Is the vertical disposition of Mycoplasma membrane proteins affected by membrane fluidity?

The influence of the physical state of the membrane lipid matrix on the vertical disposition of membrane proteins was studied with Acholeplasma laidlawii. Changes in membrane fluidity were brought about by altering the fatty acid composition of membrane lipids, by changing the growth temperature, by aging of cultures and by inducing changes in the membrane lipid-to-protein ratio through treatment with chloramphenicol. The lactoperoxidase-mediated iodination technique was used to label membrane proteins exposed to the aqueous surroundings. The degree of exposure of the iodine-binding sites of membrane proteins on the external surface of intact cells was found to undergo significant changes on varying growth conditions, but the changes could not be consistently correlated with changes in membrane fluidity, nor were they discernible on iodination of isolated membranes.     

Inner field compensation as a tool for the characterization of asymmetric membranes and Peptide-membrane interactions

Symmetric and asymmetric planar lipid bilayers prepared according to the Montal-Mueller method are a powerful tool to characterize peptide-membrane interactions. Several electrical properties of lipid bilayers such as membrane current, membrane capacitance, and the inner membrane potential differences and their changes can be deduced. The time-resolved determination of peptide-induced changes in membrane capacitance and inner membrane potential difference are of high importance for the characterization of peptide-membrane interactions. Intercalation and accumulation of peptides lead to changes in membrane capacitance, and membrane interaction of charged peptides induces changes in the charge distribution within the membrane and with that to changes in the membrane potential profile. In this study, we establish time-resolved measurements of the capacitance minimization potential DeltaPsi on various asymmetric planar lipid bilayers using the inner field compensation method. The results are compared to the respective ones of inner membrane potential differences DeltaPhi determined from ion carrier transport measurements. Finally, the time courses of membrane capacitances and of DeltaPsi have been used to characterize the interaction of cathelicidins with reconstituted lipid matrices of various Gram-negative bacteria.     

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.     

Elastic energy of curvature-driven bump formation on red blood cell membrane.

Model calculations were performed to explore quantitative aspects of the discocyte-echinocyte shape transformation in red blood cells. The shape transformation was assumed to be driven by changes in the preferred curvature of the membrane bilayer and opposed by the elastic shear rigidity of the membrane skeleton. The energy required for echinocyte bump formation was calculated for a range of bump shapes for different preferred curvatures. Energy minima corresponding to nonzero bump heights were found when the stress-free area difference between the membrane leaflets or the spontaneous curvature of the membrane became sufficiently large, but the calculations predict that the membrane can tolerate significant differences in the resting areas of the inner and outer leaflets or significant spontaneous curvature without visible changes in shape. Thus, if the cell is near the threshold for bump formation, the calculations predict that small changes in membrane properties would produce large changes in cellular geometry. These results provide a rational framework for interpreting observations of shape transformations in red cells and for understanding the mechanism by which small changes in membrane elastic properties might lead to significant changes in geometry.     

Phospholipase activity and plasma membrane homeostasis

Cyclic changes in the physiochemical state of the plasma membrane appear to be necessary for the normal functioning of the cell, especially with respect to division and differentiation. Such changes require a flexible membrane lipid composition to permit the necessary sequence of physicochemical changes to occur during these cellular events. This flexibility can be lost as a result of peroxidation-induced cross linking of membrane constituents, which prevents the normal physico-chemical membrane changes from occurring, resulting in abnormal cellular function. It is proposed that phospholipase A2 and C form a mutually regulatory enzyme system playing an important role with respect to the maintenance of membrane composition and flexibility.     

Interpretations of the effects of pH on the spectra of purple membrane.

The absorption and circular dichroism of the purple membrane in solution and the linear and circular dichroism of the purple membrane oriented in a film were used to detect changes in the membrane protein structure and membrane organization in the pH range of 2.4 to 12.6. Main findings are (a) the membrane protein structure is stable at every level of organization to pH changes over the range of 5.0 to 8.5. (b) Tertiary structural changes occur in the membrane protein structure in the pH range of 2.4 to 5.0 and 8.5 to 11.8 without any secondary structural involvement. (c) An irreversible change occurs in the membrane organization in the pH range of 11.8 to 12.6 involving large tertiary and secondary structural changes in the membrane protein. (d) The retinyl chromophore is influenced by a nearby ionizable group. (e) The membrane crystalline structure is highly stable to pH perturbation except at the high pH range of 11.0 to 11.8.     

