimyristoyl-l-alpha phosphatidylcholine liposomes above the phase-transition temperature (23.9 degrees C) did not change microviscosity. At 10 degrees C, an increase of propofol concentration from 0 to 1.0 x 10(-2) M for a constant lipid concentration mainly induced a decrease in microviscosity. This fluidity effect of propofol has been qualitatively confirmed using merocyanine 540 (MC540) as lipid packing probe. Above 10(-2) M propofol, no further decrease in microviscosity was observed, and the microviscosity at the studied depths (7.8, 16.95, and 27.7 A) amounted 260.21, 123.87, and 102.27 cP, respectively. The concentration 10(-2) M was identified as the saturation limit of propofol in dimyristoyl-l-alpha phosphatidylcholine liposomes.     

Contact-mediated changes in the fluidity of membrane lipids in normal and malignant transformed mammalian fibroblasts

The degree of microviscosity, gh, (fluidity/rigidity behavior) of membrane lipids of normal and transformed mammalian fibroblasts obtained from mice, hamsters and rats was quantitatively monitored by fluorescence polarization, P, analysis of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) when embedded in lipid regions of cellular membranes of intact viable cells. Analysis of membrane microviscosity of six different cell populations and of individual cells in each cell population have indicated that the membrane microviscosity of all cell types, both normal and transformed fibroblasts, changes as a function of the cell density in the growing cultures. The membrane microviscosity was found to be low (high lipid fluidity) in sparse conditions but high (high lipid rigidity) in dense conditions. The induced changes in membrane microviscosity are practically reversible for all cell types and a complete reversion can be obtained within a few hours after changing the cell density conditions from sparse to dense and vice versa.Comparative studies with normal and transformed fibroblasts have shown that transformed fibroblasts have a more rigid lipid layer in their cellular membranes than normal or untransformed fibroblasts. The difference in membrane microviscosity between transformed and normal fibroblasts is higher in confluent conditions as compared with subconfluent cultures. These differences in the degree of fluidity of membrane lipids that are controlled by possible differences in the cellto-cell contact in normal and transformed fibroblasts may play a major role in determining the growth behavior of normal and malignant cells that are growing as a solid tissue and may have a direct effect on the control mechanisms that determine the presence or absence of the “density dependent inhibition” of growth.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions: Effects of local anesthetics,cholesterol and protein

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 °C). Incorporation of cholesterol (30–50%) increased the microviscosity of lipid phases by 200–500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b₅ did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since the latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracaine and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of the anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at the 25 °C varied as follows:polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erytherocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol : phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important fuctional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Localization of the lipid probe 1,6-diphenyl-1,3,5 hexatriene (DPH) in intact cells by fluorescence microscopy

The lipophilic fluorescent probe DPH, generally used to determine the microviscosity of membrane lipids, has been visualized in intact cells by fluorescence microscopy. All lipid material of the cells, including cytoplasmic lipid droplets, was found to be labelled with DPH. The fluorescent signal from inside the cells contributes to a large extent to the total cell fluorescence. The results indicate that fluorescence polarization data obtained from intact cells, using DPH as probe, give information on the total lipid material of the cells rather than exclusive information on microviscosity and fluidity of plasma membranes of these cells, as has been repeatedly suggested.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decrease membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC) ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipoproteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80-85% of abetalipoproteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity (eta) between 5 and 42 degrees C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively at 10 degrees C; 4.63 vs 4.04 poise at 37 degrees C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.     

Differences in lipid fluidity among isolated plasma membranes of normal and leukemic lymphocytes and membranes exfoliated from their cell surface

The fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe was used to determine the lipid microviscosity, $\text{η}$, of isolated plasma membranes of mouse thymus-derived ascitic leukemia (GRSL) cells and of extracellular membraneous vesicles exfoliated from these cells and occurring in the ascites fluid. For comparison, $\text{η}$ was also determined in isolated plasma cell supernatants.For isolated plasma membranes of thymocytes and GRSL cells $\text{η}$ values at 25° C amounted to 4.67 and 3.28 P, respectively, which were higher than the microviscosities of the corresponding intact cells, 3.24 and 1.73 P, respectively.Microviscosities inextracellular membranes of thymocytes and GRSL cells were 5.96 and 5.83 P, respectively. The fluidity difference between these membranes and plasma membranes was most pronounced for the leukemic cells and was thereby correlated with a large difference in cholesterol/phospholipid molar ratio (1.19 for extracellular membranes and 0.37 for plasma membranes). It is proposed that extracellular membraneous vesicles are shed from the surface of GRSL cells similar to the budding process of viruses, that is by selection of the most rigid parts of the host cell membrane.Liposomes of total lipid extracts of plasma membranes and extracellular membranes of both cell types exhibited about the same microviscosity as the corresponding intact membranes, indicating virtually no contribution of (glyco)-protein to the lipid fluidity as measured by the fluorescence polarization technique. For both cell types $\text{η}$ (25° C) values of liposomes consisting of membrane phospholipids varied between 1.5 and 1.9 P, much lower than the values for total lipids, indicating a significant rigidizing effect of cholesterol in each type of membrane.     

