The excitable fluid mosaic

The Fluid Mosaic Model by Singer & Nicolson proposes that biological membranes consist of a fluid lipid layer into which integral proteins are embedded. The lipid membrane acts as a two-dimensional liquid in which the proteins can diffuse and interact. Until today, this view seems very reasonable and is the predominant picture in the literature. However, there exist broad melting transitions in biomembranes some 10–20 degrees below physiological temperatures that reach up to body temperature. Since they are found below body temperature, Singer & Nicolson did not pay any further attention to the melting process. But this is a valid view only as long as nothing happens. The transition temperature can be influenced by membrane tension, pH, ionic strength and other variables. Therefore, it is not generally correct that the physiological temperature is above this transition. The control over the membrane state by changing the intensive variables renders the membrane as a whole excitable. One expects phase behavior and domain formation that leads to protein sorting and changes in membrane function. Thus, the lipids become an active ingredient of the biological membrane. The melting transition affects the elastic constants of the membrane. This allows for the generation of propagating pulses in nerves and the formation of ion-channel-like pores in the lipid membranes. Here we show that on top of the fluid mosaic concept there exists a wealth of excitable phenomena that go beyond the original picture of Singer & Nicolson.¹     

A Non-classical Assembly Pathway of Escherichia coli Pore-forming Toxin Cytolysin A

Cytolysin A (ClyA) is an α-pore forming toxin from pathogenic Escherichia coli (E. coli) and Salmonella enterica. Here, we report that E. coli ClyA assembles into an oligomeric structure in solution in the absence of either bilayer membranes or detergents at physiological temperature. These oligomers can rearrange to create transmembrane pores when in contact with detergents or biological membranes. Intrinsic fluorescence measurements revealed that oligomers adopted an intermediate state found during the transition between monomer and transmembrane pore. These results indicate that the water-soluble oligomer represents a prepore intermediate state. Furthermore, we show that ClyA does not form transmembrane pores on E. coli lipid membranes. Because ClyA is delivered to the target host cell in an oligomeric conformation within outer membrane vesicles (OMVs), our findings suggest ClyA forms a prepore oligomeric structure independently of the lipid membrane within the OMV. The proposed model for ClyA represents a non-classical pathway to attack eukaryotic host cells.     

Dynamics properties of membrane proteins in native cell membranes revealed by solid-state NMR spectroscopy

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.     

Correlation between temperature range of growth and structural transitions in membranes and lipids of Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild-type Escherichia coli grown at different temperatures were labelled with 1,6-diphenyl-1,3,5-hexatriene and anlyzed using fluorescence polarization techniques. Lipids extracted from the membranes were similarly analyzed using fluorescence polarization. The thermotropic structural transition in outer membranes changed as a function of growth temperature. The structural transition in cytoplasmic membranes and lipids extracted from either cytoplasmic or outer membranes did not change with growth temperature. These data suggest that adaptive changes which occur in the outer membrane determine the temperature range of growth of E. coli. These changes apparently require alterations in outer membrane components other than phospholipids.     

Erratum

Escherichia coli cells (unsaturated fatty acid auxotroph) have been adapted to grow on branched-chain fatty acids. Membrane vesicles were isolated from cells grown on a mixture of branched-chain fatty acids isolated from the lipids of Bacillus subtilis (E. coli (B. subtilis) membranes) and on a pure synthetic anti-isononadecanoic acid (E. coli (aC19) membranes).We have shown, using wide-angle X-ray diffraction and differential scanning calorimetry, that the ordered state of the lipids is perturbed in the case of E. coli (aC19) membranes. The perturbation leads to the presence of a large wide-angle X-ray diffraction at 4.25–4.3 Å, as opposed to the presence of a sharp 4.2 Å reflection in unperturbed systems.We have shown, using freeze-fracture electron microscopy, that a protein segregation exists in the case of E. coli (aC19) membranes (at low temperature the integral membrane proteins aggregate in the membrane domains containing the disordered lipids); we do not observe such segregation in the case of E. coli (B. subtilis) membranes. We conclude that in cases where the branching of the fatty acids introduces a perturbation of the lipid order, the integral membrane proteins can still be accommodated in membrane domains containing the ‘perturbed’ ordered lipids.Finally, we have determined the rate of β-galactoside transport in E. coli (aC19) and E. coli (B. subtilis) membranes as a function of temperature. We have shown that, in both cases, the Arrhenius representations display an increased slope in the region of the disorder-to-order transition. We conclude that such an increased slope may have different origins. In the case of E. coli (aC19) membranes, it is the result of the aggregation of the β-galactoside carriers together with other integral membrane proteins which may lead to the inactivation of the carriers; in the case of E. coli (B. subtilis) membranes, it is the result of the partial immobilisation of the carriers embedded in a lipid environment, of which the fluidity, despite the perturbation of its lipid order, is still much less than that associated with lipids in a totally disordered state.     

