A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena

A fluorescence method is presented for quantitatively analyzing exocytosis phenomena and monitoring their kinetics. The method is based on the particular properties of a hydrophobic fluorescent probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) [Prendergast, F.G., Haugland, R.P., & Callahan, P.J. (1981) Biochemistry 20, 7333-7338; Kuhry, J.G., Fonteneau, P., Duportail, G., Maechling, C., & Laustriat, G. (1983) Cell Biophys. 5, 129-140; Kuhry, J.G., Duportail, G., Bronner, C., & Laustriat, G. (1985) Biochim. Biophys. Acta 845, 60-67]. When this probe is interacted with intact resting cells in aqueous suspensions, it labels solely the membranes that are in contact with the external medium and is incorporated into them according to a partition equilibrium; i.e., the amount of the probe incorporated is proportional to the available membrane surface. TMA-DPH is highly fluorescent in membranes and not at all in water. Thus, a measurement of the TMA-DPH fluorescence intensity provides a signal proportional to the membrane surface. In secretory cells, the membrane surface available for the probe is increased upon fusion of the membrane of the secretory granules with the cell plasma membranes, directly or via intergranule fusion. Thus, when these cells are stimulated, more TMA-DPH is incorporated than in resting cells since the probe is allowed to also interact with the granule membranes now connected with the external medium by pores. This process results in a proportional increase in the TMA-DPH fluorescence intensity. The response was found to be very rapid and able to follow accurately the exocytosis kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstituted membranes of tumour cells (proteoliposomes) induce specific protection to murine lymphoma cells

Summary Antigens presented on cell membranes or on liposomes are usually more immunogenic than antigens in soluble form, this being one of the reasons for the weak immunogenicity of extracted tumour-associated transplantation antigens (TATA). The main objective of this study is to solubilize TATA from tumour cells and to present them on a membrane-like structure to the immune system. Crude tumour cell membranes of SL2 lymphosarcoma cells (a spontaneously arising, weakly immunogenic tumour) were solubilized with octylglucoside or sodium deoxycholate, and reconstituted membranes (proteoliposomes) were prepared by detergent removal. Mice immunized s.c. with reconstituted membranes were protected against an i. p. challenge with tumour cells. Although octylglucoside solubilized only 41% of the membrane proteins, the reconstituted membranes were as immunoprotective as crude membranes. (Glyco)proteins were probably the major membrane components in the reconstituted membranes that induce immunoprotection, as mice immunized with preparations constituted of (glyco)lipids from SL2 cells could not reject SL2 cells. If Freund's complete adjuvant was used with the first immunization injection, no potentiation of the elicited immune responses was observed. Besides the membrane TATA, SL2 cells contained an apparently non-membrane-bound TATA, which was found in the cytoplasm. It is concluded that detergent solubilization of membranes and subsequent preparation of reconstituted membranes can be used to obtain membrane tumour-associated antigens that retain activity for induction of protective tumour immunity. The major advantage of this method is that membrane proteins are solubilized and are subsequently presented on a membrane-like structure that resembles the tumour cell membrane. On theoretical and practical grounds it provides a promising alternative for whole-cell vaccines.     

Localization in the Cell and Extraction of Alkaline Phosphatase from Bacillus subtilis

Study of protoplasts, lysed protoplasts, and cells treated with lysozyme in the absence of osmotic stabilizer suggested that the alkaline phosphatase (EC 3.1.3.1.) of Bacillus subtilis is located in the protoplasmic membrane. Cytochemical evidence in support of this view is presented. The enzyme protein was strongly bound to the membrane structure and could not be solubilized by a number of treatments known to release enzymes from membranes and other lipoprotein structures. Alkaline phosphatase was, however, solubilized by treatment of intact B. subtilis cells or isolated protoplasmic membranes with strong salt solutions at pH 7.2, suggesting that electrostatic forces are responsible for the association between membrane and enzyme protein. Dialysis of alkaline phosphatase solutions against buffer of low ionic strength resulted in precipitation of the enzyme.     

