Interplay of packing and flip-flop in local bilayer deformation. How phosphatidylglycerol could rescue mitochondrial function in a cardiolipin-deficient yeast mutant

In a previous work, we have shown that a spatially localized transmembrane pH gradient, produced by acid micro-injection near the external side of cardiolipin-containing giant unilamellar vesicles, leads to the formation of tubules that retract after the dissipation of this gradient. These tubules have morphologies similar to mitochondrial cristae. The tubulation effect is due to direct phospholipid packing modification in the outer leaflet that is promoted by protonation of cardiolipin headgroups. Here we compare the case of cardiolipin-containing giant unilamellar vesicles with that of phosphatidylglycerol-containing giant unilamellar vesicles. Local acidification also promotes formation of tubules in the latter. However, compared to cardiolipin-containing giant unilamellar vesicles the tubules are longer, exhibit a visible pearling and have a much longer lifetime after acid micro-injection is stopped. We attribute these differences to an additional mechanism that increases monolayer surface imbalance, namely inward PG flip-flop promoted by the local transmembrane pH-gradient. Simulations using a fully non-linear membrane model as well as geometrical calculations are in agreement with this hypothesis. Interestingly, among yeast mutants deficient in cardiolipin biosynthesis, only the crd1-null mutant, which accumulates phosphatidylglycerol, displays significant mitochondrial activity. Our work provides a possible explanation of such a property and further emphasizes the salient role of specific lipids in mitochondrial function.Comment: 28 pages, 10 figure

Lipid membrane deformation in response to a local pH modification: theory and experiments

We study the deformation of a lipid membrane in response to a local pH modification. Experimentally, a basic solution is microinjected close to a giant unilamellar vesicle. A local deformation appears in the zone of the membrane that is closest to the micropipette, and relaxes when the injection is stopped. A theoretical description of this phenomenon is provided. It takes fully into account the spatiotemporal evolution of the concentration of hydroxide ions during and after the microinjection, as well as the linear dynamics of the membrane. This description applies to a local injection of any substance that reacts reversibly with the membrane lipids. We compare experimental data obtained in the domain of small deformations to the results of our linear description, and we obtain a good agreement between theory and experiments. In addition, we present direct experimental observations of the pH profile on the membrane during and after the microinjection, using pH-sensitive fluorescent lipids.Comment: 11 pages, 8 figure

Dynamical membrane curvature instability controlled by intermonolayer friction

We study a dynamical curvature instability caused by a local chemical modification of a phospholipid membrane. In our experiments, a basic solution is microinjected close to a giant unilamellar vesicle, which induces a local chemical modification of some lipids in the external monolayer of the membrane. This modification causes a local deformation of the vesicle, which then relaxes. We present a theoretical description of this instability, taking into account both the change of the equilibrium lipid density and the change of the spontaneous membrane curvature induced by the chemical modification. We show that these two types of changes of the membrane properties yield different dynamics. In contrast, it is impossible to distinguish them when studying the equilibrium shape of a vesicle subjected to a global modification. In our model, the longest relaxation timescale is related to the intermonolayer friction, which plays an important part when there is a change in the equilibrium density in one monolayer. We compare our experimental results to the predictions of our model by fitting the measured time evolution of the deformation height to the solution of our dynamical equations. We obtain good agreement between theory and experiments. Our fits enable us to estimate the intermonolayer friction coefficient, yielding values that are consistent with previous measurements

On the possible structural role of single chain sphingolipids Sphingosine and Sphingosine 1-phosphate in the amyloid-β peptide interactions with membranes. Consequences for Alzheimer’s disease development

International audienceA strong interplay between the neurodegerative effects of the amyloid β (Aβ) peptides and the cell lipid composition or metabolism has been evidenced in Alzheimer’s disease. This appears to be, in part, related to Aβ-membrane interactions. Recently, an influence of the two cell fate-modulating single-chain sphingolipids, sphingosine (Sph) and sphingosine 1-phosphate (S1P), on AD-related mechanisms has been reported. We have investigated the influence of Sph and S1P on the interaction of Aβ(1–42) with lipid model membranes. A fluorescent Aβ(1–42) binds to egg phosphatidylcholine (EPC) giant unilamellar vesicles containing Sph or S1P. With Sph, gel microdomains are present at low temperature and Aβ(1–42) binds preferentially to these domains, especially at their boundaries. With S1P, which displays single lipid phase morphology, Aβ(1–42) binding is uniform. The binding of Aβ(1–42) to EPC/sphingolipid large unilamellar vesicles was investigated by spectrofluorimetry using the 2 probes Laurdan and di-ANEPPS. With most lipid compositions the binding of Aβ(1–42) to LUVs appears superficial. However, with Sph, a deeper membrane penetration is observed. This deeper interaction is reversed to superficial by the simultaneous presence of S1P. It is suggested that the influence of single-chain sphingolipids in AD might be related to a selective interaction of Aβ(1–42) with sphingosine in membranes, that is antagonized by S1P. Such interaction might occur intracellularly for Aβ(1–42) monomers or oligomers and/or extracellularly for Aβ still part of APP. Aβ(1–42) might also influence microdomains by binding to their boundaries. The influence of the Sph/S1P balance on the Alzheimer pathology might be related in part to the differential interactions of Aβ(1–42) with Sph and S1P and their effects on membrane domains

Macroscopic consequences of the action of phospholipase C on giant unilamellar liposomes.

Macroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10-100 microm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution of the microscope. The presence of N-palmitoyl-sphingomyelin (mole fraction X = 0.25) in the GUVs did not affect the outcome of the PC-PLC reaction. In addition to hydrolysis, PC-PLC induced adhesion of vicinal vesicles. When multilamellar SOPC vesicles were used only a minor decrease in their diameter was evident suggesting that PC-PLC can exert its hydrolytic activity only in the outer monolayer. A series of stepwise shrinkages was observed also for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) GUVs above their main phase transition temperature, T(m), i.e., when the bilayer is in the liquid crystalline state. However, this process was not observed for DMPC GUVs in the gel state, below T(m). These results are supported by the enhanced activity of PC-PLC upon exceeding T(m) of DMPC large unilamellar vesicles (diameter approximately 0.1 microm) used as a substrate. Studies on SOPC monolayers revealed that PC-PLC can exert its hydrolytic activity only at surface pressures below approximately 30 mN/m. Accordingly, the lack of changes in the gel state DMPC GUVs could be explained by the equilibrium lateral pressure in these vesicles exceeding this critical value

The Alzheimer's disease amyloid-β peptide affects the size-dynamics of raft-mimicking Lo domains in GM1-containing lipid bilayers

International audience† Electronic supplementary information (ESI) available: Films illustrating the effect of A(1-42) on photoinduced spinodal decomposition in GUVs, speed acceletated x 12: Movie_1_GUVs_PC-SM-Chol_10%GM1_withoutAb_20Cx12.avi Movie_2_GUVs_PC-SM-Chol_10%GM1_withAb_20Cx12.avi, Movie_3_GUVs_PC-SM-Chol_10%GM1_withoutAb_14Cx12.avi Movie_4_GUVs_PC-SM-Chol_10%GM1_withAb_14Cx12.avi Alzheimer's disease (AD) is characterized by the overproduction of the amyloid- peptide (A) which forms fibrils under the influence of raft microdomains containing the ganglioside GM1. Raft-mimicking artificial liquid ordered (Lo) domains containing GM1 enhance amyloid- polymerization. Other experiments suggest that A binds preferably to the non-raft liquid disordered (Ld) phase rather than to the Lo phase in the presence of GM1. Here, the interaction of A(1-42) with GM1-containing biphasic Lo-Ld giant vesicles was investigated. Fluorescence colocalisation experiments confirm that A(1-42) binds preferentially to the Ld phase. The effect of A(1-42) on Lo-Ld size dynamics was studied using photoinduced spinodal decomposition which mimics the nanodomain-microdomain raft coalescence. A affects the kinetics of the coarsening phase and the size of the resulting microdomains. The effect depends on which phase is majoritary: when Lo microdomains are formed inside an Ld phase, their growth rate becomes slower and their final size smaller in the presence of A(1-42), whereas when Ld microdomains are formed inside an Lo phase, growth rate becomes faster and final size larger. Fluorimetric measurements on large vesicles using the probe LAURDAN indicate that A(1-42) binding respectively increases or decreases the packing of the Ld phase in the presence or absence of GM1. The differential effects of A on spinodal decomposition are accordingly interpreted as resulting from distinct effects of the peptide on Lo-Ld line tension modulated by GM1. Such modulating effect of A on domain dynamics could be important for lipid rafts in signaling disorders in AD as well as in A fibrillation

Lipid membrane deformation in response to a local pH modification: theory and experiments

We study the deformation of a lipid membrane in response to a local pH modification. Experimentally, a basic solution is microinjected close to a giant unilamellar vesicle. A local deformation appears in the zone of the membrane that is closest to the micropipette, and relaxes when the injection is stopped. A theoretical description of this phenomenon is provided. It fully takes into account the spatiotemporal evolution of the concentration of hydroxide ions during and after the microinjection, as well as the linear dynamics of the membrane. This description applies to a local injection of any substance that reacts reversibly with the membrane lipids. We compare experimental data obtained in the domain of small deformations to the results of our linear description, and we obtain a good agreement between theory and experiments. In addition, we present direct experimental observations of the pH profile on the membrane during and after the microinjection, using pH-sensitive fluorescent lipids

Segregative Clustering of Lo and Ld Membrane Microdomains Induced by Local pH Gradients in GM1-Containing Giant Vesicles: A Lipid Model for Cellular Polarization

Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells

Verrucaria indet.

Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells