Photon correlation spectroscopy of bilayer lipid membranes

Light scattering by thermal fluctuations on simple monoglyceride bilayer membranes has been used to investigate the viscoelastic properties of these structures. Spectroscopic analysis of these fluctuations (capillary waves) permits the nonperturbative measurement of the interfacial tension and a shear interfacial viscosity acting normal to the membrane plane. The methods were established by studies of solvent and nonsolvent bilayers of glycerol monooleate (GMO). Changes in the tension of GMO/n-decane membranes induced by altering the composition of the parent solution were detected and quantified. In a test of the reliability of the technique controlled variations of the viscosity of the aqueous bathing solution were accurately monitored. The technique was applied to solvent-free bilayers formed from dispersions of GMO in squalane. The lower tensions observed attested to the comparative absence of solvent in such bilayers. In contrast to the solvent case, the solvent-free membranes exhibited a significant transverse shear viscosity, indicative of the enhanced intermolecular interactions within the bilayer.     

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.     

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

Effect of benzyl alcohol on lipid bilayers. A comparisons of bilayer systems.

The effect of the small anesthetic molecule, benzyl alcohol, on the structure of various bilayer system has been studied by optical, electrical, and x-ray diffraction techniques. We find that the modifications in bilayer thickness caused by benzyl alcohol differ dramatically for planar (or black lipid) bilayers containing solvent, planar bilayers containing little or no solvent, and vesicular bilayers. Benzyl alcohol increases the thickness of planar bilayers containing n-alkane solvents, yet decreases the thickness of "solvent-free" planar bilayers. The effect of benzyl alcohol on vesicular bilayers below the phase transition temperature also depends on whether solvent is present in the bilayers. Without solvent, gel-state bilayers are reduced in thickness by benzyl alcohol, whereas in the presence of solvent, the thickness is unchanged. Above the phase transition temperature, benzyl alcohol has no measurable effect on vesicular bilayer thickness, whether solvent is present or not. These results indicate that different model membrane systems respond quite differently to a particular anesthetic.     

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.     

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.     

Bending elasticities of model membranes: influences of temperature and sterol content

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.     

Viscoelastic relaxation of bilayer lipid membranes. Frequency-dependent tension and membrane viscosity.

Photon correlation spectroscopy has been used to study capillary waves on black lipid membranes of glycerol monooleate at temperatures above the lipid transition. For the first time the tension and viscosity of solvent-free bilayers have been observed to display a frequency dependence. The variations of both parameters can be accounted for by a Maxwell viscoelastic fluid model having a relaxation time of 37 microseconds. The equilibrium (omega = 0) tension is compatible with literature values. The present results do not suffice to precisely define the specific molecular processes involved, but relaxation times similar to the present are associated with certain phenomena in phospholipid vesicles. Bilayers containing hydrocarbon solvent do not show such relaxation, presumably due to their weaker intermolecular interactions.     

Gramicidin conformational studies with mixed-chain unsaturated phospholipid bilayer systems.

The transition of gramicidin from a nonchannel to a channel form was investigated using mixed-chain phosphatidylcholine lipid bilayers. Gramicidin and phospholipids were codispersed, after removal of the solvents chloroform/methanol or trifluoroethanol which resulted in nonchannel and channel conformations, respectively, as confirmed using circular dichroism (CD). The fluorescence emission maxima of the nonchannel form were shifted toward shorter wavelengths by heating at 60 degrees C (for 0-12 h), which converted it to a channel form, again as confirmed by CD. The channel form did not respond to heat treatment. Heat treatment also increased the fluorescence anisotropy of the nonchannel gramicidin tryptophans. The rate of transition from the nonchannel to channel conformation was found to be faster if phosphatidylethanolamine was present in combination with phosphatidylcholine compared to phosphatidylcholine alone. Also, gramicidin in bilayers of the polyunsaturated 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine converted more rapidly compared to 1-palmitoyl-2-oleoylphosphatidylcholine. Using the fluorescence anisotropy of the membrane lipid probe 1,6-diphenyl-1,3,5-hexatriene, it was also shown that the motional properties of the surrounding lipid acyl chains differed for the channel and nonchannel gramicidin conformations. The possibility that lipids tending to favor the hexagonal phase (HII) would enhance the rate of the nonchannel to channel transition was supported by 31P NMR which revealed the presence of some HII lipids in the channel preparations. The results of this study suggest that gramicidin may serve as a useful model for similar conformational transitions in other more complex membrane proteins.     

