Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study.

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

Polymyxin binding to charged lipid membranes an example of cooperative lipid-protein interaction

The binding of polymyxin-B to lipid bilayer vesicles of synthesis phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescene probes to study the lipid phase transition.The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (with ΔT = 5°C) are observed. The upper transition (at T_u) is that of the pure lipid and the lower transition (at T₁) concerns the lipids bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1:1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin.With both fluorescence methods the fraction of lipid bound to polymxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction action of Ca²⁺ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca²⁺, polymyxin) interaction.A model is proposed which explains the association of polymyxing within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.     

Ca2+-induced lateral phase separations in phosphatidic acid-phosphatidylcholine membranes

The effects of Ca²⁺ on phosphatidic acid-phosphatidylcholine membranes have been studied using phospholipid spin labels. ESR spectra of spin-labeled phosphatidic acid-phosphatidylcholine membranes and phosphatidic acid-spin-labeled phosphatidylcholine membranes are exchange-broadened immediately upon addition of CaCl₂. These changes directly and conclusively indicate Ca²⁺-induced clustering of spin-labeled phosphatidylcholine and aggregation of spin-labeled phosphatidic acid bridged by Ca²⁺-chelation in the binary phopholipid membranes. In the Ca²⁺-chelated aggregates, the motions of the alkyl chains of phosphatidic acid are greatly reduced and the lipid molecules are more closely packed. The clusters and aggregates are formed in patches and the sizes are dependent on the fractions. Ba²⁺ and Sr²⁺ induce the lateral phase separations to the same extent as Ca²⁺. Mg²⁺ is also effective but to a lesser extent. In acid solutions (pH 5.5), the Ca²⁺-induced lateral phase separations are of slightly lesser extent than in alkaline solution (pH 7.9). These results are compared with those for phosphatidylserine-phosphatidylcholine membranes reported previously and necessary conditions for the lateral phase separations are discussed.     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

Cis-unsaturated fatty acids modulate the function of the cell-surface cAMP receptor of Dictyostelium discoideum

The binding of cAMP to the chemotactic cAMP receptor in intact Dictyostelium discoideum cells and isolated membranes is strongly inhibited by unsaturated fatty acids. In isolated membranes, cis-unsaturated fatty acids decreased the number of accessible cAMP binding sites, without significantly altering their affinity. Most potent were C₁₈ and C₂₀ cis-poly unsaturated fatty acids, like arachidonic acid, linoleic acid and linolenic acid. Trans-unsaturated fatty acid was less potent than its cis isomer, while saturated fatty acids did not affect the binding of cAMP to receptors at all. Oxidation reactions were not important for the effect of unsaturated fatty acids. When membranes were preincubated with millimolar concentrations of Ca²⁺, the effect of unsaturated fatty acids was strongly diminished. Mg²⁺ was ineffective. Ca²⁺, if presented after the incubation of membranes with unsaturated fatty acids, did not reverse the inhibitory effect. The specificity of the fatty acid effect, and the interference with Ca²⁺, but not Mg²⁺, suggest that the properties of the cAMP receptor are changed as a result of alterations in the lipid bilayer structure of the membrane.     

Alterations in Ca2+/Mg2+ ATPase activity upon treatment of heart sarcolemma with phospholipases

In order to examine the role of phospholipids in the activation of membrane bound Ca²⁺/Mg²⁺ ATPase, the activities of Ca²⁺ ATPase and Mg²⁺ ATPase were studied in heart sarcolemma after treatments with phospholipases A, C and D. The Mg²⁺ ATPase activity was decreased upon treating the sarcolemmal membranes with phospholipases, A, C and D; phospholipase A produced the most dramatic effect. The reduction in Mg², ATPase activity by each phospholipase treatment was associated with a decrease in the V_max value without any changes in the K_a value. The depression of Mg²⁺ ATPase in the phospholipase treated preparations was not found to be due to release of fatty acids in the medium and was not restored upon reconstitution of these membranes by the addition of synthetic phospholipids such as lecithin, lysolecithin or phosphatidic acid. In contrast to the Mg²⁺ ATPase, the sarcolemmal Ca²⁺ ATPase was affected only slightly by phospholipase treatments. The greater sensitivity of Mg^- ATPase to phospholipase treatments was also apparent when deoxycholate-treated preparations were employed. These results indicate that glycerophospholipids are required for the sarcolemmal Mg²⁺ ATPase activity to a greater extent in comparison to that for the Ca²⁺ ATPase activity and the phospholipids associated with Mg²⁺ ATPase are predominantly exposed at the outer surface of the membrane.     

