Molecular order and fluidity of the plasma membrane of human platelets from time-resolved fluorescence depolarization

The ability of seven fluorescence polarization probes (1,6-diphenyl-1,3,5-hexatriene, 1-[(4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene, 16(9-anthroyloxy)palmitic acid, CIS-parinaric acid, trans-parinaric acid and perylene) to report changes induced by temperature and Ca²⁺ in the plasma membrane of human platelets has been examined. The steady-state fluorescence anisotropy of the probes was compared after being incorporated into whole resting platelets, fragments of platelet plasma membrane and multilayers of lipids extracted from these membranes. In addition, we have investigated the molecular order and dynamics of the three preparations by time-resolved fluorescence depolarization of DPH and CE-DPH as a function of temperature and Ca²⁺ concentration. The high values of the order parameters found in intact platelets (S_{DPH, 36.c}=0.70) were almost identical to those in membrane fragments and lipid vesicles, suggesting that lipid-lipid interactions and, therefore, the lipid composition are the main factors influencing the probe order parameter. Other lipid interactions such as those with membrane proteins and intracellular components have little effect on the SDP, in platelets. These measurements also showed that the stationary fluorescence anisotropy of DPH and CE-DPH in platelets is largely determined (80%) by the structural order of the lipid bilayer. Therefore, the previous microviscosity values based on stationary anisotropy data reflect the alignment and packing rather than the mobility of the bilayer components. The dynamic component of the anisotropy decay of these probes was analyzed in terms of the wobbling-in-cone model, allowing an estimation of the apparent viscosity of platelet plasma membrane (DPH, 36°C =–0–5 P) that is similar to that of the erythrocyte membrane. This value decreased substantially in multilayers of native lipids, indicating a large effect of the lipidprotein interactions on the probe dynamics within the bilayer. When the temperature was raised from 25° to 36°C a pronounced decrease was observed in the order parameter and apparent viscosity, followed by a tendency to level-off in the 36°-40°C interval. This may be related to the end-point of the lipid phase separation reported by Gordon et al. (1983). Finally, the rigidifying (lipid ordering) effect of Ca²⁺ on the platelet plasma membrane could also be observed by the fluorescence anisotropy measurements, in the form of an increase (2%) of the order parameter of CE-DPH for Ca²⁺ concentrations in the millimolar range.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - TMA-DPH 1-[(4-trimethyl-amino)phenyl]-6-phenyl-1,3,5-hexatriene - CE-DPH (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene - 16AP 16-(9-anthroyloxy)-palmitic acid; c-PnA, CIS-parinaric acid; t-PnA, trans-parinaric acid - PER perylene - POPOP p-bis[2(5-phenyl-oxazolyl)benzene] - ESR electron spin resonance Offprint requests to: A. U. Acuña     

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.     

Reconstitution of the GalP galactose transport activity of Escherichia coli into liposomes made from soybean phospholipids

Galactose transport activity from Escherichia coli was solubilized with octyl glucoside, and reconstituted into liposomes made from soybean or E. coli lipid. Galactose counterflow in the proteoliposomes was inhibited by glucose, talose, 2-deoxygalactose and 6-deoxygalactose, confirming that it was due to GalP and not one of the other E. coli galactose transport systems.     

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca²⁺ flux into ciliary membrane vesicles of Paramecium as measured with the Ca²⁺ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca²⁺ remained unaffected by temperature. The differences in temperature dependency of Ca²⁺ conductance and membrane fluidity indicate that the Ca²⁺ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.