Liposomes fuse with sperm cells and induce activation by delivery of impermeant agents

Sperm cell activation is a critical step in fertilization. To directly investigate the cell signaling events leading to sperm activation it is necessary to deliver membrane impermeant agents into the cytoplasm. In this study, the use of liposomes as possible agent-loading vectors was examined using (1) the octadecylrhodamine B (R18) and NBD phosphatidylethanolamine (NBD DHPE)/rhodamine phosphatidylethanolamine (rhod DHPE) fusion assays in bulk samples, (2) membrane transfer of fluorescence from liposome membranes labeled with R18 and rhodamine-tagged phosphatidylethanolamine (TRITC DHPE), and (3) lumenal transfer of impermeant calcium ions from liposomes to sperm cells, a process that stimulated sperm cell activation. Intermediate-sized unilamellar liposomes (98.17+/-15.34 nm) were prepared by the detergent-removal technique using sodium cholate as the detergent and a phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) lipid composition. In the R18 fusion assays, self-quenching increased logarithmically with increasing concentrations of R18 in the liposome membranes; addition of unlabeled sperm to R18-labeled liposomes lead to a rapid release of self-quenching. In the NBD DHPE/rhod DHPE resonance energy transfer (RET) fusion assay, RET was rapidly reduced under similar conditions. In addition, individual sperm became fluorescent when TRITC DHPE-labeled liposomes were incubated with unlabeled sperm cells. Incubation of sperm cells with empty liposomes did not significantly affect sperm cell activation and did not alter cell morphology. However, incubation with Ca (10 mM)-loaded liposomes resulted in a time-dependent increase in sperm cell activation (7.5-fold over controls after 15 min). We conclude that liposomes can be used for direct loading of membrane-impermeant agents into sea squirt sperm cell cytoplasm, and that delivery occurs via fusion and content intermixing.     

Phosphatidylcholine and phosphatidylinositol-specific phospholipases C of bull and rabbit spermatozoa.

Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine-specific phospholipase C (PC-PLC) that hydrolyzes L-alpha-dipalmitoyl-(choline-methyl-14C-153.0 Ci/mmol and a phosphatidylinositol-specific phospholipase C (PI-PLC) that hydrolyzes L-alpha-(Myo-Inositol-2-3H (N)-5.2 Ci mmol. PI-PLC from bull sperm acrosome has been purified 568 x fold with a specific activity 6.25 +/- 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC-PLC remained unaffected by varying concentrations of Ca2+, whereas PI-PLC activity was significantly increased. The bulk of PI-PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC-PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell.     

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.     

Fusion of artificial lipid membranes induced by synthetic "fusion peptide" of arenaviruses

The fusing capacity of lipid membranes of a synthetic 23-member peptide was studied. This hydrophobic peptide represents an analog of a predicted functional site ("fusion peptide") of the GP2 envelope protein of the Lassa virus (family Arenaviridae). Fusion of small monolayer liposomes was detected by the method of resonance energy transfer between the fluorescent derivatives of the lipid, NBD-PE (donor) and Rd-PE (acceptor). Using this peptide, the pH-dependent fusing activity was found in liposomes having different phospholipid composition. The rate and efficiency of liposome fusion increased with a decrease in pH and the lipid/peptide ratio as well as with a temperature increase. The increase in the ionic strength and Ca2+ concentration in the reaction mixture led to the inhibition of the peptide-induced fusion of liposomes. Neither the phospholipid charge, nor the transmembrane proton gradient of liposomes had any appreciable effect on the kinetics of the peptide-induced fusion. Neutralization of the medium in the course of the fusion reaction sharply decelerated, whereas repeated acidification activated this process. This finding suggests that peptide protonation plays a role in fusion reactions. It was suggested that acidification causes conformational changes in the peptide structure, thus activating the peptide-induced fusion of liposomes. The fusing capacity of the predicted Lassa virus fusion peptide is similar to that of viruses characterized by a pH-dependent step at the initial stages of the viral infection.     

