Lipid dynamics in exocytosis.

Regulated exocytosis of neurotransmitter- and hormone-containing vesicles underpins neuronal and hormonal communication and relies on a well-orchestrated series of molecular interactions. This in part involves the upstream formation of a complex of SNAREs and associated proteins leading to the eventual fusion of the vesicle membrane with the plasma membrane, a process that enables content release. Although the role of lipids in exocytosis is intuitive, it has long been overlooked at least compared to the extensive work on SNAREs. Here, we will present the latest advances in this rapidly developing field revealing that lipids actually play an active role in exocytosis by focusing on cholesterol, 3'-phosphorylated phosphoinositides and phosphatidic acid.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis.

BACKGROUND: The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model. RESULTS: In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes. CONCLUSION: This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

A stage-specific preparation to study the Ca(2+)-triggered fusion steps of exocytosis: rationale and perspectives.

Despite groundbreaking work to identify numerous proteins and to focus attention on molecular interactions, the mechanism of calcium-triggered membrane fusion remains unresolved. A major difficulty in such research has been the many overlapping and interacting membrane trafficking steps in the secretory pathway, including those of membrane retrieval. Identifying the specific role(s) of a given protein, beyond its general involvement in exocytosis, has therefore proven problematic. Furthermore, the power of time-resolved optical and electrophysiological assays can be best applied to testing the function of known proteins rather than to the identification of unknown, critical membrane components. The identification of essential membrane constituents requires combined biochemical (molecular) and functional (physiological) analyses. A fully functional, stage-specific physiological membrane preparation would be one direct approach to dissecting the calcium-triggered fusion steps of regulated exocytosis. Herein we review our use of specific minimal membrane preparations consisting of fully primed and docked secretory vesicles, or the isolated vesicles themselves, and characterize the late events of exocytosis, with an aim towards identification of essential molecular components. We have established a functional definition of the fusion complex and its activation by calcium, based on our kinetic analyses. Together with a variety of biochemical and alternate functional assays, we have tested whether the SNARE core complex that is present in our vesicle membranes satisfies the criteria of the functionally defined fusion complex. Rather than a direct fusogenic role, the SNARE complex may promote the calcium sensitivity of fusion, possibly by defining or delimiting a localized, focal membrane fusion site that ensures rapid and efficient exocytosis in vivo.

Sea urchin egg preparations as systems for the study of calcium-triggered exocytosis.

This paper reviews recent work in our laboratory on the mechanism of calcium-triggered exocytosis. Upon echinoderm egg fertilization, cortical secretory vesicle exocytosis is massive and synchronous. By combining physiological and molecular analyses with a variety of purified membrane isolates containing secretory vesicles that fuse to the plasma membrane or each other, we have characterized the final steps of this calcium-triggered exocytosis. Our kinetic analysis led to a functional definition of the fusion complex whose activation by calcium follows Poisson statistics. The properties of this complex are compared with the properties of the heterotrimeric SNARE protein complex that is present in the cortical vesicle system. Our data do not support the hypothesis that this particular heterotrimeric complex is by itself the biological fusogen.

Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion.

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (approximately 70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca2+]free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca2+-triggering step, blocks complex disruption by Ca2+. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca2+-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca2+ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca2+ binding removes a "fusion-clamp."

Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca2+ sensitivity.

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV-PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, Max, was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV-PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion.

Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms.

Using the patch-clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12-myristate 13-acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated approximately 2-fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 microM also resulted in a 2-fold acceleration of pore expansion; this effect was not prevented by staurosporine, indicating that intracellular Ca2+ and activation of PKC accelerate fusion pore expansion via distinct mechanisms. However, fusion pores can expand fully even when PKC is inhibited. In contrast, the phosphatase inhibitor alpha-naphthylphosphate inhibits exocytotic fusion and slows fusion pore expansion. These results demonstrate that, subsequent to its formation, fusion pore expansion is under control of proteins subject to functional changes based on their phosphorylation states.

Ca2+ triggers massive exocytosis in Chinese hamster ovary cells.