Evidence for a transition in the cortical membranes of Paramecium

Ever since the pioneering studies in the 1960s and 70s, the importance of order transitions for cell membrane functions has remained a matter of debate. Recently, it has been proposed that the nonlinear stimulus-response curve of excitable cells, which manifests in all-or-none pulses (action potentials (AP)), is due to a transition in the cell membrane. Indeed, evidence for transitions has accumulated in plant cells and neurons, but studies with other excitable cells are expedient in order to show if this finding is of a general nature. Herein, we investigated intact, motile specimens of the “swimming neuron” Paramecium. The cellular membranes were labelled with the solvatochromic fluorophores LAURDAN or Di-4-ANEPPDHQ. Subsequently, a cell was trapped in a microfluidic channel and investigated by fluorescence spectroscopy. The generalized polarization (GP) of the fluorescence emission from cell cortical membranes (probably plasma and alveolar membranes) was extracted by an edge-finding algorithm. The thermo-optical state diagram, i.e. the dependence of GP on temperature, exhibited clear indications for a reversible transition. This transition had a width of ~10–15 °C and a midpoint that was located ~4 °C below the growth temperature. The state diagrams with LAURDAN and Di-4-ANEPPDHQ had widely identical characteristics. These results suggested that the cortical membranes of Paramecium reside in an order transition regime under physiological growth conditions. Based on these findings, membrane potential fluctuations, spontaneous depolarizing spikes, and thermal excitation of Paramecium was interpreted.     

An estimate of the minimum amount of fluid lipid required for the growth of escherichia coli

The lipid phase transition of Escherichia coli was studied by high sensitivity differential scanning calorimetry. A temperature sensitive unsaturated fatty acid auxotroph was used to obtain lipids with subnormal unsaturated fatty acid contents. From these studies it was concluded that E. coli can grow normally with as much as 20% of its membrane lipids in the ordered state but that if more than 55% of the lipids are ordered, growth ceases. Studies with wild-type cells show that the phase transition ends more than 10°C below the growth temperature when the growth temperature when the growth temperature is either 25°C or 37°C.     

Particle aggregation in photosynthetic membranes of the blue-green alga Anacystis nidulans

Freeze-fracture electron microscopy demonstrates that in photosynthetic membranes of the blue-green alga Anacystis nidulans quenched from a temperature below growth temperature, areas devoid of membrane particles occur. We suggest that this phenomenon is related to phase transitions in the photosynthetic membrane.     

A spin label study of E. coli membrane vesicles

The phase transition in E. coli membrane vesicles has been investigated by the spin labeling technique. N-oxyl-4′,4′-dimethyloxazolidine derivatives of stearic acid were incorporated into the vesicles. The results suggest that there are two phase transitions in these bacterial membrane vesicles (one at ≈20°C and the other at ≈30°C). These two phase transitions may be related to some of the functional properties of the membranes.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.     

The effect of alterations in the physical state of the membrane lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect on both the temperature range within which Acholeplasma laidlawii B cells can grow and on growth rates within the permissible temperature ranges. The minimum growth temperature of 8 °C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting, unsaturated fatty acids. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 $\text{kcal}\text{mol}$, but this value increases abruptly to 40 to 45 $\text{kcal}\text{mol}$ at temperatures below the phase transition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.     

Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy

Under physiological conditions, multicomponent biological membranes undergo structural changes which help define how the membrane functions. An understanding of biomembrane structure-function relations can be based on knowledge of the physical and chemical properties of pure phospholipid bilayers. Here, we have investigated phase transitions in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) bilayers. We demonstrated the existence of several phase transitions in DPPC and DOPC mica-supported bilayers by both atomic force microscopy imaging and force measurements. Supported DPPC bilayers show a broad L(beta)-L(alpha) transition. In addition to the main transition we observed structural changes both above and below main transition temperature, which include increase in bilayer coverage and changes in bilayer height. Force measurements provide valuable information on bilayer thickness and phase transitions and are in good agreement with atomic force microscopy imaging data. A De Gennes model was used to characterize the repulsive steric forces as the origin of supported bilayer elastic properties. Both electrostatic and steric forces contribute to the repulsive part of the force plot.     