Changes in the cellular composition of the deciduous membrane in physiologic pregnancy and late toxicosis

Cytochemical characteristics of the decidual membrane at a physiologically normal pregnancy and in cases of late toxicosis are presented. Three main cell types of the decidual membrane are defined: large decidual cells (LDC), small decidual cells (SDC) and granular cells of endometrium, or K-cells. Part of each cell type in the decidual membrane is determined. At physiologically normal pregnancy the part of the LDC makes 50-60% in the membranes and 80-85% in the basal plate of the placenta; SDC--10-18% in the fetal membranes and 1-2% in the basal plate of the placenta; K-cells--0.5-1%. At late toxicosis of pregnancy there is a change in relative and absolute amount of the decidual cells: the part of the LDC decreases up to 26-40% in the fetal membranes and up to 55% in the basal plate of the placenta; part of the K-cells at a slight form of preeclampsia rises up to 3-4%, at a severe form--up to 11-12%. The change in cell composition results in certain disturbances of physiological equilibrium of biologically active substances produced by the decidual cells. This correlates with the severity and clinical manifestations of the late toxicosis of pregnancy. Correlation of the decidual cells disfunction, directed to regulation of their reproduction and functioning, can become one of the elements of pathogenic treatment of the late toxicosis of pregnancy.     

Filipin as a cholesterol probe. II. Filipin-cholesterol interaction in red blood cell membranes

Filipin, a mixture of polyene antibiotics which form complexes with cholesterol, perturbs membrane lipid organization, and causes hemolysis of erythrocytes, is increasingly used as a cytochemical probe for the distribution of cholesterol in cell membranes. We used light (phase-contrast, dark-field and fluorescence) and electron microscopical techniques (whole-mount shadowing, negative staining, and freeze-fracture) to study the interaction of filipin with unfixed and glutaraldehyde-fixed human red blood cell (RBC) membranes. Lysis time and extent depended upon the cholesterol:filipin (C:F) ratio. Lysis was prevented by osmotic protection with high MW dextran. Filipin treated cells fluoresced, but variation in fluorescence intensity among unfixed as well as among fixed cells was evident both at low and high C:F ratios. Negatively stained preparations of unfixed cells lysed on grids or in suspension revealed ring- or C-shaped filipin-induced lesions (FIL) equipped with a veil-like appendage; single FIL, and FIL fused by their veils into aggregates, were shed from membranes. FIL at the surface proper of shadowed whole-mounts and of freeze-etched preparations of prefixed cells appeared as single, dispersed or aggregated cylinders protruding to variable heights above the membrane's plane; aggregated FIL were shed from cells. The freeze-fracture appearance of FIL differed in membranes fixed before or after filipin treatment. E- and P-faces of post-fixed membranes exhibited cylindrical protrusions and depressions, respectively; in essence, the reverse was found in pre-fixed RBC. Both pre- and post-fixed membranes showed considerable variation in the number of FIL on individual cells whether incubated at high (1:1) or low (1:5) C:F ratios, or for a short (10 min) or a long (80-180 min) time. Aggregation and shedding of FIL was evident in all preparations. Thin layer chromatography of the incubation fluid after sedimentation of cells showed that membrane cholesterol was shed from incubated cells. The presented data question the feasibility of filipin as a probe for the topographical distribution of cholesterol in cell membranes.     

Mechanosensitivity of cell membranes

Physical and biophysical mechanisms of mechano-sensitivity of cell membranes are reviewed. The possible roles of the lipid matrix and of the cytoskeleton in membrane mechanoreception are discussed. Techniques for generation of static strains and dynamic curvatures of membrane patches are considered. A unified model for stress-activated and stress-inactivated ion channels under static strains is described. A review of work on stress-sensitive pores in lipid-peptide model membranes is presented. The possible role of flexoelectricity in mechano-electric transduction, e.g. in auditory receptors is discussed. Studies of flexoelectricity in model lipid membranes, lipid-peptide membranes and natural membranes containing ion channels are reviewed. Finally, possible applications in molecular electronics of mechanosensors employing some of the recognized principles of mechano-electric transduction in natural membranes are discussed.Abbreviations BLM Layer lipid membrane - SAC stress-activated channel - SIC stress-inactivated channel - MCYST microcystin-LR - DPhL diphytanoyl lecithin - CME condenser microphone effect Dedicated to Professor Alexander Derzhanski on the occasion of his 60th birthday Correspondence to: A. G. Petrov     

Dissociation of insulin-stimulated glucose transport from the translocation of glucose carriers in rat adipose cells