Incorporating lipids into boar sperm decreases chilling sensitivity but not capacitation potential

Fresh boar sperm were incubated with small unilamellar liposomes composed of either the total lipids extracted from head plasma membranes (HPM) of fresh boar sperm or selected lipids (SL) of five defined phospholipids with specific acyl chains. To optimize fusion, liposomes with 2 mol% octadecyl rhodamine fluorophore in Beltsville Thawing Solution +/- 1 mM CaCl(2) were incubated at 35 degrees C with 1;ts 10(7) or 10(8) spermatozoa/ml and monitored over 60 min, using flow cytometry and fluorescence microscopy. The HPM fused to both sperm concentrations faster than SL but was equivalent by 30 min (10(8) sperm/ml) or 60 min (10(7) sperm/ml; 57.5 +/- 3% and 67.1 +/- 8% sperm fused to HPM and SL, respectively) +/- Ca(2+). Neither HPM nor SL affected onset of capacitation or spontaneous or ionophore-induced acrosome reactions at 0 or 3 h (chlortetracycline and fluorescein isothiocyanate-Pisum sativum agglutinin; n = 3). During cooling and after cryopreservation (n = 4 ejaculates), SL but not HPM significantly improved sperm motility and viability (Sybr14/propidium iodide staining) +/- 20% egg yolk, but egg yolk alone was more effective than SL alone. Liposomes of complex composition can fuse to boar sperm without harming in vitro capacitation or acrosome reaction and reduce sperm chilling sensitivity.     

Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ channels from the sea urchin sperm plasma membrane

Ca2+ influx across the sea urchin sperm plasma membrane is a necessary step during the egg jelly-induced acrosome reaction. There is pharmacological evidence for the involvement of Ca2+ channels in this influx, but their presence has not been directly demonstrated because of the small size of this cell. Sea urchin sperm Ca2+ channels are being studied by fusing isolated plasma membranes into planar lipid bilayers. With this strategy, a Ca2+ channel has been detected with the following characteristics: (a) the channel exhibits a high mainstate conductance (gamma MS) of 172 pS in 50 mM CaCl2 solutions with voltage-dependent decaying to smaller conductance states at negative Em; (b) the channel is blocked by millimolar concentrations of Cd2+, Co2+, and La3+, which also inhibit the egg jelly-induced acrosome reaction; (c) the gamma MS conductance sequence for the tested divalent cations is the following: Ba2+ greater than Sr2+ greater than Ca2+; and (d) the channel discriminates poorly for divalent over monovalent cations (PCa/PNa = 5.9). The sperm Ca2+ channel gamma MS rectifies in symmetrical 10 mM CaCl2, having a maximal slope conductance value of 94 pS at +100 mV applied to the cis side of the bilayer. Under these conditions, a different single-channel activity of lesser conductance became apparent above the gamma MS current at positive membrane potentials. Also in 10 mM Ca2+ solutions, Mg2+ permeates through the main channel when added to the cis side with a PCa/PMg = 2.9, while it blocks when added to the trans side. In 50 mM Ca2+ solutions, the gamma MS open probability has values of 1.0 at voltages more positive than -40 mV and decreases at more negatives potentials, following a Boltzmann function with an E0.5 = -72 mV and an apparent gating charge value of 3.9. These results describe a novel Ca2(+)-selective channel, and suggest that the main channel works as a single multipore assembly.     

Synexin-like proteins from human polymorphonuclear leukocytes. Identification and characterization of granule-aggregating and membrane-fusing activities

We have used Ca2+-dependent binding to a phospholipid vesicle affinity column to isolate a mixture of three synexin-like proteins from the cytosol of human polymorphonuclear leukocytes (PMN), with relative molecular weights of approximately 67,000, 47,000, and 28,000. Rabbit antibodies raised against bovine liver synexin recognized the 47,000 molecular weight PMN protein. These PMN proteins, like bovine liver synexin, promoted aggregation of isolated PMN specific granules in the presence of Ca2+ and increased the overall rate of Ca2+-induced fusion of liposomes composed of phosphatidate (PA)/phosphatidylethanolamine (PE) (1:3) and phosphatidylserine/PE (1:3), but decreased the rate of spermine-induced fusion of PA/PE (1:3) liposomes. Using fluorescent lipid probes, rapid fusion of PA/PE liposomes with PMN specific granules (50% maximum signal within a few minutes) was observed when 1 mM Ca2+ was added in the presence of both synexin and free arachidonic acid. Dilution of the aqueous contents of liposomes was also observed under the same conditions. The rate of fusion increased monotonically with Ca2+ and arachidonic acid concentrations, but synexin exhibited an optimum concentration. Lack of any one of the components precluded rapid fusion. These results suggest that PMN contain a protein similar to, or identical with, synexin that may be involved in calcium-dependent fusion of intracellular membranes.     

Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing

Diluents containing sonicated liposomes of purified phosphatidylserine (PS), phosphatidylcholine (PC) with varying fatty acyl chain lengths and double bonds and cholesterol (CH) alone or in combination, or egg yolk lecithin were evaluated for protection of bull sperm during cold shock produced by rapid cooling from 25 to 0 degrees C and during freezing and thawing. Bull semen was washed twice and diluted to 50 X 10(6) sperm/ml in diluents containing no lipid, 0.5 or 5 mM sonicated lipid or 20% egg yolk and plunged into ice water to cold shock the sperm. Sperm so treated were frozen using conventional methods. The percentage of progressively motile sperm (MS) was estimated prior to cooling, after cold shock, and after freezing and thawing. Lipids with fatty acyl chains of less than 12 carbons were toxic to sperm cells. Phosphatidylserine alone or in combination with PC or CH, but not PC or CH alone, protected sperm from cold shock as well as did egg yolk lecithin liposomes or egg yolk. Liposomes of PS/PC or PS/CH were not better than PS in protecting sperm from cold shock. Lipid concentrations of 0.5 mM were more effective than liposomes at 5 mM in protecting sperm during freezing and thawing. During freezing, PS alone or in combination with PC partially protected sperm, but only PS/CH was as effective as egg yolk in protecting sperm from freeze-thaw damage. It is concluded that defined diluents, particularly those containing PS, may be useful in studies of cryobiology of spermatozoa.     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

Comparison of two liposome fusion assays monitoring the intermixing of aqueous contents and of membrane components

Divalent cation-induced fusion of large unilamellar vesicles (approx. 0.1 micron diameter) made of phosphatidylserine (PS) or phosphatidylglycerol (PG) has been studied. Intermixing of aqueous contents during fusion was followed by the Tb/dipicolinic acid fluorescence assay, and intermixing of membrane components by resonance energy transfer between fluorescent lipid probes. Both assays gave identical threshold concentrations for Ca2+, which were 2 mM for PS and 15 mM for PG. The dependencies of the initial rate of fusion on the concentration of PG vesicles determined by either assay were identical, the order of this dependence being 1.2 in the concentration range of 5-200 microM lipid. For PS liposomes, this order was found to be 1.5 in the fluorescent lipid assay. No leakage of contents was detected during the fusion of PG vesicles. Mg2+ inhibited the Ca2+-induced fusion of PS vesicles, but did not cause any fusion by itself, consistent with previous results with the Tb/dipicolinic acid assay.     

Response of isolated sperm plasma membranes from sea urchin to egg jelly

The acrosome reaction in sea urchin sperm is induced by a glycoprotein jelly surrounding the egg and is accompanied by changes in ion permeability of sperm plasma membrane. In an attempt to learn what membrane components are involved in the response to jelly, we have begun to reassemble sperm membrane components into artificial membranes and assay for permeability changes mimicking those that occur in sperm. Jelly in sea water at concentrations that induce the acrosome reaction did not significantly change 45Ca2+ uptake of sonicated unilamellar vesicles made with soybean lipid only (ratio jelly:control uptake = 1.08 +/- 0.36 SD, n = 21). Experiments with pure lipid planar bilayers made with soybean lipid or a lipid extract from sperm and held at various voltages, also did not reveal substantial permeability changes at comparable jelly concentrations. Thus, jelly by itself does not change the conductance of a pure lipid bilayer. In contrast, significant (P----0.0005, t test for two sample means) 45Ca2+ uptake was observed with vesicles made by cosonicating soybean phospholipids and Strongylocentrotus purpuratus sperm membranes isolated by the method of Cross, N. L. [1983, J. Cell Sci. 59, 13-25] (ratio jelly: control uptake = 1.51 +/- 0.75, n = 20, 16 positive out of 20 experiments). The calcium uptake response of the mixed vesicles was also species-specific: it did not occur with jelly from Arbacia punctulata (ratio Arbacia jelly: control = 1.18 +/- 0.51; ratio Strongylocentrotus jelly: control = 1.71 +/- 0.97, n = 10; P----0.025, paired t statistic). Vesicles made with soybean lipid and an octyl glucoside extract of sperm membranes also responded to jelly with increased 45Ca2+ uptake. Our results indicate that we have the starting conditions to isolate and characterize the sperm membrane components that participate in the egg jelly induced permeability changes.     