Membrane deenergization by colicin K affects fluorescence of exogenously added but not biosynthetically esterified parinaric acid probes in Escherichia coli

Fluorescence of the conjugated polyene fatty acid, parinaric acid (PnA), was studied in membranes of Escherichia coli during deenergization by colicin K. The free fatty acid and biosynthetically esterified forms of cis-PnA (9,11,13,15-cis,trans,trans,cis-octadecatetraenoic acid), both of which are sensitive to E. coli lipid-phase transitions, were compared. When free cis-PnA was added exogenously to respiring bacteria, dissipation of the energized state of the membrane resulted in a dramatic increase in cis-PnA fluorescence; all-trans-PnA was much less sensitive. Neither spectral shifts nor a change in cis-PnA fluorescence polarization were observed. Analysis of the PnA content of extracellular fractions of deenergized and control cells revealed a difference in probe distribution: the membranes of energy-poisoned E. coli bound about 77% of exogenously added cis-PnA, whereas membranes of actively respiring controls bound only about 44%. No fluorescence enhancement was observed in cells centrifuged to remove unbound cis-PnA before colicin treatment. When cis-PnA was biosynthetically esterified to phospholipids of an unsaturated fatty acid auxotroph of E. coli, the fluorescence did not change during membrane deenergization. In double-probe experiments, membrane deenergization resulted in fluorescence enhancement of exogenously added N-phenyl-1-naphthylamine, without change in esterified PnA fluorescence. We conclude that deenergization of E. coli membranes leads to increased binding and fluorescence of exogenously added PnA and cannot be detected from within the inner and outer membranes by PnA esterified in vivo.     

Pressure and Temperature Dependence of Growth and Morphology of Escherichia coli: Experiments and Stochastic Model

We have investigated the growth of Escherichia coli, a mesophilic bacterium, as a function of pressure (P) and temperature (T). Escherichia coli can grow and divide in a wide range of pressure (1–400 atm) and temperature (23–40°C). For T > 30°C, the doubling time of E. coli increases exponentially with pressure and exhibits a departure from exponential behavior at pressures between 250 and 400 atm for all the temperatures studied in our experiments. The sharp change in doubling time is followed by a sharp change in phenotypic transition of E. coli at high pressures where bacterial cells switch to an elongating cell type. We propose a model that this phenotypic change in bacteria at high pressures is an irreversible stochastic process, whereas the switching probability to elongating cell type increases with increasing pressure. The model fits well the experimental data. We discuss our experimental results in the light of structural and thus functional changes in proteins and membranes.     

Pressure and Temperature Dependence of Growth and Morphology of?Escherichia coli: Experiments and Stochastic Model

We have investigated the growth of Escherichia coli, a mesophilic bacterium, as a function of pressure (P) and temperature (T). Escherichia coli can grow and divide in a wide range of pressure (1–400 atm) and temperature (23–40°C). For T > 30°C, the doubling time of E. coli increases exponentially with pressure and exhibits a departure from exponential behavior at pressures between 250 and 400 atm for all the temperatures studied in our experiments. The sharp change in doubling time is followed by a sharp change in phenotypic transition of E. coli at high pressures where bacterial cells switch to an elongating cell type. We propose a model that this phenotypic change in bacteria at high pressures is an irreversible stochastic process, whereas the switching probability to elongating cell type increases with increasing pressure. The model fits well the experimental data. We discuss our experimental results in the light of structural and thus functional changes in proteins and membranes.     