Curvature elasticity of multilamellar lipid bilayers close to the chain-melting transition

We directly measured curvature elasticity of dipalmitoylphosphatidylcholine multilamellar bilayers close to the chain-melting transition using the method of electric-field-induced bending deformation of the cylindrical tubes. The result shows that the bending modulus, kappa(c), decreases remarkably at temperatures close to the melting transition temperature. This reflects a softening of the bilayer resulted from the area fluctuations as predicted theoretically. However, the decrease of kappa(c) near the transition is far smaller than that predicted. This is due to the experimental method and the narrow transition width of the multilamellar bilayers. Nevertheless, the result obtained gives direct evidence of the kappa(c) reduction predicted for multilamellar membranes in the transition regime. Below about 41 degrees C, almost of all cylindrical tubes cannot response to the electric field, indicating a very large bending rigidity.     

Artificial black membranes from bipolar lipids of thermophilic Archaebacteria

The membrane of thermophilic archaebacteria is characterized by the presence of unusual isoprenoid bipolar lipids. The molecular organization of these lipids is still a matter of study. Important information could come from forming artificial black membranes. Black films can be formed from n-alkane or squalene dispersions of bipolar lipids extracted from the membrane of Caldariella acidophila. Membrane formation occurred only above a critical temperature (approximately 70 degrees C) corresponding to the physiological one. At lower temperatures, special solvent systems (n-alkanes or squalene, butanol and n-alkanes or squalene, butanol chloroform) were required. To characterize the physical parameters of these membranes, conductance and capacitance measurements were performed. Conductance was in the range of 10(-8) - 10(-7) omega -1 cm -2 , where specific capacitance at T = 72 degrees C was Cs = 0.685 +/- 0.004 microF/cm2 and Cs = 0.658 +/- 0.08 microF/cm2, corresponding to a dielectric thickness of 27 and 29 A for squalene and dodecane dispersions, respectively. Capacitance was shown to vary as the square of membrane potential, as usual in lipid bilayers. Values of the proportionality constant alpha have been compared to those of solvent-containing and solvent-free bilayers. The behavior of capacitance as a function of temperature is also shown by lowering temperature; the occurrence of complex structural changes was indicated. All the experimental data suggest that the presence of solvent is very low. Two possible molecular configurations of the films are discussed.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Thermally induced proliferation of pores in a model fluid membrane.

The growth of thermally induced pores in a two-dimensional model fluid membrane is investigated by Monte Carlo simulation. Holes appear in the membrane via an activated process, and their subsequent growth is controlled by an edge energy per unit length or line tension. The barrier height and line tension, together with a lateral tension, are the independent parameters of the model. In the resulting phase diagram, a rupture transition separates an intact membrane from a disintegrated state. The approach to the ruptured state shows distinct regimes. Reducing the barrier height at large line tension produces multiple, quasi-independent, small holes whose behavior is dominated by their edge energy, whereas at lower line tensions shape fluctuations of the holes facilitate their coalescence into a single large hole. At a small value of line tension and large barrier height, a single hole spontaneously permeabilizes the membrane in an entropically driven phase transition. Entropy dominates pore growth for line tensions not far below those measured for artificial vesicles. Permeabilization of lipid bilayers by certain peptides involves perturbing lipid-lipid cohesive energies, and our simulations show that at small line tensions the entropy of hole shape fluctuations destroys the model membrane's stability.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Pretransitional effects in dimyristoylphosphatidylcholine vesicle membranes: optical dynamometry study