Phosphatidic acid integrates calcium signaling and microtubule dynamics into regulating ABA-induced stomatal closure in Arabidopsis

Specific cellular components have been identified to function in abscisic acid (ABA) regulation of stomatal apertures, including calcium, the cytoskeleton, and phosphatidic acid. In this study, the regulation and dynamic organization of microtubules during ABA-induced stomatal closure by phospholipase D (PLD) and its product PA were investigated. ABA induced microtubule depolymerization and stomatal closure in wide-type (WT) Arabidopsis, whereas these processes were impaired in PLD mutant (pldα1). The microtubule-disrupting drugs oryzalin or propyzamide induced microtubule depolymerization, but did not affect the stomatal aperture, whereas their co-treatment with ABA resulted in stomatal closure in both WT and pldα1. In contrast, the microtubule-stabilizing drug paclitaxel arrested ABA-induced microtubule depolymerization and inhibited ABA-induced stomatal closure in both WT and pldα1. In pldα1, ABA-induced cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) elevation was partially blocked, and exogenous Ca²⁺-induced microtubule depolymerization and stomatal closure were impaired. These results suggested that PLDα1 and PA regulate microtubular organization and Ca²⁺ increases during ABA-induced stomatal closing and that crosstalk among signaling lipid, Ca²⁺, and microtubules are essential for ABA signaling.     

Fusion of phosphatidic acid-phosphatidylcholine mixed lipid vesicles

Ca²⁺-induced transformation of phosphatidylcholine-phosphatidic acid vesicles to larger bilayer structures has been examined using nuclear magnetic resonance, electron microscopy, gel permeation and radioisotope tracer techniques. For concentrated vesicle preparations where phosphatidic acid content remains less than 50% of total lipid, transformation to larger well defined unilamellar structures can be induced. The size of the product formed is dependent on phosphatidic acid content and on Ca²⁺ content when Ca²⁺ levels are between 0.3 and 1.0 mol ratios with respect to phosphatidic acid. During transformation bilayer composition remains unchanged and internal contents are retained in the final structure. These properties are indicative of concerted two vesicle and multiple vesicle fusions. The controllable and concerted fusions make the phosphatidic acid system suitable for further mechanistic studies.     

Ryanodine receptor 1 mediates Ca2+ transport and influences the biomechanical properties in RBCs

Ryanodine receptors (RyRs) are a family of Ca²⁺ channel proteins that mediate the massive release of Ca²⁺ from the endoplasmic reticulum into the cytoplasma. In the present study, we manipulated the incorporation of RyR1 into RBC membrane and investigated its influences on the intracellular Ca²⁺ ([Ca²⁺]_in) level and the biomechanical properties in RBCs. The incorporation of RyR1 into RBC membranes was demonstrated by both immunofluorescent staining and the change of [Ca²⁺]_in of RBCs. In the presence of RyR1, [Ca²⁺]_in showed biphasic changes, i.e., it increased with the extracellular Ca²⁺ ([Ca²⁺]_ex) up to 5 μM and then decreased with the further increase of [Ca²⁺]_ex. However, [Ca²⁺]_in remained constant in the absence of the RyR1. The results of biomechanical measurements on RBCs, including deformability, osmotic fragility, and membrane microviscosity, reflected similar biphasic changes of [Ca²⁺]_in mediated by RyR1 with the increases of [Ca²⁺]_ex. Therefore, it is believed that RyR1 can incorporate into RBC membrane in vitro, and mediate Ca²⁺ influx, and then regulate RBC biomechanical properties. This information suggests that RBCs may serve as a model to study the function of RyR1 as a Ca²⁺ release channel.     

