A correlation between membrane fluidity and the critical temperature for cell adhesion.

BHK 21 cells can adhere to a protein-coated plastic dish in the presence of Ca2+ at temperatures above 12 degrees C. However, they cannot adhere below 8 degrees C. The ESR spectrum of cells spin-labeled with a stearic acid label indicated that the membrane fluidity changed characteristically at 10 degrees C, 20 degrees C, and 30 degrees C. The critical temperature for cell adhesion coincided well with one of the characteristic temperatures for the membrane fluidity change. In the case of adhesion in the presence of Mg2+, no such correlation was observed.

Effect of bilayer membrane curvature on activity of phosphatidylcholine exchange protein.

Effect of bilayer membrane curvature of substrate phosphatidylcholine and inhibitor phosphatidylserine on the activity of phosphatidylcholine exchange protein has been studied by measuring transfer of spin-labeled phosphatidylcholine between vesicles, vesicles and liposomes, and between liposomes. The transfer rate between vesicles was more than 100 times larger than that between vesicles and liposomes. The transfer rate between liposomes was still smaller than that between vesicles and liposomes and nearly the same as that in the absence of exchange protein. The markedly enhanced exchange with vesicles was ascribed to the asymmetric packing of phospholipid molecules in the outer layer of the highly curved bilayer membrane. The inhibitory effect of phosphatidylserine was also greatly dependent on the membrane curvature. The vesicles with diameter of 17 nm showed more than 20 times larger inhibitory activity than those with diameter of 22 nm. The inhibitory effect of liposomes was very small. The size dependence was ascribed to stronger binding of the exchange protein to membranes with higher curvatures. The protein-mediated transfer from vesicles to spiculated erythrocyte ghosts was about four times faster than that to cup-shaped ghosts. This was ascribed to enhanced transfer to the highly curved spiculated membrane sites rather than greater mobility of phosphatidylcholine in the spiculated ghost membrane.

Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes.

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.

Interaction of polyene antibiotics with sterols in phosphatidylcholine bilayer membranes as studied by spin probes.

Interaction of filipin and amphotericin B with sterols in phosphatidylcholine membranes has been studied using various spin probes; epiandrosterone, cholestanone, phosphatidylcholine with 12-nitroxide or 5-nitroxide stearate attached to 2 position and also with tempocholine at the head group. Filipin caused increase in the fluidity of cholesterol-containing phosphatidylcholine membranes near the center, while it rather decreased the fluidity near the polar surface. On the other hand, amphotericin B did not apparently affect the fluidity. In the electron spin resonance spectrum of steriod spin probes in the antibiotic-containing membranes, both bound and free signals were observed and the association constant was calculated from the siganal intensity. In the binding of steriods with filipin, both 3 and 17 positions were involved, while the 17 positions was less involved in the binding with amphotericin B. Phase change in the host membrane markedly affected the interaction of filipin with epiandrosterone probe. The bound fraction jumped from 0.4 to 0.8 on going to the crystalline state and increased further with decrease in temperature. The overall splitting of the bound signal also increased on lowering the temperature below phase transition. This change was attributed to aggregate formation of filipin-steriod complexes in the crystalline state. On the other hand, effect of phase transition was much smaller on the interaction of amphotericin B with the steriod probe.

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current.

Disappearance of Ca2+-induced phase separation in phosphatidylserine-phosphatidylcholine membrane has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the pre parations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca2+-treated membrane was transferred to acidic salt solutions (less than or equal to pH 2.5) or to low ionic strength media (less than or equal to mM) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca2+ by proton, proton-induced separation, followed by disappearance of the phase separation in the buffered salt solution. Biological significance of the competition between Ca2+ and proton for the phase separation or domain formation in the membranes was emphasized.

Virus-induced fusion of human erythrocyte ghosts. I. Effects of macromolecules on the final stages of the fusion reaction.

Human erythrocyte ghosts prepared by hypotonic hemolysis can be fused by Sendai virus, provided that certain macromolecules (bovine serum albumin, dextran and others) are sequestered in the ghosts. Since fusion of the ghosts is dependent on intactness of the F(fusion)-glycoprotein of the virion, and since the other requirements for this reaction are also similar to those for the Sendai virus-induced fusion of intact erythrocytes, this system can be used as a model for the Sendai virus-induced cell fusion reaction. Sequestered macromolecules seem to be required for rounding of locally fused ghosts. Under low osmotic swelling conditions, such as use of ghosts sealed without macromolecules or using bovine serum albumin-loaded ghosts sealed in the presence of external macromolecules, no apparently complete cell fusion (large spherical polyghost formation) could be observed. Even under these conditions, however, occurrence of local cell fusion could be demonstrated either by transfer of fluorescent-labeled albumin from one ghost to an other, or by observation of polyghost formation after osmotic swelling in the cold. Thus, final stages of the fusion reaction can be divided into local cell-cell fusion which could not be observed by phase-contrast microscopy, and rounding (i.e. formation of spherical polyghosts). For the observation of fusion of ghosts, the last step seems to be important.