We used micron-sized latex spheres to probe the phase state and the viscoelastic properties of dimyristoylphosphatidylcholine (DMPC) bilayers as a function of temperature. One or two particles were manipulated and stuck to a DMPC giant vesicle by means of an optical trap. Above the fluid-gel main transition temperature, T(m) congruent with 23.4 degrees C, the particles could move on the surface of the vesicle, spontaneously (Brownian motion) or driven by an external force, either gravity or the laser beam's radiation pressure. From the analysis of the particle motions, we deduced the values of the membrane hydrodynamic shear viscosity, eta(s), and found that it would increase considerably near T(m). Below T(m), the long-distance motion of the particles was blocked. We performed experiments with two particles stuck on the membrane. By optical dynamometry, we measured the elastic resistance of the membrane to a variation in the interparticle distance and found that it would decrease considerably (down to zero) when the temperature was increased to T(m). We propose an interpretation relating the elastic response to the membrane curvature modulus, k(C). In this scheme, the two-bead dynamometry experiments provide a direct measurement of k(C) in the P'(beta) phase of lipid bilayers.     

Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations.

Differential polarized phase fluorometry has been used to investigate the depolarizing rotations of 1,6-diphenyl-1,3,5-hexatriene (DPH) in isotropic solvents and in lipid bilayers. For DPH dissolved in isotropic solvents, there is a precise agreement between the observed and predicted values for maximum differential tangents, indicating that in these media DPH is a free isotropic rotator. In lipid bilayers the tangent defects (i.e., the differences between the calculated and the observed maximum differential tangents) are too large to be explained by anisotropy in the depolarizing rotations but are accounted for by hindered isotropic torsional motions for the fluorophore [Weber, G (1978) Acta Phys. Pol A 54, 173]. This theory describes the depolarizing rotations of the fluorophore by its rotational rate R (in radians/second) and the limiting fluorescence anisotropy (r) at times long compared with the fluorescence lifetime. Through the combined use of both steady-state anisotropy measurements and differential phase measurements, we have demonstrated that one may obtain unique solutions for both R and r. For DPH embedded in vesicles prepared from dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholines, the depolarizing motions are highly hindered at temperatures below the transition temperature (Tc) but are unhindered above Tc. The apparent rotational rates of the probe do not change significantly at Tc. These data suggest that the changes observed in the steady-state anisotropy near Tc derive primarily from changes in the degree to which the probe's rotations are hindered, and only to a small extent from changes in rotational rate. For DPH embedded in bilayers that contained 25 mol % cholesterol, no clear transition occurred and the rotations appeared to be hindered at all temperatures. The rotational motions of DPH embedded in dioleolyphosphatidylcholine were found to be far less hindered, but the rotational rates were similar to those obtained in the saturated phosphatidylcholines. Finally, the data show that in an anisotropic environment, such as that of a lipid bilayer, steady-state fluorescence anisotropy measurements alone cannot yield quantitatively meaningful rotational rates. Extrapolation of steady-state aniosotropy data to the quantitation of membrane viscosity is therefore difficult, if not invalid; however, qualitative comparisons can be useful.     

Cholesterol-induced effects on the viscoelasticity of monoglyceride bilayers.

Changes in the viscoelastic properties of glycerol monooleate bilayers resulting from the incorporation of cholesterol into the membranes have been measured. The interface tension increases with the cholesterol concentration, reaching saturation for a 4.2:1 mole ratio of cholesterol:lipid in the film-forming solution. Incorporation of cholesterol in the membrane causes the appearance of a large intrinsic viscosity; this also increases with the sterol content of the membrane. Molecular models of lipid-sterol interactions and packing are considered to explain both the observed changes in membrane properties and similarities with comparable lipid systems.     

Investigation of secondary and tertiary structural changes of cytochrome c in complexes with anionic lipids using amide hydrogen exchange measurements: an FTIR study.