Fusion of Human Sperm to Prostasomes at Acidic pH

Prostasomes are membranous vesicles (150–200 nm diameter) present in human semen. They are secreted by the prostate and contain large amounts of cholesterol, sphingomyelin and Ca²⁺. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. The possibility that they may fuse to sperm has never been proved. In this work, we studied the fusion of sperm to prostasomes by using various methods (relief of octadecyl Rhodamine B fluorescence self-quenching, fluorescence microscopy and flow cytometry) and we found that it occurs at acidic pH (4–5), but not at pH 7.5 pH-dependent fusion relies on the integrity of one or more proteins and is different from the Ca²⁺-stimulated fusion between rat liver liposomes and spermatozoa that does not require any protein and occurs at neutral pH. We think that the H⁺-dependent fusion of prostasomes to sperm may have physiological importance by modifying the lipid and protein pattern of sperm membranes. Received: 19 June 1996/Revised: 4 September 1996     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: II. A fertility assay for frozen-thawed semen

Frozen-thawed sperm from five bulls with fertility rates ranging from 48% to 77% were treated with seven concentrations of dilauroylphosphatidylcholine (PC12) liposomes to induce an acrosome reaction (AR) that enabled sperm to penetrate eggs. Treated sperm were incubated with liposomes for 7 min prior to insemination of zona-free hamster eggs in vitro. Sperm and eggs were incubated 3 hr at 39°C prior to fixation, staining, and examination for sperm penetration and nuclear decondensation. The percentage of motile sperm immediately after thawing as well as after treatment with liposomes had a low correlation with sire fertility (r = .39 and ?.63, respectively). The percentage of sperm exhibiting an AR was more highly correlated with fertility (r ? ?.85). Similar correlations were found between fertility and the penetration rates of zona-free hamster eggs or the total number of penetrating sperm. When data for two high and for two lower fertility buils were each grouped to increase information per data point the correlation between the PC12 concentration giving the maximum proportion of eggs penetrated and fertility was r = .92 (P ≤ .05). The correlation between the PC12 concentration producing the most total sperm penetrating the eggs and fertility r = .97 (P ≤ .05). It was concluded that PC12 liposomes induced an AR in bull sperm frozen-thawed in egg yolk extender. Frozen-thawed sperm from low fertility bulls require less PC12 to induce the AR and to penetrate zona-free hamster eggs than do sperm from higher fertility bulls. These differences in lipid requirements may help to provide a quick, direct laboratory assay method to estimate the fertility of frozen bull semen.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Artificial induction of exocytosis in bull sperm.

We have investigated an exocytotic event, the acrosome reaction (AR), induced by treatment of bovine sperm with vesicles composed of dilauroyl phosphatidylcholine (PC12). Cell membrane permeability barriers (dye exclusion), acrosomal status (pisum sativum (PSA) lectin binding), and intracellular Ca2+ (Fluo3 fluorescence) were evaluated utilizing flow cytometry and fluorescence microscopy. By these methods the AR is resolved into four kinetically distinct steps: (a) PC12 transfer to the sperm plasma membrane (PM); (b) increased permeability of the PM to extracellular Ca2+; (c) localized leakage of acrosomal contents at the anterior tip of the sperm; and (d) vesiculation of sperm membranes and complete exposure of acrosomal contents. Evidence for PC12 transfer to sperm includes transfer of a fluorescent PC12 analogue from vesicles to cells and the absence of detectable vesicle--cell fusion. The fusion inducing properties of PC12 appear to reside in the lipid head group as neither dilauroylphosphatidylethanolamine nor dilauroylphosphatidylglycerol stimulated the AR. The effect of PC chain length on AR induction closely parallels the aqueous phase solubility of the lipid tested. The rate and extent of the AR depend on the extracellular calcium concentration. Cells treated in the absence of calcium do not undergo the AR, but do so rapidly (less than 1 min) upon subsequent addition of calcium. This role of Ca2+ is partially filled by Sr2+, but not by Ba2+ or Mg2+. The rate of the AR decreases with decreasing temperature and the AR occurs very slowly below 27 degrees C. Simultaneous evaluation of intracellular calcium and acrosomal status reveals the kinetic relationship between Ca2+ influx and the exposure of acrosomal contents. N-Ethylmaleimide preincubation arrests PC12-treated sperm at an intermediate stage in the AR, characterized by punctate PSA binding over the tip of the sperm head. The AR, a developmentally regulated, receptor-mediated fusion event, synchronously induced here in vitro, provides a useful model for mechanistic studies of exocytosis.     