A rapid stimulation of phosphatidylinositol metabolism in rabbit leukocytes by pseudomonal leukocidin

The time course experiments of ³²P_i-labelling and breakdown of phospholipids in rabbit leukocytes exposed to leukocidin from Pseudomonas aeruginosa suggested that the initial action of this toxin was to stimulate phosphatidic acid production, presumably by causing a rapid metabolic change of phosphatidylinositol (PI response) correlating with phosphatidylinositol-specific phospholipase C and 1,2-diacylglycerol kinase. It appears that a rapid formation of phosphatidic acid and degradation of polyphosphoinositides in leukocytes treated with the toxin might be related a Ca²⁺-movement from extra- and intracellular spaces, resuling in the activation of Ca²⁺-dependent enzymes involved in the leukocidic process.     

Kinetics of a Ca2+-triggered membrane aggregation reaction of phospholipid membranes

Ca²⁺ and other divalent cations can trigger aggregation of phospholipid vesicles containing phosphatidic acid or phosphatidylserine. The reaction, which can be detected by an increase in light scattering, has a critical dependence on the Ca²⁺ concentration, with a threshold near 4 mM Ca²⁺. This is the concentration for half-saturation of the polar head groups and for full neutralization of the membrane surface charge. The aggregation proceeds as a “polymerization” reaction, eventually forming such large aggregates that the vesicles precipitate. The stopped-flow rapid mixing technique was used to study the vesicle dimerization reaction which is the first step in the overall aggregation process. Vesicle dimerization resulted in a doubling of light scattering and had a vesicle concentration-dependent time constant ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) which varied between 0.4 and 2.0 s under the conditions of the study. Analysis of the dependence of the reaction amplitude and $\text{1}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ on the concentrations of vesicles and Ca²⁺ showed that the Ca²⁺ binding is fast, and that the dimerization proceeds by a mechanism in which the vesicles first collide to form an encounter complex followed by a slower conversion of the encounter complex to a stable complex. For phosphatidic acid vesicles, about 200–700 collisions are necessary to achieve a stable dimer. The rate-limiting step in the overall reaction in thus the transformation of the encounter complex into a stable complex, requiring 0.5 and 1.0 ms. The above-mentioned results are relatively insensitive to the type of divalent cation or to the choice of negatively charged lipid (phosphatidic acid or phosphatidylserine).Evidence is given that the stable complex is effected by Ca²⁺-mediated salt bridges between the two membranes and that the rate constant of the transformation step derives from the statistics of the distribution and the rate of redistribution of Ca²⁺-occupied polar head groups on the membrane surfaces. The relevance of these results to the problem of Ca²⁺-induced fusion of biological membranes is discussed.     

Dynamic fluorescence polarization studies on lipid mobilities in phospholipid vesicles in the presence of calcium mediators

The influence of Ca²⁺ mediators (nifedipine, verapamil and prostaglandin F_2α) on fluorescence polarization of l-anilino-8-napthalene-sulphonate in dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine liposomes was studied at various temperatures to understand the dynamic behaviour of membrane lipids. We also studied the effect of change in calcium concentration on the fluorescence polarization of the dye in the liposomes. Our results show increase in polarization (indicative of stiffening of the membrane) in the presence of Ca²⁺ ions. In the case of dimyristoyl phosphatidylcholine liposomes, all 3 drugs caused decrease in fluorescence polarization (increase in fluidity of the membrane) with or without Ca²⁺ ions in the medium. Contrary to this, in the case of dipalmitoyl phosphatidylcholine liposomes, the fluidization effect is observed for all the 3 drugs in the absence of Ca²⁺ ions; in the presence of Ca²⁺ ions stiffening is observed upon addition of nifedipine and verapamil which are antagonists, and fluidization is observed upon addition of prostaglandin F_2α. The role of drug-induced fluidity changes in membranes in therapy planning is discussed in the paper.     