The structure of cytochrome c bound to anionic lipid membranes composed of dimyristoyl, dipalmitoyl, or dioleoyl phosphatidylglycerols, or of bovine heart cardiolipin, has been investigated by Fourier transform infrared spectroscopy. Only small changes in secondary structure, as registered by the amide I band of cytochrome c, were observed upon binding at temperatures below that of denaturation of the protein, and these were not coupled to the thermotropic phase transitions of the lipid. The denaturation temperature of the protein decreased by approximately 25-30 degrees upon binding, in a progression which correlated with that of the lipid phase transition temperatures, being approximately 7 degrees lower for complexes with dioleoyl than with dipalmitoyl phosphatidylglycerol. Large changes in the amide proton exchange characteristics, as monitored by the spectral shifts in the amide I band of the protein in D2O, were observed on binding cytochrome c to the lipid membranes. For the slowly exchanging population, the amide deuteration rates of the free protein were nearly independent of temperature, whereas those of the bound protein increased by up to two orders of magnitude over the temperature range from 10 to 40 degrees C. In addition, the extent of exchange differed between the bound and unbound protein. A structural transition in the bound protein was detected as a discontinuous step in Arrhenius plots of the deuterium exchange rates which occurred at a temperature in the region of 22 to 29 degrees C, depending on the lipid, far below that of denaturation. The temperature of this transition was determined by the physical state of the lipid, being 7 degrees lower for the lipids in the fluid state than for those in the gel state, and, for complexes with dimyristoyl phosphatidylglycerol, occurred at an intermediate temperature, being controlled by the lipid chain-melting transition at 27-28 degrees C. These results provide evidence for a coupling of the tertiary structure of the membrane-bound protein with the physical state of the membrane lipids.     

The effects of viscosity on gramicidin tryptophan rotational motion.

The rotational amplitude of gramicidin tryptophans was investigated as a function of temperature and viscosity in a variety of solvents using fluorescence spectroscopy. In 80% glycerol-ethanol, gramicidin behavior was similar to that of alpha helical globular proteins. In dioleoyl-phosphatidylcholine (DOPC) and egg-phosphatidylcholine bilayers, the rotational amplitude of the tryptophans remained constant from 5 degrees to 40 degrees C due to the large number of tryptophans participating in intermolecular aromatic ring stacking. In gel phase dimyristoyl-phosphatidylcholine (DMPC), the tryptophan rotations likewise do not respond to temperature and viscosity changes, presumably because of a combination of Trp 9 and 15 stacking and the high viscosity of the membrane. In fluid phase DMPC, stacking becomes disrupted as the temperature increases causing the change in tryptophan amplitude with temperature to be greater than allowed by the membrane. In n-octylglucoside micelles, ring interactions are also broken with heat. We conclude that membrane viscosity regulates both inter- and intramolecular gramicidin interactions but not in a straightforward manner.     

Rotational relaxation of 1,6-diphenylhexatriene in membrane lipids of cells acclimated to high and low growth temperatures.

Measurement of the time-resolved fluorescence depolarization of 1,6-diphenylhexatriene (DPH) in artificial bilayers of microsomal membrane lipids from Tetrahymena gives detailed information concerning the molecular motion of this probe and fluid properties of the membrane lipids which are obscured with steady-state methods. The rotational motion of DPH in these lipids from cells acclimated to 15 and 39.5 degrees C growth temperatures was anisotropic, which agrees with recent time-resolved studies of this probe in synthetic phospholipid systems. Evaluation of DPH polarization data obtained from these lipid fractions at their respective growth temperatures showed differences in physical properties which suggest that "viscosity", per se, of the microsomal lipids is not a strictly regulated as it is in prokaryotic systems. Rotational relaxation of DPH in 39.5 degrees C microsomal lipids measured at 15 degrees C is more complex than that of either lipid fraction measured at its actual growth temperature, suggesting that the probe has partitioned into two dissimilar environments within the bilayer. Similar effects are observed in the microsomes of 39.5 degrees C cells by freeze-fracture electron microscopy following rapid cooling to 15 degrees C. Under these conditions, two distinct regions are observed on the fracture faces, suggesting a correlation between lipid phase changes and alterations in membrane structure.