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S Cribier

Publications and source records attributed to S Cribier.

At least 19 recordsLinked to original sources
PubMed2005

Indirect evidence of submicroscopic pores in giant unilamellar [correction of unilamelar] vesicles.

Formation of pore-like structures in cell membranes could participate in exchange of matter between cell compartments and modify the lipid distribution between the leaflets of a bilayer. We present experiments on two model systems in which major lipid redistribution is attributed to few submicroscopic transient pores. The first kind of experiments consists in destabilizing the membrane of a giant unilamellar vesicle by inserting conic-shaped fluorescent lipids from the outer medium. The inserted lipids (10% of the vesicle lipids) should lead to membrane rupture if segregated on the outer leaflet. We show that a 5-nm diameter pore is sufficient to ease the stress on the membrane by redistributing the lipids. The second kind of experiments consists in forcing giant vesicles containing functionalized lipids to adhere. This adhesion leads to hemifusion (merging of the outer leaflets). In certain cases, the formation of pores in one of the vesicles is attested by contrast loss on this vesicle and redistribution of fluorescent labels between the leaflets. The kinetics of these phenomena is compatible with transient submicroscopic pores and long-lived membrane defects.

Lipid Bilayers↗
PubMed2004

Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance.

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of "hemifusion" is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

Cell Surface Extensions↗
PubMed2001

Short-range specific forces are able to induce hemifusion.

Working with pure lipidic systems (giant unilamellar vesicles, 10-150 microm in diameter) as models for biological membranes, we have considered possible structures of the contact area of two adherent membranes by investigating the diffusion of fluorescent lipid analogues from one vesicle to another. Two bilayers in close contact can almost be seen as a lamellar structure in equilibrium. This is the usual configuration of two adherent vesicles, in which the interbilayer distance is estimated to be 3 nm. We have increased the attraction between the membranes by either adding depletion forces or by using a trick, inspired from the interaction between nucleic bases in nucleosides (herein adenosine and thymidine). The nucleosides were attached to the polar head of amphiphilic molecules that behave like phospholipids and were incorporated in the model membrane. The extra attraction between two membranes, resulting from base pairing, strongly decreased the interbilayer distance down to about 1 nm. This change of the water content induced lipid rearrangements, which could also be viewed in terms of a phase transition at low water content. These rearrangements were not observed in the case of depletion forces. We conclude that the introduction of an additional attractive force in the system modifies the equilibrium state, leading to a drastic change in the membrane behavior, which will tentatively be related to hemifusion.

Adenosine↗
PubMed2000

Interactions between beta-enolase and creatine kinase in the cytosol of skeletal muscle cells.

We studied interactions in vivo between the cytosolic muscle isoform of creatine kinase (M-CK) and the muscle isoform of 2-phospho-D-glycerate hydrolyase (beta-enolase) in muscle sarcoplasm by incubating glycerol-skinned fibres with FITC-labelled beta-enolase in the presence or absence of free CK. A small amount of bound beta-enolase was observed in the presence of large concentrations of CK. The mobility of enolase was measured in cultured satellite cells by modulated-fringe-pattern photobleaching. FITC-labelled beta-enolase was totally mobile in both the presence and the absence of CK but its diffusion coefficient was slightly lower in the presence of CK. This suggests a weak interaction in vivo between enolase and CK.

Animals↗
PubMed2000

Translational diffusion of globular proteins in the cytoplasm of cultured muscle cells.

Modulated fringe pattern photobleaching (MFPP) was used to measure the translational diffusion of microinjected fluorescein isothiocyanate (FITC)-labeled proteins of different sizes in the cytoplasm of cultured muscle cells. This technique, which is an extension of the classical fluorescence recovery after photobleaching (FRAP) technique, allows the measurement of the translational diffusion of macromolecules over several microns. Proteins used had molecular masses between 21 and 540 kDa. The results clearly indicated that the diffusivity of the various proteins is a decreasing function of their hydrodynamic radius. This decrease is more rapid with globular proteins than with FITC-labeled dextrans (, Biophys. J. 70:2327-2332), most likely because, unlike globular proteins, dextrans are randomly coiled macromolecules with a flexible structure. These data do not exclude the possibility of a rapid diffusion over a short distance, unobservable with our experimental set-up, which would take place within the first milliseconds after bleaching and would correspond to the diffusion in restricted domains followed by impeded diffusion provoked by the network of microtubules, microfilaments, and intermediate filaments. Thus our results may complement rather than contradict those of Verkman and collaborators (, J. Cell Biol. 138:1-12). The biological consequence of the size-dependent restriction of the mobility of proteins in the cell cytoplasm is that the formation of intracellular complexes with other proteins considerably reduces their mobility.