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca channels in the membrane

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca²⁺ homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca²⁺ in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca²⁺polyP) complexes. PHB(Ca²⁺polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA⁻ strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

Series of Concentration-Induced Phase Transitions in Cholesterol/Phosphatidylcholine Mixtures

In lipid membranes, temperature-induced transition from gel-to-fluid phase increases the lateral diffusion of the lipid molecules by three orders of magnitude. In cell membranes, a similar phase change may trigger the communication between the membrane components. Here concentration-induced phase transition properties of our recently developed statistical mechanical model of cholesterol/phospholipid mixtures are investigated. A slight (<1%) decrease in the model parameter values, controlling the lateral interaction energies, reveals the existence of a series of first- or second-order phase transitions. By weakening the lateral interactions first, the proportion of the ordered (i.e., superlattice) phase (A_reg) is slightly and continuously decreasing at every cholesterol mole fraction. Then sudden decreases in A_reg appear at the 0.18–0.26 range of cholesterol mole fractions. We point out that the sudden changes in A_reg represent first- or second-order concentration-induced phase transitions from fluid to superlattice and from superlattice to fluid phase. Sudden changes like these were detected in our previous experiments at 0.2, 0.222, and 0.25 sterol mole fractions in ergosterol/DMPC mixtures. By further decreasing the lateral interactions, the fluid phase will dominate throughout the 0.18–0.26 interval, whereas outside this interval sudden increases in A_reg may appear. Lipid composition-induced phase transitions as specified here should have far more important biological implications than temperature- or pressure-induced phase transitions. This is the case because temperature and pressure in cell membranes are largely invariant under physiological conditions.     

Lipid crystallization in senescent membranes from cotyledons

Lipid transition temperatures for rough and smooth microsomal membranes isolated from bean (Phaseolus vulgaris) cotyledon tissue at various stages of germination were determined by wide angle x-ray diffraction. The transition temperatures were established by recording diffraction patterns through a temperature series until a sharp x-ray reflection centered at a Bragg spacing of 4.15 Å and denoting the presence of crystalline lipid was discernible. For rough and smooth microsomes from 2-day-old tissue, the transitions occurred at 0 C and 3 C, respectively, indicating that at this early stage in the germination sequence the membrane lipid is entirely liquid-crystalline at physiological temperature. By the 4th day of germination, the transition temperatures had increased to 32 C for smooth microsomes and 35 C for rough microsomes, indicating that at 29 C, which was the growth temperature, portions of the membrane lipid were crystalline. During the later stages of germination, the transition temperature for smooth microsomes continued to rise through 44 C at day 7 to 56 C at day 9, by which time the cotyledons were extensively senescent and beginning to abscise. There was also a dramatic increase in the proportion of membrane lipid in the crystalline phase at 29 C. By contrast, the rough microsomes showed little change in transition temperature and only a slight increase in the proportion of crystalline lipid during this late period in germination. The data indicate that substantial amounts of the lipid is senescing membranes are crystalline even at physiological temperature. Moreover, there is a temporal correlation between the appearance of this crystallinity and loss of membrane function, suggesting that the two may be causally related.     

Acylation of monoacylglycerophosphoethanolamine in the inner and outer membranes of the envelope of an Escherichia coli K12 strain and its phospholipase A-deficient mutant

The site of the Escherichia coli envelope of the conversion of 1-acylglycero-3-phosphoethanolamine to diacylglycerophosphoethanolamine was explored, using two K12 strains with a wild-type phospholipid-degradative apparatus and a K12 mutant lacking detectable phospholipase A₁ and A₂ activity.Experiments with various radioactively labeled substrates show that acylation by crude envelope preparations as well as isolated inner and outer membranes of parent and mutant strains involves neither exogenous fatty acids nor a transacylation reaction with added monoacylglycerophosphoethanolamine. Furthermore, acylation exhibits no absolute requirement for added ATP and coenzyme A.Specific activity of acylating activity is the same in inner membrane preparations of parent and mutant strain and in outer membrane preparations of the mutant deficient in phospholipase A. Although clearly evident, net diacylglycerophosphoethanolamine formation by outer membranes of the parent strain, however, was about 6-fold less. This lower conversion may be attributed to activation during incubation of phospholipases A within the outer membrane, resulting in breakdown of the diacylcompound formed.Reacylation of lysophospholipids formed in the E. coli envelope by the action of endogenous or exogenous phospholipases A provides the organism with the potential of biochemically inexpensive repair and modification of the envelope phospholipids. Moreover, major phospholipids hydrolyzed in the outer membrane of E. coli can be resynthesized in the same location, without need for the transport of the products of hydrolysis to the lipid biosynthetic apparatus associated with the cytoplasmic membrane.