Extent Of Exocytosis Research Articles

Overcoming presynaptic effects of VAMP2 mutations with 4-aminopyridine treatment.

Clinical and genetic features of five unrelated patients with de novo pathogenic variants in the synaptic vesicle-associated membrane protein 2 (VAMP2) reveal common features of global developmental delay, autistic tendencies, behavioral disturbances, and a higher propensity to develop epilepsy. For one patient, a cognitively impaired adolescent with a de novo stop-gain VAMP2 mutation, we tested a potential treatment strategy, enhancing neurotransmission by prolonging action potentials with the aminopyridine family of potassium channel blockers, 4-aminopyridine and 3,4-diaminopyridine, in vitro and in vivo. Synaptic vesicle recycling and neurotransmission were assayed in neurons expressing three VAMP2 variants by live-cell imaging and electrophysiology. In cellular models, two variants decrease both the rate of exocytosis and the number of synaptic vesicles released from the recycling pool, compared with wild-type. Aminopyridine treatment increases the rate and extent of exocytosis and total synaptic charge transfer and desynchronizes GABA release. The clinical response of the patient to 2 years of off-label aminopyridine treatment includes improved emotional and behavioral regulation by parental report, and objective improvement in standardized cognitive measures. Aminopyridine treatment may extend to patients with pathogenic variants in VAMP2 and other genes influencing presynaptic function or GABAergic tone, and tested in vitro before treatment.

The cysteine-rich domain of synaptosomal-associated protein of 23 kDa (SNAP-23) regulates its membrane association and regulated exocytosis from mast cells

Synaptosomal-associated protein of 23 kDa (SNAP-23) plays an important role during regulated exocytosis of various inflammatory mediators, stored in secretory granules, from mast cells in response to physiological triggers. It is however synthesized as a soluble protein, and the mechanisms by which free SNAP-23 gets peripherally associated with membrane for the regulation of exocytosis, are poorly defined. SNAP-23 contains a hydrophobic domain with five closely spaced cysteines which get palmitoylated, and we show that SNAP-23 cysteine mutants show differential membrane association when transfected in rat basophilic leukemia (RBL) mast cells. SNAP-23 Cys− mutant, devoid of all five cysteines, and SNAP-23 P119A (proline to alanine) mutant, that likely interferes with palmitoylation of SNAP-23 by palmitoyl transferases are completely cytosolic. Mutating specific cysteines (Cys; C) to leucine or phenylalanine (L or F; retains hydrophobicity but lacks palmitoylation) partially decreases the membrane association of SNAP-23 which is further hampered by alanine (A; has lesser hydrophobicity, and lacks palmitoylation) mutation at C79, C80 or C83 position. Cloning a transmembrane domain MDR31–145 from multidrug resistance protein into SNAP-23 Cys− mutant is able to partially restore its membrane association. Regulated exocytosis studies using co-transfected human growth hormone (hGH) secretion reporter plasmid revealed that overexpression of SNAP-23 Cys− and P119A mutants significantly inhibits the overall extent of exocytosis from RBL mast cells, whereas expression of SNAP-23 Cys−-MDR31–145 fusion protein is able to restore exocytosis. These results establish that the cysteine-rich domain of SNAP-23 regulates its membrane association and thereby also regulates exocytosis from mast cells.

Quantification of exocytosis kinetics by DIC image analysis of cortical lawns

Cortical lawns prepared from sea urchin eggs have offered a robust in vitro system for study of regulated exocytosis and membrane fusion events since their introduction by Vacquier almost 40years ago (Vacquier in Dev Biol 43:62-74, 1975). Lawns have been imaged by phase contrast, darkfield, differential interference contrast, and electron microscopy. Quantification of exocytosis kinetics has been achieved primarily by light scattering assays. We present simple differential interference contrast image analysis procedures for quantifying the kinetics and extent of exocytosis in cortical lawns using an open vessel that allows rapid solvent equilibration and modification. These preparations maintain the architecture of the original cortices, allow for cytological and immunocytochemical analyses, and permit quantification of variation within and between lawns. When combined, these methods can shed light on factors controlling the rate of secretion in a spatially relevant cellular context. We additionally provide a subroutine for IGOR Pro® that converts raw data from line scans of cortical lawns into kinetic profiles of exocytosis. Rapid image acquisition reveals spatial variations in time of initiation of individual granule fusion events with the plasma membrane not previously reported.