Tension sensitivity of prestin: comparison with the membrane motor in outer hair cells

The membrane motor in outer hair cells undergoes conformational transitions involving charge displacement of approximately 0.8 e across the membrane and changes of approximately 4 nm(2) in its membrane area. Previous reports have established that the charge transfer in the membrane motor and that in prestin, a membrane protein in the plasma membrane of outer hair cells, are approximately equal. Here, we determine the membrane area changes based on its sensitivity to membrane tension. We found that prestin does undergo area changes and that the magnitude is approximately 1 nm(2), smaller than the value 4 nm(2) for outer hair cell motor. This result confirms that prestin is a protein that functions as a membrane motor based on piezoelectricity. The discrepancy in the magnitude could suggest a prestin-containing complex in outer hair cells.     

Apical membrane rupture and backward bile flooding in acetaminophen-induced hepatocyte necrosis

Morphological changes of hepatocyte death have so far only been described on cells in culture or in tissue sections. Using a high-resolution and high-magnification multiphoton microscopic system, we recorded in living mice serial changes of acetaminophen (APAP)-induced hepatocyte necrosis in relevance to metabolism of a fluorogenic bile solute. Initial changes of hepatocyte injury included basal membrane disruption and loss of mitochondrial membrane potential. An overwhelming event of rupture at adjacent apical membrane resulting in flooding of bile into these hepatocytes might ensue. Belbs formed on basal membrane and then dislodged into the sinusoid circulation. Transmission electron microscopy disclosed a necrotic hepatocyte depicting well the changes after apical membrane rupture and bile flooding. Administration of the antidote N-acetylcysteine dramatically reduced the occurrence of apical membrane rupture. The present results demonstrated a hidden but critical step of apical membrane rupture leading to irreversible APAP-induced hepatocyte injury.     

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.     

2-D gel comparison of membrane proteins from freshly isolated and cultured fetal and adult hepatocytes

Labelled membrane proteins from freshly isolated or over-night cultured rat hepatocytes were compared using 2-D gel electrophoresis. The membrane protein patterns changed rapidly after culturing, some changes being apparent after 5 hours in culture. These changes included the disappearance of a number of membrane proteins and the apparent altered glycosylation of others. Stability of hepatocyte membrane proteins in culture did not appear to be related to the stage of hepatocyte differentiation. A recognition that rapid changes in the membrane protein composition may occur after culturing is important for those wishing to use cultured cells to study membrane-associated functions.     

Effect of lipid composition changes on carbocyanine dye fluorescent response

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.     

The membrane potential of the cellular slime mold Dictyostelium discoideum is mainly generated by an electrogenic proton pump

Trans membrane potential or ionic current changes may play a role in signal transduction and differentiation in the cellular slime mold dictyostelium discoideum. Therefore, the contribution of electrogenic ion pumps to the membrane potential of D. discoideum cells was investigated. the (negative) peak-value of the rapid potential transient, seen upon microelectrode impalement, was used to detect membrane potential changes upon changes in the external pH in the range of 5.5 to 8.0. The membrane potential was close to the Nernstian potential for protons over the pH range 5.5 to 7.5. The acid-induced changes in membrane potential were consistent with outward-proton pumping. The maximal membrane potential was at pH 7.5. Furthermore, the proton pump inhibitors diethylstilbestrol, miconazole and zearalenone directly depolarize the membrane. Cyanide and temperature decrease cause membrane depolarization as well. During recovery from cyanide poisoning a H+ efflux is present. From these measurements we conclude that the membrane potential of d. discoideum cells is mainly generated by an electrogenic proton pump. Measurements in cells with different extracellular potassium and H+ concentrations suggest a role for potassium in the function of the electrogenic proton pump. These results provide a framework for future research towards a possible role for the proton pump in signal transduction and differentiation.     

Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence

In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.     

The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane

The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.     

PROTONATION AND CHLOROPLAST MEMBRANE STRUCTURE

Light changes the structure of chloroplasts. This effect was investigated by high resolution electron microscopy, photometric methods, and chemical modification. (a) A reversible contraction of chloroplast membrane occurs upon illumination, dark titration with H⁺, or increasing osmolarity. These gross structural changes arise from a flattening of the thylakoids, with a corresponding decrease in the spacing between membranes. Microdensitometry showed that illumination or dark addition of H⁺ resulted in a 13–23% decrease in membrane thickness. Osmotically contracted chloroplasts do not show this effect. (b) Rapid glutaraldehyde fixation during actual experiments revealed that transmission changes are closely correlated with the spacing changes and therefore reflect an osmotic mechanism, whereas the light scattering changes have kinetics most similar to changes in membrane thickness or conformation. (c) Kinetic analysis of light scattering and transmission changes with the changes in fluorescence of anilinonaphthalene sulfonic acid bound to membranes revealed that fluorescence preceded light scattering or transmission changes. (d) It is concluded that the temporal sequence of events following illumination probably are protonation, changes in the environment within the membrane, change in membrane thickness, change in internal osmolarity accompanying ion movements with consequent collapse and flattening of thylakoid, change in the gross morphology of the inner chloroplast membrane system, and change in the gross morphology of whole chloroplasts.     