We have tracked the cell surface area of CHO cells by measuring the membrane capacitance, Cm. An increase in cytosolic [Ca2+], [Ca2+]i, increased the cell surface area by 20-30%. At micromolar [Ca2+]i the increase occurred in minutes, while at 20 microM or higher [Ca2+]i it occurred in seconds and was transient. GTPgammaS caused a 3% increase even at 0.1 microM [Ca2+]i. We conclude that CHO cells, previously thought capable only of constitutive exocytosis, can perform Ca2+-triggered exocytosis that is both massive and rapid. Ca2+-triggered exocytosis was also observed in 3T3 fibroblasts. Our findings add evidence to the view that Ca induces exocytosis in cells other than known secretory cells.

Phospholipase activation and secretion: evidence that PLA2, PLC, and PLD are not essential to exocytosis.

Numerous studies have identified phospholipase metabolites as membrane fusogens, and phospholipase D (PLD) (J.R. Coorssen and R.J. Haslam. FEBS Lett. 316: 170-174, 1993), C (PLC), and A2 (PLA2) activities correlate with secretion. Do these enzymes have essential or modulatory roles? This study confirms that secretion does not require Ca2+ or PLC (Coorssen et al. Cell Regul. 1: 1027-1041, 1990). Arachidonic acid (AA), phosphatidic acid (PA) and analogues, exogenous metabolites of PLA2 and PLD, were tested in electropermeabilized human platelets. AA potentiated guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-induced secretion, and eicosanoids were not essential. Endogenous [3H]AA formation correlated with GTP gamma S-induced secretion, and phorbol 12-myristate 13-acetate (PMA) promoted these effects. Inhibitors were used to probe phospholipase influences on secretion. Only PLD inhibitors blocked secretion. However, PMA blocked inhibition of protein kinase C (PKC) and secretion by quercetin, suggesting that PA formed by PLD supports PKC activation and GTP gamma S-induced secretion. Thus PA analogues had no effect alone but enhanced GTP gamma S-induced PKC activity and secretion. Slower PLD activation compared with secretion also indicates a nonessential role. This is the first report of a Ca(2+)-independent PLA2 activity in human platelets, use of quercetin as a PLD inhibitor, and dissociation of PLA2, PLC, and PLD activities from secretion. No major phospholipase activities are essential to the final steps in exocytosis, but modulatory roles are indicated.

Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells.

Ca(2+)-triggered exocytosis was studied in single rat melanotrophs and bovine chromaffin cells by capacitance measurements. Sustained exocytosis required MgATP, but even in the absence of MgATP, Ca2+ could trigger exocytosis of 2700 granules in a typical melanotroph and of 840 granules in a chromaffin cell. Granules undergoing ATP-independent exocytosis were similar in number to those appearing docked to the plasmalemma in quickly frozen unfixed sections (3300 in a melanotroph and 830 in a chromaffin cell). Most exocytosis required tens of seconds, but a small pool of granules was released in tens of milliseconds. Evidently, only a small subset of docked granules is rapidly releasable. We suggest that, temporally, the last ATP-dependent step in exocytosis is closely associated with docking and that docked granules reach fusion competence only after subsequent steps.

Structural effects of neutral lipids on divalent cation-induced interactions of phosphatidylserine-containing bilayers.

Ca2+ is known to induce the adhesion and collapse of phosphatidylserine (PS) bilayers into dehydrated multilamellar structures. The aim of this study was to examine how that interaction and the resultant structures might be modified by neutral lipid species. A combination of rapid mixing, x-ray diffraction, thin-layer chromatography, density gradient centrifugation, and freeze-fracture electron microscopy was used in conjunction with osmotic stress techniques to characterize the structures formed by the Ca(2+)-induced interaction of multilamellar liposomes and of large unilamellar vesicles. The results showed that dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine at concentrations of up to approximately 30 mol % are accommodated in a single dehydrated multilamellar structure. Similar results were obtained using mixed PS species isolated from bovine brain. Principally, the data indicate that neutral lipid is both dehydrated during the rapid collapse process of Ca(PS)2 formation and accommodated within this dehydrated structure. The large energies available on formation of the Ca(PS)2 bilayers contribute to the dehydration of neighboring neutral lipids that likely form continuous bilayers with them. Higher concentrations of these neutral lipids modify Ca(2+)-induced bilayer interactions, leading to progressively weaker interactions, larger bilayer separations, and in some cases separation into two structures; phosphatidylethanolamine species favoring nonbilayer structures tended to promote such separation at lower concentrations than bilayer lipids.

Evidence that activation of phospholipase D can mediate secretion from permeabilized platelets.