Direct evidence for Ca++-induced lateral phase separation in black membranes of lipid mixtures by the analysis of gramicidin A single-channels

Single-channel conductance fluctuations are analysed for gramicidin A incorporated into binary-mixed black lipid membranes of charged phosphatidic acid and neutral lecithin in different molar ratios. At very low Ca⁺⁺ concentrations in the electrolyte (i.e. in the presence of EDTA) homogeneous lipid mixtures are identified through their conductance and life time probability distributions for integral gramicidin pores. As for the pure lipid components, the conductance histograms each show a single maximum with regular width and for all channels a single mean lifetime is found.For Ca⁺⁺-levels (10^-6–10^-5 M) that are close to the critical demixing concentration (10^-4 M) unusually broad conductance distributions and reduced lifetimes are found provided the PC content, x, of the membrane is close to the critical mixture (x _crit0.5). We interpret this as a first example of the coupling of a membrane function (the transport of ions) to a lipid matrix with locally fluctuating composition close to a critical demixing point.For the conductance histogram of gramicidin A in an equimolar mixture of PA and PC shows two well-separated maxima. A correlation analysis between conductance and lifetime of the single pores shows that the two channel populations also differ significantly in their mean channel lifetime, ^*. This finding is interpreted as being direct evidence for Ca⁺⁺-induced lateral phase separation in black lipid membranes, as has been postulated recently.Abbreviations used HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid - EDTA ethylenediaminetetraacetic acid     

EFFECTS OF ACETYLCHOLINE ON THE IN CORPORATION OF [32P]ORTHOPHOSPHATE IN VITRO INTO THE PHOSPHOLIPIDS OF SUBSYNAPTOSOMAL MEMBRANES FROM GUINEA-PIG BRAIN

-Synaptosomes prepared from guinea-pig cerebral cortex were incubated with ³²P₁ in a medium with or without 10^?4 M-acetylcholine and 10^?4 M-eserine. They were then subjected to osmotic shock and density-gradient centrifugation for the preparation of subsynaptosomal fractions and the phospholipids of each fraction were separated by two-dimensional thin-layer chromatography. The fraction containing synaptic vesicles and that containing mitochondria were the most highly labelled of the sub-synaptosomal fractions. Phosphatidic acid followed by phosphatidylinositol had the highest specific activity of the phospholipids studied. Acetylcholine caused a marked increase in the specific activity of the vesicular but not of the mitochondrial phosphatidic acid. Phosphatidylinositol specific activity also increased in the presence of acetylcholine but the increase was more reproducible in the fraction containing microsomal membranes than in the vesicle fraction. The other phospholipids were relatively poorly labelled and no effect of acetylcholine on the incorporation of ³²P₁ into these lipids could be detected. Acetylcholine also caused a decrease in the amount of phosphatidic acid in the synaptic vesicles.     

Cation binding to brain plasma membranes: An evaluation of the use of anionic fluorescent probes

Cation binding to brain plasma membranes has been studied using anionic sulfonate fluorescent probes. Ion affinity sequences follow the order Mg²⁺ > Ca²⁺ ? K⁺ > Cs⁺ > Na⁺ > Li⁺. The order of effectiveness, in increasing probe fluorescence, is the reverse of the affinity sequence for ions of the same charge. The affinity orders for erythrocyte membranes and dipalmitoyl lecithin are Mg²⁺ > Ca²⁺ ? Cs⁺ > K⁺ > Na⁺ > Li⁺ and Mg²⁺ > Ca²⁺ ? Li⁺ > Na⁺ > K⁺ > Cs⁺. These sequence variations are related to the differences in the nature of the ion binding sites. Heterogeneity in ion binding sites is demonstrated. Evidence is presented for the role of proteins in binding hydrophobic probes. The problem of separating specific conformational effects on ion binding from nonspecific charge neutralization effects is discussed. Pyrene excimer fluoresence rules out the possibility of extensive changes in mobility in the lipid phase on cation binding. Tetrodotoxin has been shown to inhibit Li⁺-, Na⁺-, and K⁺-induced fluorescence enancements of 1-anilino-8-naphthalene sulfonate bound to brain membranes.     

Ca2+-dependent and Ca2+-independent mechanisms for phosphatidylinositol hydrolysis and 32P-labeling during cholinergic stimulation of the rat submaxillary gland in vitro

Ca²⁺ was required for carbachol-induced decreases in phosphatidylinositol (PI) and increases in phosphatidic acid (PA) concentrations during incubation of rat submaxillary gland fragments, but was not required for increases in [³²P]P_i incorporation into these phospholipids. Like carbachol, A23187 provoked a Ca²⁺-dependent decrease in PI mass. These results suggest concomitant operation of two separate mechanisms for stimulating PI hydrolysis and ³²P labeling of PA and PI during carbachol action: one mechanism is not dependent on external Ca²⁺ and is manifested by rapid labeling in a relatively small PA-PI pool; the other mechanism is dependent on Ca²⁺ and involves a large PA-PI pool which appears to have a relatively slow renewal (labeling) rate.     