The role of calcium/calmodulin‐dependent protein phosphatase (PP2B) isoforms in exocytosis in Paramecium tetraurelia

Paramecium tetraurelia are ciliated protozoa that contain membrane‐docked vesicles called trichocysts. Trichocysts fuse with the membrane in a calcium‐dependent manner and release proteins that form long crystalline spines, which help Paramecium escape predators. A bacteria‐mediated RNA interference (RNAi) protocol was used to determine if members of the calcium/calmodulin‐dependent protein phosphatase family (PP2B) present in Paramecium were involved in trichocyst discharge as suggested by previous work. After treatment, Paramecium cells were selected and exocytosis triggered by the addition of a saturated picric acid solution. After normalization, the extent of exocytosis was compared and indicated that RNAi treatment with one of the isoforms of Paramecium PP2B resulted in a reduced efficiency of exocytosis. The number of cells exhibiting full exocytosis was reduced from 76% in control cells (n=129) to 25% in PP2B treated cells (n=190). Cells exhibiting dramatically reduced exocytosis (<50% full discharge) increased from 2% (control treatment) to 41% (PP2B treatment). Based upon these results, we conclude that PP2B has a role in the regulation of calcium‐dependent exocytosis in Paramecium tetraurelia. Funding for this project was provided by the Wilson Research Awards and the Howard Hughes Medical Institute.

Amisyn Regulates Exocytosis and Fusion Pore Stability by Both Syntaxin-dependent and Syntaxin-independent Mechanisms

Amisyn and tomosyn are related by the possession of a C-terminal vesicle-associated membrane protein-like domain that allows them to bind to syntaxin 1 and assemble into SNARE complexes. The formation of inactive complexes may sequester syntaxin and allow tomosyn and amisyn to act as inhibitors of exocytosis. We aimed to use adrenal chromaffin and PC12 cells to probe this possible mode of action of amisyn and tomosyn in dense core granule exocytosis. Although tomosyn is expressed by adrenal chromaffin and PC12 cells, amisyn expression could not be detected allowing examination of the effect of introduction of amisyn expression onto a neuronal-like background. Overexpression of m-tomosyn1 and expression of amisyn both inhibited Ca2+-induced exocytosis in transfected PC12 cells. Surprisingly, this inhibition was not removed when amisyn and tomosyn constructs were used in which key residues required for efficient binding to syntaxin1 were mutated. The effect of amisyn was further characterized using carbon fiber amperometry in chromaffin cells. Expression of amisyn had no effect on the basic characteristics of the amperometric spikes but reduced the number of spikes elicited. This inhibitory action on the extent of exocytosis was also seen with the amisyn mutant deficient in syntaxin1 binding. In addition, expression of amisyn resulted in an increase in the lifetime of the prespike foot, and this effect was abolished by the mutations. These results show that tomosyn and amisyn can negatively regulate exocytosis independently of syntaxin and also that amisyn can regulate the stability of the fusion pore.

Munc18-1 Regulates Early and Late Stages of Exocytosis via Syntaxin-independent Protein Interactions

Sec1/Munc18 (SM) proteins are involved in various intracellular membrane trafficking steps. Many SM proteins bind to appropriate syntaxin homologues involved in these steps, suggesting that SM proteins function as syntaxin chaperones. Organisms with mutations in SM genes, however, exhibit defects in either early (docking) or late (fusion) stages of exocytosis, implying that SM proteins may have multiple functions. To gain insight into the role of SM proteins, we introduced mutations modeled on those identified in Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae into mammalian Munc18-1. As expected, several mutants exhibited reduced binding to syntaxin1A. However, three mutants displayed wild-type syntaxin binding affinities, indicating syntaxin-independent defects. Expression of these mutants in chromaffin cells either increased the rate and extent of exocytosis or altered the kinetics of individual release events. This latter effect was associated with a reduced Mint binding affinity in one mutant, implying a potential mechanism for the observed alteration in release kinetics. Furthermore, this phenotype persisted when the mutation was combined with a second mutation that greatly reduced syntaxin binding affinity. These results clarify the data on the function of SM proteins in mutant organisms and indicate that Munc18-1 controls multiple stages of exocytosis via both syntaxin-dependent and -independent protein interactions.