Dynamic states of phospholipids in Escherichia coli B membrane. Electron spin resonance studies with biosynthetically generated phospholipid spin labels.

Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label. For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography. DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically. ESR spectra of these E. coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane. These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ.

Spin-labeling of Escherichia coli membrane by enzymatic synthesis of phosphatidylglycerol and divalent cation-induced interaction of phosphatidylglycerol with membrane proteins.

Escherichia coli membrane particulate fraction has been spin-labeled by incubating with sn-glycerol-3-phosphate, CTP, palmitoyl CoA and 12-nitroxide stearoyl CoA. Incorporation of the spin-labeled acyl chain into phosphatidyl-glycerol was confirmed. ESR spectrum of the spin-labeled phosphatidylglycerol in E. coli membrane consisted at least of two components; one due to the labels undergoing rapid anisotropic motions and the other due to strongly immobilized labels (the overall splitting value, approx. 58 G). The relative intensity of the two components was dependent on the concentration of divalent cations. The immobilized component decreased on treatment of the membrane with EDTA and increased on addition of Mg2+ or Ca2+. The spectrum at 1 mM Mg2+ or Ca2+ consisted almost only of the immobilized component. Spin-labeled phosphatidylglycerol in total lipid membrane showed ESR spectrum due to mobile labels and the spectrum was not affected appreciably by the divalent cations. The results suggest the divalent cation-mediated interaction of phosphatidylglycerol with proteins in E. coli membrane. Phosphoenolpyruvate-dependent uptake of methyl-alpha-D-glucoside was markedly accelerated by Mg2+. Ca2+ was not effective for the enhancement. The divalent cation-induced interaction of phosphatidylglycerol with proteins was discussed in relation to the sugar transport.

Outer membrane of Salmonella typhimurium. Electron spin resonance studies.

The supramolecular structure of the outer membrane of Salmonella typhimurium that produces an Rc-type lipopolysaccharide was studied by adding spin-labeled fatty acid probes to membranes as well as model bilayers. Lipopolysaccharide of this organism apparently formed a bilayer structure in 0.2 M NaCl/0.01 M MgCl2, and the electron spin resonance spectra suggested that the motion of the segments of hydrocarbon chains near the carboxyl end was quite restricted even at high temperature; this is presumably due to the anchoring of more than a dozen fatty acid residues to a single backbone structure. In the presence of Mg2+, we could produce lipoplysaccharide-phospholipid mixed bilayers contining up to 50% (by weight) lipoplysaccharide. Their spectra showed no sign of major heterogeneity, and the maximum hyperfine splitting values were considerably larger than in phospholipid-only liposomes; these results suggest that the two components are finely interspersed and that the mobility of phospholipid hydrocarbons is severely restricted by the hydrocarbon chains of lipopolysaccharide. In spite of the presence of lipoplysaccharide in an amount equal to or exceeding that of phospholipids, the outer membrane produced spectra remarkably similar to those of the inner membrane, which does not contain lipoplysaccharide, and there was little sign of immobilization by lipopolysaccharides. Signals corresponding to the pure lipoplysaccharide phase were not detected, either. These results suggest that the phospholipids and lipopolysaccharides are segregated into separate domains in the outer membrane, and the fatty acid probes enter almost exclusively into the phospholipid domains. This conclusion was fully corroborated by determining, through the exchange broadening of line width, the total area of the domains that accommodated the spin label probes.

The endocytic process in CHO cells, a toxic pathway of the polyene antibiotic amphotericin B.

We describe the fate of the polyene antibiotic amphotericin B (AmB) after its interaction with Chinese hamster ovary (CHO) cells. The global uptake of AmB by these cells was measured at 37 degrees C after a 1-h incubation in the presence of 5% fetal bovine serum. It increased with the total concentration of drug and reached a plateau of approximately 1 nmol/mg of cell protein for an external concentration of 25 microM. The same experiment performed at 5 degrees C revealed a drastic decrease in uptake. The distribution of the drug among plasma membranes, endosomes, and lysosomes was then investigated after the separation of the postnuclear fractions by a Percoll gradient. After a 10-min incubation, AmB was found only in the plasma membrane fraction, regardless of the drug concentrations used (5 to 100 microM). After 60 min, at low drug concentrations (5 and 10 microM) AmB was found to be incorporated mainly in plasma and lysosomal fractions. At high concentrations (50 microM) AmB accumulated in endosomal fractions and plasma membranes. At intermediate concentrations (25 microM) AmB was distributed among the three fractions. When the same experiment was carried out at 5 degrees C, AmB was associated only with the plasma membrane even after 60 min, which was consistent with the absence of endocytotic process at low temperature. The effect of AmB on the endocytic process resulted in the increased uptake of sulforhodamine B, a fluid-phase marker of endocytosis, as well as by the accumulation of sulforhodamine in spots scattered in the cytoplasms of AmB-treated cells, in contrast to the accumulation around the nuclei observed in the control cells. These results are interpreted as indicating that AmB is internalized by the cells through endocytosis and that high concentrations of the drug block the fusion between endosomes and/or the fusion between endosomes and lysosomes.