THE EFFECT OF ELECTRICAL STIMULATION ON THE TURNOVER OF PHOSPHATIDIC ACID IN SYNAPTOSOMES FROM GUINEA-PIG BRAIN

Synaptosomes prepared from guinea-pig cerebral cortex were suspended in a medium containing [³²P]orthophosphate and subjected to electrical stimulation. When the synaptosomal phospholipids were subsequently separated, the most highly labelled was phosphatidic acid and electrical stimulation over a 10 min period increased incorporation of ³²P₁ into this lipid. Stimulated synaptosomes were osmotically lysed and subsynaptosomal fractions isolated. The electrically stimulated increase in phosphatidic acid labelling was localized in a fraction enriched in synaptic vesicles. This phospholipid effect was not merely a reflection of an increased specific radioactivity of synaptosomal ATP, due to the electrically stimulated increase in respiration. The time course of the phosphatidic acid effect suggests that it is synchronous with release of transmitter.     

A lipid requirement for the (Ca2+ + Mg2+)-activated ATPase of erythrocyte membranes.

The lipid requirement of the (Ca²⁺ + Mg²⁺)-stimulated ATPase of human erythrocytes has been studied. The enzyme activity was lost after removal of the phospholipids using phospholipase A₂ from Naja naja and serum albumin. Optimal restoration of the (Ca²⁺ + Mg²⁺)-ATPase activity in the partially lipid-depleted membranes was obtained with oleate. The reactivation was not due to the removal of a permeability barrier for ATP, since lysolecithin or cholate did not show latent activity. Reactivation was also obtained with several negatively charged phospholipids. Among the ones normally found in the erythrocyte membranes, only phosphatidyl serine reactivated significantly.     

The effect of exogenous glycerophospholipids on the fluorescence polarization ratios of Escherichia coli cells labelled with diphenylhexatriene

Saturated, unsaturated, and short acyl chain analogues of phosphatidylcholine and phosphatidylcholine and phosphatidylethanolamine were incorporated into a deep heptoseless mutant of Escherichia coli, strain D21F2, and into the parent wild-type strain, K12. Normal and lipid-treated cells or lipid extracts from such cells were labelled with diphenylhexatriene and their fluorescence polarization ratios were measured as a function of temperature. Incorporations of dipalmitoyl analogues of phosphatidylethanolamine and/or phosphatidylcholine in the presence of Ca²⁺ caused an increase in polarization ratios over a wide temperature range and the appearance of new phase transitions at 25–30°C as measured in whole D21F2 cells. Incorporation into D21F2 of the dioleoyl analogues of these glycerophospholipids under similar conditions had the opposite effect on the polarization ratios and, in the case of dioleoylphosphatidylethanolamine, caused the occurrence of a new phase transition at 20°C. Incorporation of these same lipids in K12 cells, in the presence of Ca²⁺, caused changes in the polarization ratios similar to those recorded for D21F2 cells when measurements were made on whole cells. Furthermore incorporation of didecanoyl-phosphatidylcholine in wild-type cells, in the presence of Ca²⁺, substantially decreased the polarization ratio and broadened the phase transition as could be measured with cell preparations. Since Ca²⁺ stimulates incorporation of lipid, the changes in polarization ratio were always greater when cells had been exposed to exogenous lipid in the presence of this cation. However, even in cells not treated with lipid, Ca²⁺ caused increases in the polarization ratio and affected the thermotropic structural transitions. The polarization ratios of extracted lipids were always reduced when compared to whole cells. Generally there was an attenuation of any differences in polarization ratio between normal and glycerophospholipid-treated samples. Extracted lipids also displayed broadened phase transitions. The results as a whole indicated that E. coli cells respond to the uptake of lipid and to the presence of Ca²⁺ by changes in their thermotropic mesomorphic behaviour. These changes reflect to a large extent the fluidity of the incorporated lipid and are exerted on a structural system the phase transitions of which are strongly influenced by the presence of non lipid components in the membrane.