Animals↗
PubMed1997

Mobility of creatine phosphokinase and beta-enolase in cultured muscle cells.

The diffusion of beta-enolase and creatine phosphokinase in muscle cells has been studied by modulated fringe pattern photobleaching. Beta-enolase is mobile in the sarcoplasm. At 20 degrees C, the diffusion coefficient is 13.5 +/- 2.5 microm2 s(-1) in the cytosol and 56 microm2 s(-1) in aqueous media. As in the case of dextrans of the same hydrodynamic radius, its mobility is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments. A fraction of creatine phosphokinase is mobile in the sarcoplasm. Its diffusion coefficient in the cytosol, 4.5 +/- 1 microm2 s(-1), is lower than that of the dextran of equivalent size. The other fraction (20 to 50%) is very slightly mobile, with an apparent diffusion coefficient varying from 0.0035 to 0.043 microm2 s(-1). This low mobility might be attributed to exchange between free and bound creatine phosphokinase. The bound fraction of the endogenous enzyme was localized by immunocytofluorescence on the cultured muscle cells. Our results favor a localization of bound cytosolic creatine phosphokinase on the M-line and a diffuse distribution in all myotubes.

Animals↗
PubMed1996

Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field.

Giant unilamellar vesicles with diameters ranging from 10 to 60 microns were obtained by the swelling of phospholipid bilayers in water in the presence of an AC electric field. This technique leads to a homogeneous population of perfectly spherical and unilamellar vesicles, as revealed by phase-contrast optical microscopy and freeze-fracture electron microscopy. Freshly prepared vesicles had a high surface tension with no visible surface undulations. Undulations started spontaneously after several hours of incubation or were triggered by the application of a small osmotic pressure. Partially deflated giant vesicles could undergo further shape change if asymmetrical bilayers were formed by adding lyso compounds to the external leaflet or by imposing a transmembrane pH gradient that selectively accumulates on one leaflet phosphatidylglycerol. Fluorescence photobleaching with 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled phospholipids or labeled dextran trapped within the vesicles enabled the measurement of the membrane continuity in the dumbbell-shaped vesicles. In all instances phospholipids diffused from one lobe to the other, but soluble dextran sometimes was unable to traverse the neck. This suggests that the diameter of the connecting neck may be variable.

4-Chloro-7-nitrobenzofurazan↗
PubMed1996

Diffusion of fluorescently labeled macromolecules in cultured muscle cells.

Myotubes were obtained from culture of satellite cells. They had a sarcomeric organization similar to that of muscle. The diffusion in the direction perpendicular to the fibers of microinjected fluorescein isothiocyanate-dextrans of molecular weight ranging from 9500 to 150,000 was examined by modulated fringe pattern photobleaching. On the time scale of the observation, 10-30 S, all of the dextrans were completely mobile in the cytoplasm. The diffusion coefficients were compared to the values obtained in water. The ratio D(cytoplasm)/D(w) decreased with the hydrodynamic radius R(h) of the macromolecules. The mobility of inert molecules in muscle cells is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments: D(cytoplasm)/D(w) = (D/D(w)) protein crowding x (D/D(w))(filament screening). The equation (D/D(w))filament screening = exp(-K(L)RCh) was used for the contribution of the filaments to the restriction of diffusion. A free protein concentration of 135 mg/ml, a solvent viscosity of cytoplasm near that of bulk water, and a calculated K(L) of 0.066 nm(-1), which takes into account the sarcomeric organization of filaments, accurately represent our data.

Animals↗
PubMed1993

Quantitative comparison between aminophospholipid translocase activity in human erythrocytes and in K562 cells.

Spin-labeled phospholipids were used to determine the transbilayer movement of phospholipids in human erythrocytes, in K562 cells and in human neonatal red cells. The erythroleukemia cell line, K562, as well as human neonatal red cells, which are rich in reticulocytes, were considered as representative of human erythrocyte precursor cells. In the nucleated cells, the difference between outside-inside movement of aminophospholipids and that of phosphatidylcholine or sphingomyelin analogues allowed us to discriminate between lipid internalization due to aminophospholipid translocase activity and to endocytosis. From the initial rates of aminophospholipid inward movement, we inferred that the activity of the aminophospholipid translocase is higher in the precursor cells than in mature erythrocytes.