The blocking effect of aliphatic hydrocarbons on Ca2+ leakage channels formed by aggregates of Ca2+-ATPase of the sarcoplasmic reticulum

The effects of aliphatic hydrocarbons within the liposomes on the Ca2+ transport function of isolated sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle, vesiculate preparation of Ca2+ dependent ATPase and proteoliposomes reconstituted from Ca2+-ATPase and egg phosphatidylcholine, were studied. It was shown that liposomes prepared from dipalmitoyl phosphatidylcholine containing aliphatic hydrocarbons increase 2 to 3 times Ca2+ accumulation by Ca2+-dependent ATPase from rabbit skeletal muscle SR. Ca2+ transport by SR vesicles increases in the presence of hydrocarbons by 15--20%. The activating effect of hydrocarbons on Ca2+ transport by proteoliposomes depends on the lipid/protein ratio. The proteoliposomes with a high lipid/protein ratio are practically insensitive to the effects of hydrocarbons. It was suggested that activation of Ca2+ transport by hydrocarbons is due to blocking of Ca2+ leakage channels formed during the aggregation of Ca2+-ATPase molecules. Treatment of membranes by formaldehyde results in the oligomerization of Ca2+-ATPase and decreases 2--4-fold the ATP-dependent accumulation of Ca2+. Subsequent addition of decane restores Ca2+ transport practically completely.     

Role of Ca2+ and Ca2+-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Twenty-eight-kilodalton phosphorylatable calcium- and lipid-binding proteins purified from Physarum plasmodium.

Two 28-kDa calcium- and lipid-binding proteins were isolated from a detergent-insoluble fraction of the Physarum plasmodium. Both proteins have molecular masses of approximately 28 kDa by SDS-PAGE. The protein designated 28K-I has a slightly lower mobility than that designated 28K-II. The purified 28K-I has a dissociation constant of 1.0 microM for Ca2+ ions, while the 28K-II has two different dissociation constants: one of 0.32 microM and the other of 3.2 mM. The 28K-I binds to liposomes at Ca2+ concentrations higher than 1.0 microM and has a dissociation constant for lipid of 34 micrograms/ml at 10 microM Ca2+. The 28K-II binds to liposomes at concentrations of Ca2+ above the mM range and has a dissociation constant of 36 micrograms/ml for lipid at 2 mM Ca2+. There is no evidence of actin-binding activity by either of the 28-kDa (28K) proteins. The 28K proteins crossreacted with an antiserum against chicken brush border calpactin I. The two proteins have quite different phosphorylation levels between a fraction prepared from the cytosolic endoplasm and a fraction prepared from the whole cell. The 28K proteins may play some role in the membrane structure dynamics of the cortical gel layer.     

聚乙二醇和金属离子诱导脂质体与细胞的融合

用荧光菜振能量转移技术检测ＰＥＧ和金属离子诱导脂质体和细胞的融合,发现有ＴＥＧ参与诱导时,虽然Ｃａ＾２＋对膜融合的促进作用仍专一地依赖于ＰＳ的存在,但其对ＰＳ的依赖性降低;Ｍｎ＾２＋促进含ＰＳ和ＰＥ的脂质体与细胞的融合,而Ｍｇ＾２＋无作用。以ＰＣ：ＣＬ：Ｃｈｏｌ为０．５：０．５：１的脂质体包埋天花粉蛋白,经ＰＥＧ诱导与骨髓瘤细胞ＳＰ２０融合,提高了天花粉蛋白对骨髓瘤细胞的杀伤力。