Lipid composition determines interaction of liposome membranes with Pluronic L61

Triblock copolymers of ethylene oxide (EO) and propylene oxide (PO) of EO(n/2)PO(m)EO(n/2) type (Pluronics) demonstrate a variety of biological effects that are mainly due to their interaction with cell membranes. Previously, we have shown that Pluronics can bind to artificial lipid membranes and enhance accumulation of the anti-tumor drug doxorubicin (DOX) inside the pH-gradient liposomes and transmembrane migration (flip-flop) of NBD-labeled phosphatidylethanolamine in the liposomes composed from one component-lecithin. Here, we describe the effects caused by insertion of other natural lipids in lecithin liposomes and the significance of the lipid composition for interaction of Pluronic L61 with the membrane. We used binary liposomes consisting of lecithin and one of the following lipids: cholesterol, phosphatidylethanolamine, ganglioside GM1, sphingomyelin, cardiolipin or phosphatidic acid. The influence of the additives on (1) membrane microviscosity; (2) binding of Pluronic L61; (3) the copolymer effect on lipid flip-flop and membrane permeability towards DOX was studied. The results showed that insertion of sphingomyelin and cardiolipin did not influence membrane microviscosity and effects of Pluronic on the membrane permeability. Addition of phosphatidic acid led to a decrease in microviscosity of the bilayer and provoked its destabilization by the copolymer. On the contrary, cholesterol increased microviscosity of the membrane and decreased binding of Pluronic and its capacity to enhance flip-flop and DOX accumulation. Analogous tendencies were revealed upon incorporation of egg phosphatidylethanolamine or bovine brain ganglioside GM1. Thus, a reverse dependence between the microviscosity of membranes and their sensitivity to Pluronic effects was demonstrated. The described data may be relevant to mechanisms of Pluronic L61 interaction with normal and tumor cells.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Correlation between cell density, membrane fluidity, and the availability of transferrin receptors in Friend erythroleukemic cells

Undifferentiated Friend erythroleukemic cells (FL-cells) acquire membrane microviscosity (eta), in accord with the culture cell density. At low cell density eta (21 degrees) approximately 2.8 poise, whereas at confluency it increases to eta (21 degrees) approximately 5.3 poise. Concomitantly, the total number of available transferring receptors per cell decreases by about 80% upon increase in cell density. Modulation of membrane microviscosity, by artificial alteration of the membrane cholesterol level, mediates similar modulations of the availability of the transferrin receptors. The correlation between the availability of the transferring receptors and the membrane lipid fluidity may take part in the overt decrease in iron uptake by erythroid cells along the erythropoiesis pathway.     

Membrane-potential-dependent changes of the lipid microviscosity of mitochondria and phospholipid vesicles.

The effects of a transmembrane potential difference upon the lipid microviscosity of cytochrome oxidase vesicles (COVs) and rat liver mitochondria (RLM) were investigated. COVs and RLM were labelled with the fluorescent probe 1,6-diphenylhexa-1,3,5-triene (DPH). The fluorescence polarization of the probe was then measured when potentials of different magnitudes were induced across the membranes of these particles. It was shown that the absolute value of the microviscosity changes to quite a significant extent, owing to the imposition of large membrane potentials. On relaxation of the membrane potential the lipid microviscosity was also shown to return to the value before the induction of the potential. The largest change in lipid microviscosity was observed when coupled respiration was initiated. This occurred in both the COV system and the RLM system. The absolute value of the lipid microviscosity was shown to change by as much as 22% with the induction of membrane potentials, owing to respiration. To confirm the viscosity measurements made with DPH, lipid microviscosity was also measured with the spin-labelled fatty acid 5-doxyl stearate. Measurements of the order parameters indicated that, in agreement with the results of fluorescence experiments, viscosity changes occurred that were due to the induction of a membrane potential. The significance of these findings to the regulation of metabolism is briefly discussed, the main conclusion being that, although there is certainly a significant variation of lipid microviscosity with electric field, mechanistic interpretations will require further studies.     

Effects of cholesteryl esters on the accessibility of LH/hCG receptors and membrane lipid fluidity in rat testes

Incubation of rat testicular membranes with cholesteryl hemisuccinate resulted in an increase in both membrane lipid microviscosity and 125I-labelled hCG specific binding. The purpose of this investigation was to establish which functional groups of cholesteryl hemisuccinate are important for the stimulatory effects. The data obtained showed that only esters of cholesterol with dicarboxylic acids, not those of monocarboxylic acids, increase the accessibility of LH/hCG receptors and membrane rigidity. Experiments with cholesteryl sulfates showed that there are polar groups on C3 carbon of cholesterol having no stimulatory effect on receptors, although an increase in membrane rigidity occurred. The side-chain of cholesterol is important for the stimulatory action. Androstenolone hemisuccinate was ineffective in this respect. On the other hand, partially modified side-chains (hemisuccinates of beta-sitosterol and stigmasterol) did not result in a marked reduction of the stimulatory action. The carboxyl group of cholesteryl hemisuccinate must be 'free': its esterification abolishes the stimulatory effect of cholesteryl hemisuccinate on both the LH/hCG receptor and membrane microviscosity. These results suggest that an intact carboxyl group of ester and the side-chain of cholesterol are indispensable for the stimulatory effect of cholesteryl hemisuccinate on the accessibility of LH/hCG receptors.     

Effects of native and oxidized apolipoprotein A-I on lipid bilayer microviscosity of erythrocyte plasma membrane

Using a pyrene as a fluorescent probe, we investigated the influence of native and oxidized apolipoprotein A-I (apo A-I) and their complexes with tetrahydrocortisol (THC) on the microviscosity of the erythrocyte plasma membrane. The addition of THC to isolated membranes led to a 17% increase in the membrane microviscosity. In contrast, native apo A-I reduced the microviscosity (i.e., increased the fluidity) of the membranes by 15%. A more pronounced increase (by 25%) in the membrane fluidity was found in the presence of the complex of apo A-I with THC. Unlike native apo A-I, oxidized apo A-I and its complex with THC did not change the membrane viscosity. In view of the fact that apo A-I plays an important role in the binding of membrane cholesterol we suggest that the observed increase in the membrane fluidity under the influence of the native apo A-I is associated with the cholesterol efflux from plasma membrane. Oxidative modification of apo A-I likely disturbs the mechanisms of the cholesterol efflux and prevents the decrease in the membrane microviscosity.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes.

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.     

Chronic changes in plasma triglyceride levels do modify platelet membrane microviscosity in rats

Lipid metabolism disorders were proposed to mediate numerous cell membrane alterations in various forms of hypertension. Elevated plasma triglycerides were found to be associated with changes in membrane structure and function related to altered microviscosity in particular domains of the cell membrane. The aim of our study was to determine if an abnormal triglyceride metabolism might play a causal role in these alterations of membrane dynamics. Using genetically hypertensive rats of the Prague hereditary hypertriglyceridemic (HTG) strain we investigated whether the elevation of circulating triglycerides induced by high fructose intake and/or their lowering by chronic gemfibrozil treatment (for 10 weeks starting at the age of 6 weeks) are followed by reciprocal changes in membrane microviscosity. Two different fluorescent probes exploring either the outer membrane leaflet (TMA-DPH anisotropy) or the membrane lipid core (DPH anisotropy) were used in platelets of HTG rats. DPH (diphenylhexatriene) fluorescence anisotropy was decreased in platelets of fructose-treated HTG animals with highly elevated plasma triglyceride levels, whereas it was increased in gemfibrozil-treated HTG rats in which triglyceride levels were almost normalized. On the contrary, TMA-DPH (trimethylamino-diphenylhexatriene) anisotropy was not substantially altered in platelets from HTG rats by the above modifications of circulating triglycerides. No changes of plasma cholesterol or blood pressure were associated with the triglyceride-dependent modifications of membrane core microviscosity. Our interventional study demonstrates a major causal role of circulating triglycerides in the control of the microviscosity of membrane lipid core.     

Membrane microviscosity regulates endothelial cell motility

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.