Adult↗
PubMed1993

Cubic phases of lipid-containing systems. A translational diffusion study by fluorescence recovery after photobleaching.

The lateral diffusion coefficient of fluorescent lipid analogues incorporated in four cubic phases of lipid-water systems was determined by the modulated fringe pattern photobleaching technique. In two of the phases, Q230 and Q224, whose structure is bicontinuous, the diffusion is almost as fast as in the fluid lipid bilayers, and is essentially independent of the chemical nature of the probe. In the other two phases, whose structure consists of disjointed hydrocarbon micelles embedded in a water matrix (phase Q223, type I) and of water-containing micelles embedded in a hydrocarbon matrix (phase Q227, type II), the diffusion coefficient is strongly dependent on the chemical structure of the probe and on the topological type (I or II) of the structure. The conclusion is drawn that in the micellar phases the apparent diffusion mirrors the ability of the probe to hop from micelle to micelle.

Diffusion↗
PubMed1993

Phospholipid transmembrane domains and lateral diffusion in fibroblasts.

The lateral diffusion of fluorescent phospholipids in cultured Chinese hamster lung fibroblasts was examined by modulated fringe pattern photobleaching. When cells were labeled and maintained at 7 degrees C, the fluorescence remained localized at the plasma membrane. N-[6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] sphingosylphosphocholine (C6-NBD-SphPCho) and 1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylcholine (C6-NBD-PtdCho) both diffused with the same apparent lateral diffusion coefficient (D1 approximately 0.3 x 10(-9) cm2/s). By contrast, the phosphatidylserine derivative (1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylserine (C6-NBD-Ptd-Ser)) gave rise to two diffusional components: a slow component, D1, analogous to that measured with the choline-containing lipids, and a fast component (D2 approximately 2 x 10(-9) cm2/s). The fast component only exists in ATP-containing cells. It was shown to be associated with C6-NBD-PtdSer translocated to the inner leaflet. This indicates that the two leaflets form very different membranous domains. At higher temperature, the same difference in mobility was observed between the choline-containing lipids and the aminolipid. However, with C6-NBD-SphPCho, a fraction of very slowly diffusing or quasi-immobilized probes gradually appeared with time. This could be attributed to sphingomyelin located in small organelles after internalization. From the amplitude of this component registered at different intervals, we calculated that approximately 50% of the plasma membrane sphingomyelin is recycled in less than 30 min in Chinese hamster fibroblasts by an ATP- and microtubule-dependent process.

Animals↗
PubMed1993

Dynamics of the membrane lipid phase.

The lipid bilayer of a membrane is sometimes seen as an inert hydrophobic phase allowing the 'solubility' of transmembrane proteins and acting as a barrier between two compartments. However, the bilayer is, in fact, a highly organized system subjected to many movements leading to a dynamically equilibrated structure. A lipid within a membrane experiences intramolecular motions (movement of some segments of the molecule) and moves or diffuses in and across each monolayer. In plasma membranes, transverse diffusion is either passive (cholinecontaining phospholipids, fatty acids ...) or active via a carrier protein (amino-phospholipids). The known asymmetric transverse distribution of phospholipids between the two plasma membrane leaflets is a stationary state resulting from all these motions, especially the active transport. Nevertheless, recent studies have shown that it is also possible to obtain an uneven distribution of some lipids (e.g. fatty acid, phosphatidic acid) across a membrane via a pH gradient. Lateral diffusion within a monolayer depends on the composition of the monolayer and not on the nature of the diffusing lipid. The phospholipid asymmetry, based on the polar head groups, exists also for the corresponding fatty acids, as the nature of the acyl chains differs according to the head group. A consequence is that the cytoplasmic leaflet of plasma membranes has a different 'fluidity' from that of the outer leaflet.

Biological Transport, Active↗
PubMed1990

Lateral diffusion of erythrocyte phospholipids in model membranes comparison between inner and outer leaflet components.

