A new approach to polarized fluorescence using phase and modulation fluorometry

In the preceding paper, an alternative method is described for obtaining information about the reorientational behavior of a fluorophore in a membrane system from frequency domain measurements. To demonstrate this new analysis procedure, we present data for the probe-molecule 1,6-diphenyl-1,3,5-hexatriene (DPH) in l--dimyristoyl- and l--dipalmitoylphosphatidylcholine (DMPC and DPPC) obtained with two different phase fluorometers: the SLM 4800A Subnanosecond Spectrofluorometer which has only three fixed frequencies available (6, 18 and 30 MHz) and the recently constructed continuously variable multifrequency phasefluorometer (Gratton and Limkeman 1983).It will be shown that reasonable information about the anisotropy behavior of a fluorophore can be obtained even if only three frequencies are used. The phase modulation technique was also used to check the new expression for the anisotropy, r(t), called the general model and introduced by Van der Meer et al. (1984). The parameters P ₂, P ₄ and D, obtained from the nonlinear least squares fit (Bevington 1969) for this general model, confirm the results from the pulse technique of Ameloot and coworkers (Ameloot et al. 1984; Pottel et al. 1986).     

Fluidity of detergent micelles plays an important role in muscarinic receptor solubilization

In order to find a suitable reagent for extracting the muscarinic receptor from rat brain membranes 14 different detergents were tested. Only the plant glycoside digitonin efficiently solubilized the receptor protein in its native form. At the same time microviscosity of detergent micelles was determined by measuring the fluorescence polarization of a hydrophobic fluorescent probe diphenylhexatriene incorporated into the micelles. In the case of digitonin the polarization value was close to the corresponding value obtained for rat brain membrane fragments, while for the other detergents studied it remained considerably lower. The results obtained indicate that the fluidity of detergent micelles may play an important role in retaining the active conformation of the solubilized muscarinic receptor.     

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles

Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains most strongly stabilized by 7-dehydrocholesterol (7DHC) and cholesterol, stabilized to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, in asymmetric vesicles desmosterol stabilized ordered domain almost as well as cholesterol, and to a much greater degree than epicholesterol, so that the ability to support ordered domains decreased in the order 7-DHC > cholesterol > desmosterol > lanosterol > epicholesterol > 4-cholesten-3-one. This contrasts with values for intermediate stabilizing sterols in symmetric vesicles in which the ranking was cholesterol > lanosterol ~ desmosterol ~ epicholesterol or prior studies in which the ranking was cholesterol ~ epicholesterol > lanosterol ~ desmosterol. The reasons for these differences are discussed. Based on these results, we re-evaluated our prior studies in cells and conclude that endocytosis levels and bacterial uptake are even more closely correlated with the ability of sterols to form ordered domains than previously thought, and do not necessarily require that a sterol have a 3β-OH group.     

Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes

The purpose of this study was to generate the equivalent of a cholesterol/temperature phase map for a biological membrane using fluorescence spectroscopy. The pseudo-phase map was created using human erythrocytes treated with various concentrations of methyl-beta-cyclodextrin to remove defined amounts of cholesterol and a trio of fluorescent probes that assess different membrane properties (laurdan, diphenylhexatriene, and merocyanine 540). Parallel experiments with two-photon microscopy suggested that changes in cellular cholesterol content affected the entire membrane rather than being localized to specific macroscopic domains. The various regions of the composite erythrocyte pseudo-phase map were interpreted using analogous data acquired from multilamellar vesicles that served as simplified models of cholesterol-dependent phases. The vesicles consisted of various concentrations of cholesterol (0 to 50 mol%) with either palmitoyl sphingomyelin, 1:1 dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine, or phospholipid mixtures intended to simulate either the inner or outer leaflet of erythrocyte membranes. Four distinguishable regions were observed in sphingomyelin phase maps corresponding to the traditional solid-ordered and liquid-disordered phases and two types of liquid-ordered behavior. Physical properties were less diverse in the mixed phospholipid vesicles, as expected, based on previous studies. Erythrocytes displayed five regions of different combinations of membrane properties along the phase map. Some of the observations identified similarities between the cells and liquid-ordered behavior observed in the various types of liposomes as well as some interesting differences.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

Thermotropic behavior and lateral distribution of very long chain sphingolipids

Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The T_m was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties.     

Fluorinated cholesterol retains domain-forming activity in sphingomyelin bilayers

Lipid rafts are cholesterol (Chol)-rich microdomains floating in a sea of lipid bilayers. Chol is thought to interact preferentially with sphingolipids such as sphingomyelin (SM) rather than with glycerophospholipids, and this putative SM–Chol interaction is generally recognized as a requirement for raft formation. However, the presence of the specific interaction is still controversial, primarily because of the lack of useful molecular probes for scrutinizing this interaction. Recently, we reported that the dynamic properties of 6-F-Chol in DMPC bilayers are similar to those of unmodified Chol. Hence, in the present study, we first compared the roles of 6-F-Chol and Chol in SM bilayers through detergent insolubility, fluorescence polarization, and ²H NMR experiments. The results demonstrated that 6-F-Chol and Chol behave similarly in SM bilayers, whereas, in SM–DOPC membranes, 6-F-Chol is less effective in domain formation. Then, we analyzed the molecular orientation of 6-F-Chol in SM bilayers using solid-state NMR, and found that the dynamics and orientation of 6-F-Chol in SM bilayers are almost identical to those in DMPC bilayers. This supports the notion of the lack of a putative specific interaction between SM and Chol. Thus, this study demonstrates the utility of 6-F-Chol as a molecular probe for understanding molecular recognition in lipid rafts.     

Fusion and lipid exchange in vesicles containing lipophilic spin labels

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.     

Rat brain microsome fluidity as modified by prenatal ethanol administration

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) and of trimethylamino-diphenylhexatriene (TMA-DPH) as a function of temperature (10° to 54°C) was measured in brain microsomes of newborn rats prenatally exposed to ethanol. In this temperature range, the relationship between r and T was linear. The addition of ethanol in vitro to microsomal suspensions influenced the slope of the line of r versus T only when DPH was used as a probe and with high concentrations of the alcohol (0.3 M).The administration of ethanol (18% of total energy intake) in vivo to pregnant dams affected the slope of the lines of r versus T of the microsomes of pups, either using DPH or TMA-DPH as probes. The slope was also affected in brain microsomes obtained from dams, yet, only with TMA-DPH and in the opposite sense than in pups. We conclude that the effect of prenatal exposure to ethanol depended on metabolic alterations induced by the alcohol and not on its detergent properties for the following reasons: (a) The effects in vitro and in vivo were different and (b) in vitro effects could be obtained only with high concentrations (0.3 M), whereas in vivo effects were produced by small doses of ethanol. Besides, the effects of the administration of the alcohol in vivo were different in adult and intrauterine life.Abbreviations DPH 1,6-diphenyl hexa-1,3,5-triene - HEPES 4-(2-hydroxyethyl-1-piperazineethansulfonic) acid - SHB sucrose-HEPES-buffer (0.32 M sucrose, 2 mM HEPES, pH 7.0) - TMA-DPH 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene, p-toluensulfonate