Label-free imaging of membrane potential using membrane electromotility

Electrical activity may cause observable changes in a cell's structure in the absence of exogenous reporter molecules. In this work, we report a low-coherence interferometric microscopy technique that can detect an optical signal correlated with the membrane potential changes in individual mammalian cells without exogenous labels. By measuring milliradian-scale phase shifts in the transmitted light, we can detect changes in the cells' membrane potential. We find that the observed optical signals are due to membrane electromotility, which causes the cells to deform in response to the membrane potential changes. We demonstrate wide-field imaging of the propagation of electrical stimuli in gap-junction-coupled cell networks. Membrane electromotility-induced cell deformation may be useful as a reporter of electrical activity.     

A histochemical study about changes in rat liver plasma membrane enzyme activities after galactosamine administration.

In rats changes in plasma membrane enzyme activities due to Gal-N intoxication were studied by enzymehistochemical methods. The bile canalicular 5'-nucleotidase and nucleoside polyphosphatase activities decreased; the sinusoidal 5'-nucleotidase remained unchanged. The bile canalicular leucyl-beta-naphthyl-amidase showed an increase in activity; the alkaline phosphatase activity remained unchanged. In contrast to the spotty necrosis, changes in plasma membrane enzyme activities were seen in all liver cells, suggesting that changes of these activities, occurring after Gal-N treatment, do not correlate with cell death. The conclusion was drawn that the deviations of the enzyme activities might be due to changes in the lipid environment of the enzyme proteins in the membrane. With the exception of alkaline phosphatase, partial hepatectomy caused the same changes in enzyme activities as did Gal-N intoxication. Nevertheless Gal-N administration to partial hepatectomized rats did not lead to hepatic necrosis. Galactose given simultaneously or within two hours after Gal-N prevented both changes in plasma membrane enzyme activities and hepatocellular damage. This suggests an important role of galactolipids and galactoproteins in the plasma membrane alterations.     

Characteristics of the effect of implantation of the human ApoA1 gene on the condition of hepatocytes in rats of varying age

The results obtained show the essential changes in functional state of hepatocyte's plasmatic membrane due to the implantation of human ApoA1 gene to the rat liver. The changes in phospholipid composition, hyperpolarization, increase in activity of membrane bound enzymes, cytochrome P-450 and biosynthesis of liver total proteins have been found. The essential changes characterizing cell effect were more marked in the adult rats, and membrane effect in the old ones.     

Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors

This study examines whether changes in cGMP concentration initiated by illumination of frog rod photoreceptors occur rapidly enough to implicate cGMP as an intermediate between rhodopsin activation in the disc membrane and permeability changes in the plasma membrane. Previous studies using whole retinas or isolated outer segments have provided conflicting evidence on the role of cGMP in the initial events of phototransduction. The rod photoreceptor preparation employed in this work consists of purified suspensions of outer segments still attached to the mitochondria-rich ellipsoid portion of the inner segment. These photoreceptors are known to retain normal electrophysiological responses to illumination and have cGMP levels comparable to those measured in the intact retina. When examined under several different conditions, changes in cGMP concentrations were found to occur as rapidly or more rapidly than the suppression of the membrane dark current. Subsecond changes in cGMP concentration were analyzed with a rapid quench apparatus and confirmed by comparison with a rapid freezing technique. In a 1 mM Ca2+ Ringer's solution, cGMP levels decrease to 65% of their final extent within 200 ms after bright illumination; changes in membrane dark current follow a similar time course. When the light intensity is decreased to 8000 rhodopsins bleached per rod per s, the light-induced cGMP decrease is completed within 50 ms, with 7 X 10(5) cGMP molecules hydrolyzed per rhodopsin bleached. During this time the dark current has not yet begun to change. Thus, under physiological conditions it is clear that changes in cGMP concentration precede permeability changes at the plasma membrane. The correlation of rapid changes in cGMP levels with changes in membrane current leave open the possibility that changes in cGMP concentration may be an obligatory step in the reaction sequence linking rhodopsin activation by light and the resultant decrease in sodium permeability of the plasma membrane.     

Molecular mechanism of general anesthesia: I. Fluorescence studies in mitochondrial membranes

We have tested the working hypothesis that anesthetics, by labilizing lipid-protein interactions, induce conformational changes in membrane proteins involved in the transmission of neural impulses. In the first communication of this series we report that general anesthetics induce changes in the fluorescence of the probes ANS and NPN in model membranes, lipid vesicles and mitochondria. The changes observed concern the quantumyield but not the position of the emission maximum. Such changes may be interpreted as due to fluidization of the membrane core (NPN), accompanied by variable effects in the membrane surface(ANS).