The physical properties of lipid bilayers with a similar composition to the outer and inner leaflets of the human erythrocyte membrane have been examined in protein-free model systems. The outer leaflet (OL) was represented by a phospholipid mixture containing phosphatidylcholine and sphingomyelin extracted from human erythrocytes, while a mixture of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine represented the inner leaflet (IL). The ratio of cholesterol to phospholipid was varied in both mixtures. The lateral diffusion coefficient of fluorescent phospholipids diluted in such lipid mixtures was determined by the modulated fringe pattern photobleaching technique. Contrast curves with a single exponential decay, indicative of homogeneous samples, were obtained only for temperatures above 15 degrees C and for a cholesterol to phospholipid molar ratio below 0.8. The rate of lateral diffusion was approximately five times faster in IL than in OL multilayers, in agreement with former results obtained in human erythrocytes (Morrot et al. 1986). Varying the cholesterol to phospholipid ratio from 0 to 0.8 (mol/mol) enabled us to decrease the diffusion constant by only a factor of approximately 2 for both IL and OL mixtures. The order parameter of a spin-labeled phospholipid was determined in the different systems and found to be systematically smaller in IL mixtures than in OL mixtures. The present study indicates that the difference in lipid diffusivity of the two erythrocyte leaflets may be accounted for solely by a difference in phospholipid composition, and may be independent of cholesterol and protein asymmetry.

Diffusion↗
PubMed1990

Control of the transmembrane phospholipid distribution in eukaryotic cells by aminophospholipid translocase.

The aminophospholipid translocase is a plasma membrane Mg2(+)-ATPase which selectively pumps the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the outer to the inner monolayer in eukaryotic cells and is predominantly responsible for the asymmetric phospholipid distribution of the plasma membrane. Similar ATP-dependent transport of phospholipid takes place in some organelles such as chromaffin granules. On the other hand, the phospholipid flippase of rat liver endoplasmic reticulum does not require ATP and has a low lipid specificity. The biological implications of these phospholipid flippases are discussed.

Carrier Proteins↗
PubMed1986

Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme.

When human erythrocytes are incubated with spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, with a short beta chain (C5) bearing a doxyl group at the fourth carbon position, the labeled lipids incorporate readily in the outer monolayer. The incorporation is followed in fresh erythrocytes by a selective inward diffusion of the amino derivatives. This observation led us to postulate the existence of a selective ATP-dependent system that would flip aminophospholipids from the outer to the inner monolayer [Seigneuret, M., & Devaux, P. F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3751-3755]. This study further examines the nature of this selective transport and demonstrates that it is mediated by a specific membrane protein. By measurement of the initial rate of transverse diffusion of spin-labeled lipids incorporated at various concentrations in the membrane outer leaflet of packed erythrocytes, apparent Km values were determined for the phosphatidylserine and phosphatidylethanolamine analogues. A ratio of approximately equal to 1/9.4 [corrected] was obtained (KmPS/KmPE). Using spin-labels bearing either a 14N or a 15N isotope, we have carried out competition experiments allowing us to measure simultaneously the transport of two different phospholipids. By this procedure, we show that phosphatidylserine and phosphatidylethanolamine compete for the same transport site but that phosphatidylserine has a higher affinity, in agreement with a lower apparent Km. On the other hand, the slow diffusion of the phosphatidylcholine or sphingomyelin analogues has no influence on the transport of phosphatidylserine or phosphatidylethanolamine. Experiments carried out in ghosts loaded with ATP enabled us to determine the activation energies for phosphatidylserine and phosphatidylcholine transverse diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed1986

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane.

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non-specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6-times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP-depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.

Adenosine Triphosphate↗
PubMed1986

[Energy-dependent translocation of amino-phospholipids in the erythrocyte membrane].

In this review, we show how the stability of the asymmetric transverse distribution of phospholipids and the physiological role of the asymmetric distribution can be explained. Experiments with paramagnetic or fluorescent lipids enabled us to show that in fresh red blood cells, i.e. containing ATP, and in resealed ghosts containing ATP (1 mM) the amino derivatives (phosphatidylserine and phosphatidylethanolamine) are selectively transported from the outer monolayer to the inner monolayer of the membranes. On the other hand, phosphatidylcholine and sphingomyelin are not carried and diffuse spontaneously with a very long characteristic time. The ATP-dependent carrier mechanism can be inhibited by protein reacting groups (N-ethyl maleimide and ortho-vanadate), which very probably implies a transmembrane protein specific for amino phospholipids. The affinity for phosphatidylserine seems slightly higher than that for phosphatidylethanolamine. In addition we show the close parallel between the transverse distribution of phospholipids and cell shape. This leads us to suggest that the phospholipid translocation would be used to maintain the natural discoid shape of red blood cells. A possible generalisation of this mechanism to other cells and its implications for endocytosis are discussed.

Adenosine Triphosphate↗