Studies on electropermeabilized human platelets indicated that any two of three distinct factors must be present for marked secretion of dense or alpha-granule constituents to occur. These factors are Ca2+, activation of protein kinase C (PKC) and activation of an unidentified GTP-binding protein ('GE'). Thus, in the absence of Ca2+, phorbol ester and GTP[S] acted synergistically to promote secretion, whereas in the presence of Ca2+, either activation of PKC or addition of GTP[S] was sufficient. In all cases, secretion correlated with the activation of phospholipase D (PLD), as detected by the formation of [3H]phosphatidic acid (PA) in the absence of ethanol or of [3H]phosphatidylethanol (PEt) in the presence of ethanol. Secretion did not correlate with phospholipase C (PLC) activity or with the accumulation of 1,2-diacylglycerol (DAG), both of which required Ca2+ and were inhibited by phorbol ester. Ethanol partially inhibited secretion in the absence of Ca2+. BAPTA, a known inhibitor of Ca(2+)-independent secretion in permeabilized cells, caused parallel inhibitions of secretion and PLD activity. GTP[S] enhanced PKC activity, as indicated by pleckstrin phosphorylation, apparently by stimulating the formation of PA in the absence of Ca2+, as well as of DAG in the presence of Ca2+. PA and stable analogues, including PEt, stimulated the Ca(2+)-independent phosphorylation of pleckstrin and other proteins in platelet supernatant fraction. The results suggest that PA formed by activation of PLD may mediate secretion from permeabilized platelets by PKC-dependent and independent mechanisms. However, in intact platelets stimulated by thrombin, PLD accounted for only 10-20% of the total PA formed and can only play a major role in secretion if this PA fraction is distinct from that formed by the combined actions of PLC and DAG kinase.

GTP gamma S and phorbol ester act synergistically to stimulate both Ca(2+)-independent secretion and phospholipase D activity in permeabilized human platelets. Inhibition by BAPTA and analogues.

We have tested the hypothesis that phospholipase D (PLD) is the effector of the unidentified G protein (GE) mediating Ca(2+)-independent exocytosis in platelets. Although GTP gamma S, and to a lesser extent phorbol 12-myristate 13-acetate (PMA), caused some secretion of 5-HT from electropermeabilized human platelets in the effective absence of Ca2+ (pCa > 9), these stimuli had much more potent synergistic effects when added together. In all cases, secretion of 5-HT was closely correlated to the stimulus-induced formation of [3H]phosphatidic acid ([3H]PA) from [3H]arachidonate-labelled phospholipids. Addition of ethanol inhibited both secretion and [3H]PA formation and led to the accumulation of [3H]phosphatidylethanol ([3H]PEt), indicating that [3H]PA was formed largely by activation of PLD. BAPTA and analogues caused dose-dependent inhibitions of both GTP gamma S-induced secretion and PLD activity in the permeabilized platelets. This action of BAPTA did not appear to be mediated by chelation of Ca2+ or by direct inhibition of protein kinase C (PKC). The results suggest that PLD is the target of GE in platelets and that BAPTA can block PLD activation.

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.

Effects of cholesterol on the structural transitions induced by diacylglycerol in phosphatidylcholine and phosphatidylethanolamine bilayer systems.

The transient membrane lipid diacylglycerol (DG) is known to modify and destabilize phospholipid bilayers and can lead to the formation of nonbilayer structures. Since cholesterol forms a major fraction of many plasma membranes, we have investigated how it modifies the structural effects of DG on bilayers of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE). We view these systems as modelling the behaviour of local, DG-containing sites in membranes. Using X-ray diffraction, we have characterized the lamellar (L alpha) and inverse hexagonal (HII) structures that these ternary lipid mixtures form in excess aqueous solution. As the DG level increases, the lipid progresses from a single L alpha structure to a mixture of L alpha and HII, and then to a pure HII structure. This allows determination of the DG levels at which the HII transition begins, which we interpret as those levels that destabilize bilayers. In both PC and PE bilayers, the presence of 30 mol% cholesterol reduces the amounts of DG required to destabilize the bilayer structure. The destabilization can be translated into the number of neighbouring lipid molecules that a DG molecule perturbs, and of bilayer areas that it affects. The data show that the presence of cholesterol greatly enhances the perturbing effects of DG. We examine the possible role of DG in enzyme activation and membrane fusion.