Cycloheximide, a potent inhibitor of protein synthesis, has been used to examine the relationship between recruitment of hexose carriers and the activation of glucose transport by insulin in rat adipocytes. Adipocytes were preincubated +/- cycloheximide for 90 min then +/- insulin for a further 30 min. We measured 3-O-methylglucose uptake in intact cells and in isolated plasma membrane vesicles. The concentration of glucose transporters in plasma membranes and low density microsomes was measured using a cytochalasin B binding assay. Cycloheximide had no affect on basal or insulin-stimulated 3-O-methylglucose uptake in intact cells or in plasma membrane vesicles. However, the number of glucose carriers in plasma membranes prepared from cells incubated with cycloheximide and insulin was markedly reduced compared to that from cells incubated with insulin alone (14 and 34 pmol/mg protein, respectively). Incubation of cells with cycloheximide alone did not change the concentration of glucose carriers in either plasma membranes or in low density microsomes compared to control cells. When isolated membranes were analyzed with an antiserum prepared against human erythrocyte glucose transporter, decreased cross-reactivity was observed in plasma membranes prepared from cycloheximide/insulin-treated cells compared to those from insulin cells. The present findings indicate that incubation of adipocytes with cycloheximide greatly reduces the number of hexose carriers in the plasma membrane of insulin-stimulated cells. Despite this reduction, insulin is still able to maximally stimulate glucose uptake. Thus, these data suggest an apparent dissociation between insulin stimulation of glucose transport activity and the recruitment of glucose carriers by the hormone.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

Analytical isolation of plasma membranes of intestinal epithelial cells: Identification of Na,K-ATPase rich membranes and the distribution of enzyme activities

Summary A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and cole material. Sucrase specific activity in the purified brush border plasma membrane was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochromec reductase, nonspecific esterase, -glucoronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity.Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent³⁵S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein.Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E

In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of arginine-rich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 μM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically >10 μM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.     

PrP(106-126) does not interact with membranes under physiological conditions

Transmissible spongiform encephalopathies are neurodegenerative diseases characterized by the accumulation of an abnormal isoform of the prion protein PrP^Sc. Its fragment 106-126 has been reported to maintain most of the pathological features of PrP^Sc, and a role in neurodegeneration has been proposed based on the modulation of membrane properties and channel formation. The ability of PrP^Sc to modulate membranes and/or form channels in membranes has not been clearly demonstrated; however, if these processes are important, peptide-membrane interactions would be a key feature in the toxicity of PrP^Sc. In this work, the interaction of PrP(106-126) with model membranes comprising typical lipid identities, as well as more specialized lipids such as phosphatidylserine and GM1 ganglioside, was examined using surface plasmon resonance and fluorescence methodologies. This comprehensive study examines different parameters relevant to characterization of peptide-membrane interactions, including membrane charge, viscosity, lipid composition, pH, and ionic strength. We report that PrP(106-126) has a low affinity for lipid membranes under physiological conditions without evidence of membrane disturbances. Membrane insertion and leakage occur only under conditions in which strong electrostatic interactions operate. These results support the hypothesis that the physiological prion protein PrP^C mediates PrP(106-126) toxic effects in neuronal cells.     

Caveolin-2 localizes to the golgi complex but redistributes to plasma membrane, caveolae, and rafts when co-expressed with caveolin-1.

We have characterized comparatively the subcellular distributions of caveolins-1 and -2, their interactions and their roles in caveolar formation in polarized epithelial cells. In Fischer rat thyroid (FRT) cells, which express low levels of caveolin-2 and no caveolin-1, caveolin-2 localizes exclusively to the Golgi complex but is partially redistributed to the plasma membrane upon co-expression of caveolin-1 by transfection or by adenovirus-mediated transduction. In Madin-Darby canine kidney (MDCK) cells, which constitutively express both caveolin-1 and -2, caveolin-2 localized to both the Golgi complex and to the plasma membrane, where it co-distributed with caveolin-1 in flat patches and in caveolae. In FRT cells, endogenous or overexpressed caveolin-2 did not associate with low density Triton insoluble membranes that floated in sucrose density gradients but was recruited to these membranes when co-expressed together with caveolin-1. In MDCK cells, both caveolin-1 and caveolin-2 associated with low density Triton-insoluble membranes. In FRT cells, transfection of caveolin-1 promoted the assembly of plasma membrane caveolae that localized preferentially (over 99%) to the basolateral surface, like constitutive caveolae of MDCK cells. In contrast, as expected from its intracellular distribution, endogenous or overexpressed caveolin-2 did not promote the assembly of caveolae; rather, it appeared to promote the assembly of intracellular vesicles in the peri-Golgi area. The data reported here demonstrate that caveolin-1 and -2 have different and complementary subcellular localizations and functional properties in polarized epithelial cells and suggest that the two proteins co-operate to carry out specific as yet unknown tasks between the Golgi complex and the cell surface.     