Transmembrane phospholipid motions induced by F glycoprotein in hemagglutinating virus of Japan.

Transfer of phospholipid from the envelope of hemagglutinating virus of Japan (HVJ) to erythrocyte (RBC) membrane and the virus-induced transfer of phospholipid between RBC membranes were studied using spin-labeled phosphatidylcholine (PC). The transfer of PC from membranes labeled densely with PC to unlabeled membranes was followed by the peak height increase in the electron spin resonance spectrum. The two kinds of transfer reactions took place very rapidly as reported previously. To obtain further details, the transfer reactions were studied with HVJ, HVJ inactivated by trypsin, HVJ harvested early, HVJ grown in fibroblast cells, the fibroblast HVJ activated by trypsin, influenza virus, and glutaraldehyde-treated RBCs. The results demonstrated that the viral F glycoprotein played a crucial role in the transmembrane phospholipid movements as well as in the fusion and hemolysis of RBCs. The transfer from HVJ to RBC's occurred partially through an exchange mechanism not accompanying the envelope fusion. This was shown by a decrease in the exchange broadening of the electron spin resonance spectrum of released spin-labeled HVJ (HVJ) and also by an increase in the ratio of PC to viral proteins incorporated into RBC membranes. HVJ modified RBC membrane so as to be able to exchange its phospholipids with those of inactive membranes such as fibroblast HVJ, influenza virus, glutaraldehyde-treated RBC'S, and phosphatidylcholine vesicles. HVJ affected the fluidity of RBC membranes markedly, the environments around PC being much fluidized. The virus-induced fusion was discussed based on close apposition of the membranes by HANA proteins and on the destabilization and fluidization of RBC membranes by F glycoproteins.

An arrested late endosome-lysosome intermediate aggregate observed in a Chinese hamster ovary cell mutant isolated by novel three-step screening.

Chinese hamster ovary cell mutants defective in the post-uptake degradation of low-density lipoprotein (LDL) in lysosomes were selected from mutagenized cells by novel three-step screening. First, in the presence of LDL, clones sensitive to an inhibitor of the rate-limiting enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-CoA reductase, were isolated. Second, from the selected clones, those lacking in the degradation of a constituent of a fluorescent LDL were qualitatively screened by microscopy. Third, the clones were further screened by previously established quantitative analytical flow cytometry that detects the early-phase disintegration of LDL by lysosomal acid hydrolases. One of the isolated mutant clones, LEX1 (Lysosome-Endosome X 1), was a recessive mutant, and exhibited a specific disorder in the late endocytic pathway. LEX1 cells showed an unusual perinuclear aggregate of vesicles, heterogeneously positive for lysosomal glycoprotein-B/cathepsin D and rab7, yet negative for the cation-independent mannose 6-phosphate receptor. The aggregate was formed around the microtubule organizing center, and was disrupted by nocodazole treatment. Internalized octadecyl rhodamine B-labeled LDL (R18-LDL) was accumulated in the perinuclear rab7-positive vesicles. In a Percoll density gradient, neither internalized R18-LDL nor internalized horseradish peroxidase was efficiently chased into heavy lysosomal fractions positive for beta-hexosaminidase. LEX1 cells showed differences in the activity and subcellular distribution of lysosomal enzymes. These characteristics of LEX1 cells are consistent with the ideas that the perinuclear vesicle aggregate is an arrested intermediate of direct fusion or divergence between lysosomes and rab7-positive, cation-independent mannose 6-phosphate receptor-negative late endosomes, and that equilibrium between the lysosomes and the late endosomes is shifted towards the late endosomes in LEX1 cells. Such fusion or divergence between the late endosomes and the lysosomes would determine an appropriate equilibrium between them, and might thereby play an important role for proper lysosomal digestive functions. LEX1 mutant cells would be helpful for the dissection of the as yet unrevealed details of the late endocytic membrane dynamics and for the identification of factors involved in the process arrested by the mutation.