Interaction of p-aminobenzoic acid with erythrocyte membrane: photoaffinity labeling of the binding sites

p-Aminobenzoic acid (PABA) was found to prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right-side-out membrane vesicles revealed a similar number of binding sites and Kd values for both normal and sickle cell membranes. A [14C]Azide analog of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface. The azide was found to covalently incorporate into the membrane components upon irradiation; 52-35% of the label was associated with the proteins and the remaining with the lipids. Electrophoretic analysis of photolabeled membranes revealed that the azide interacts mainly with Band 3 protein in the case of intact erythrocytes and right-side-out sealed vesicles; however, if unsealed ghosts are used, other membrane proteins besides Band 3 are photolabeled. PABA was found to inhibit both high and low affinity calcium-binding sites situated on either surface of the membrane apparently in a non-competitive manner. However, calcium binding stimulated by magnesium and ATP was only slightly affected. Calcium transport into inside-out vesicles was inhibited by PABA, but it did not affect the calcium ATPase activity.     

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Distribution of EIIFru over the membranes of phototrophically grown Rps. sphaeroides

The distribution of the fructose carrier over the membranes of Rhodopseudomonas sphaeroides was studied in cells grown under light saturation and light limitation. Three types of membranes were isolated after disruption of the cells in a French press. All three types were present in the cells grown either under the high or low light intensity, but they were present in different quantities. The cytoplasmic membrane could be separated from the photosynthetic membranes by Sephacryl S-1000 chromatography. The cytoplasmic membrane has the highest specific density and fructose carrier content and does not contain the light-harvesting pigments. The photosynthetic membranes could be resolved into two types by sucrose density gradient centrifugation. Type A predominates when cells are grown under light saturation, whereas type B, the chromatophores, is synthesized abundantly under light limitation. The properties of type A are in between the properties of the cytoplasmic membrane and the chromatophores. It has a slightly lower specific density and contains four times less fructose carrier than the cytoplasmic membrane, but contains half of the light-harvesting bacteriochlorophyll of the chromatophore membrane. The fructose carrier content in the type B membranes, the chromatophores, is very low.     

Temperature-mediated processes in teleost immunity: homeoviscous adaptation by channel catfish peripheral blood cells

1. Channel catfish peripheral blood erythrocyte, thrombocyte, T cell and B cell membranes were assayed by fluorescence depolarization using the fluorescent probe 1,6-diphenyl-1,3,5 hexatriene (DPH) to determine the effects of in vivo temperature acclimation on membrane viscosity and the kinetics of homeoviscous adaptation. 2. Erythrocyte membranes did not undergo homeoviscous adaptation during the 8 week time period studied and were more rigid compared with those of the other cell types. 3. The kinetics of homeoviscous adaptation exhibited by membranes from T cells, B cells and thrombocytes differed: B cells required 1-3 weeks while T cells and thrombocytes each required 3-5 weeks. Membranes from T cells, B cells and thrombocytes from fish acclimated for relatively short times (less than or equal to 3 weeks) exhibited similar membrane fluidities. 4. T cells from channel catfish appeared not only to be sensitive to temperature but also to a factor(s) independent of temperature but correlated to long term in vivo acclimation, i.e. T cell membranes underwent additional decreases in membrane viscosity between 3 and 5 weeks. 5. In conclusion, it appears that low temperature-mediated immunosuppression of T cell functions in channel catfish is probably not due to an inherent non-adaptability or rigid nature of the T cell membranes.     

The early formation of the photosynthetic apparatus in Rhodospirillum rubrum

The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c ₂do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-¹⁴C-proteinhydrolyzate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism.Abbreviations Bchl bacteriochlorophyll - LH light harvesting - RC reaction center - R. Rhodospirillum - R. Rhodopseudomonas     

THE FINE STRUCTURE OF THE TRANSITIONAL EPITHELIUM OF RAT URETER

The fine structure of the transitional epithelium of rat ureter has been studied in thin sections with the electron microscope, including some stained cytochemically to show nucleoside triphosphatase activity. The epithelium is three to four cells deep with cuboidal or columnar basal cells, intermediate cells, and superficial squamous cells. The basal cells are attached by half desmosomes, or attachment plates, on their basal membranes to a basement membrane which separates the epithelium from the lamina propria. Fine extracellular fibres, ca. 100 A in diameter, are to be found in the connective tissue layer immediately below the basement membrane of this epithelium. The plasma membranes of the basal and intermediate cells and the lateral and basal membranes of the squamous cells are deeply interdigitated, and nucleoside triphosphatase activity is associated with them. All the cells have a dense feltwork of tonofilaments which ramify throughout the cytoplasm. The existence of junctional complexes, comprising a zonula occludens, zonula adhaerens, and macula adhaerens or desmosome, between the lateral borders of the squamous cells is reported. It is suggested that this complex is the major obstacle to the free flow of water from the extracellular spaces into the hypertonic urine. The free luminal surface of the squamous cells and many cytoplasmic vesicles in these cells are bounded by an unusually thick plasma membrane. The three leaflets of this unit membrane are asymmetric, with the outer one about twice as thick as the innermost one. The vesicles and the plasma membrane maintain angular conformations which suggest the membrane to be unusually rigid. No nucleoside triphosphatase activity is associated with this membrane. Arguments are presented to support a suggestion that this thick plasma membrane is the morphological site of a passive permeability barrier to water flow across the cells, and that keratin may be included in the membrane structure. The possible origin of the thick plasma membrane in the Golgi complex is discussed. Bodies with heterogeneous contents, including characteristic hexagonally packed stacks of thick membranes, are described. It is suggested that these are "disposal units" for old or surplus thick membrane. A cell type is described, which forms only 0.1 to 0.5 per cent of the total cell population and contains bundles of tubular fibres or crystallites. Their origin and function are not known.     

Interactions of agonists and antagonists with beta-adrenergic receptors on intact L6 muscle cells

A binding assay has been developed to characterize beta-adrenergic receptors on intact L6 muscle cells. The affinity of beta-adrenergic receptors for the radioligand iodohydroxybenzylpindolol (IHYP) was the same in membrane preparations and in intact cells when determined by either equilibrium binding or kinetic analysis. The number of specific IHYP binding sites per cell was approximately the same on intact cells as on membranes. The pharmacological properties of antagonists indicated that the receptors on intact cells were identical to those on membranes. However, the beta-adrenergic receptors on intact cells had a 100-400 fold lower affinity at equilibrium for the agonist isoproterenol than did beta-adrenergic receptors on membranes. This low affinity of the receptor for agonists as measured by inhibition of radioligand binding in intact cells has also been observed in C6 (2) and S49 (3) cells. Our results suggest that beta receptors on intact cells after a 1 minute incubation was similar to the KD value for isoproterenol measured in membranes at equilibrium in the presence of GTP. After 1-2 minutes of exposure to a low concentration of agonist, binding of IHYP was no longer inhibited. These results suggest that agonists rapidly convert the beta receptors on intact cells to a state which has a low affinity for agonists. The affinity of the receptor for antagonists did not change during the incubation.     

From mosaic to patchwork: matching lipids and proteins in membrane organization

Biological membranes encompass and compartmentalize cells and organelles and are a prerequisite to life as we know it. One defining feature of membranes is an astonishing diversity of building blocks. The mechanisms and principles organizing the thousands of proteins and lipids that make up membrane bilayers in cells are still under debate. Many terms and mechanisms have been introduced over the years to account for certain phenomena and aspects of membrane organization and function. Recently, the different viewpoints - focusing on lipids vs. proteins or physical vs. molecular driving forces for membrane organization - are increasingly converging. Here we review the basic properties of biological membranes and the most common theories for lateral segregation of membrane components before discussing an emerging model of a self-organized, multi-domain membrane or 'patchwork membrane'.     

Isolation of plasma membrane fragments and vesicles from ascites fluid of lymphoma-bearing mice and their possible role in the escape mechanism of tumors from host immune rejection

Summary Plasma membranes, generated in vivo by actively growing YAC lymphoma cells, were isolated from cell-free ascites fluid of lymphoma-bearing mice. Partial purification of the ascites fluid (AF) by means of ultracentrifugation resulted in the identification of two main fractions: (a) membrane fragments (AFM _s ) and (b) membrane vesicles (AFM _p ). Electron microscopy studies, polyacrylamide gel electrophoresis, marker enzymes, and binding capacity of radioactive lectins, have indicated that these membranes are released from the cell surface of YAC lymphoma cells, presumably by a shedding-off mechanism.In vitro studies have demonstrated that the isolated membranes can specifically inhibit the association of normal macrophages and YAC lymphoma cells. In vivo studies have shown that these membranes can immunize against YAC tumors if injected intramuscularly or subcutaneously into adult mice. The results indicate that the ascites fluid membranes bear tumor-specific antigenic determinants.Our results suggest that in vivo shedding of plasma membrane fragments or of membrane vesicles by actively growing YAC lymphoma cells may induce a self-protection of ascites tumors from host immune rejection.Abbreviations YAC= Moloney-virus-induced lymphoma cells grown in A-strain mice - AF= ascites fluid of YAC lymphoma-bearing mice - AFM_s and AFM_p= membrane fragments and vesicles isolated from AF - PBS= phosphate-buffered saline - Con A= Concanavalin A