A single amino acid near the C terminus of the synaptosomeassociated protein of 25 kDa (SNAP-25) is essential for exocytosis in chromaffin cells.

Amperometry in chromaffin cells expressing green fluorescent protein (GFP) fused to synaptosome-associated protein of 25 kDa (SNAP-25) have been used to test the involvement of single amino acids in exocytotic function, overcoming some of the limitations of studies based on Botulinum neurotoxin cleavage, as this occurs at defined sites of the protein. Constructs containing either the whole SNAP-25 polypeptide or several deleted forms lacking its C-terminal domain were heavily overexpressed in transfected cells. All GFP-fusions were located in both the cytoplasm and the plasma membrane. Although a construct containing complete SNAP-25 sustained normal secretion, removal of four or more amino acids of its C terminus greatly altered the overall rate and extent of exocytosis. Further mutational analysis proved that Leu203, the fourth residue from the C terminus, is critical for secretion. Kinetics of single granule fusions from cells expressing truncated forms showed slow onset and decay times when compared with control cells expressing full SNAP-25. Thus, these data provide direct evidence for the involvement of a specific residue of SNAP-25 in exocytosis and show that overexpression of GFP-SNAP contructs combined with single vesicle fusion measurements constitutes a powerful approach to dissect the structural elements playing a role in individual steps of the exocytotic cascade.

Specific role for the PH domain of dynamin-1 in the regulation of rapid endocytosis in adrenal chromaffin cells.

Dynamin plays a key role in the scission event common to various types of endocytosis. We demonstrate that the pleckstrin homology (PH) domain of dynamin-1 is critical in the process of rapid endocytosis (RE) in chromaffin cells. Introduction of this isolated PH domain into cells at concentrations as low as 1 microM completely suppressed RE. PH domains from other proteins, including that from the closely related dynamin-2, were ineffective as inhibitors, even at high concentrations. Mutational studies indicated that a pair of isoform-specific amino acids, located in a variable loop between the first two beta-strands, accounted for the differential effect of the two dynamin PH domains. Switching these amino acids in the dynamin-2 PH domain to the equivalent residues in dynamin-1 (SL-->GI) generated a molecule that blocked RE. Thus, the PH domain of dynamin-1 is essential for RE and exhibits a precise molecular selectivity. As chromaffin cells express both dynamin-1 and -2, we speculate that different isoforms of dynamin may regulate distinct endocytotic processes and that the PH domain contributes to this specificity.

Patch-clamp capacitance analysis of the effects of alpha-SNAP on exocytosis in adrenal chromaffin cells.

We have examined the effect of alpha-SNAP on exocytosis in adrenal chromaffin cells by direct assay of exocytosis using patch-clamp capacitance analysis. Cells were recorded using the whole cell patch-clamp configuration and the cells dialysed with control pipette solution or with a pipette solution containing alpha-SNAP or the deletion mutant alpha-SNAP(41-295). The deletion mutant was found to be unable to bind to syntaxin allowing a test of the requirement for syntaxin-binding for any effect of alpha-SNAP on exocytosis. Following cell dialysis for 10 minutes, cells were depolarised five times at 2 minute intervals. At each depolarisation step cells dialysed with alpha-SNAP showed a significant increase in both the initial rate and extent of exocytosis which was seen as a rise in membrane capacitance. This increase in exocytosis was not observed with alpha-SNAP(41-295) which instead produced some inhibition of the extent but had no effect on the initial rate of exocytosis. These results show directly that alpha-SNAP has a specific and marked stimulatory effect on exocytosis in chromaffin cells.

Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.

Proteases Stimulate Fertilization-like Responses in Starfish Eggs

Application of proteases to eggs of the starfish, Asterina miniata, caused several responses like those seen at fertilization. Cortical granule exocytosis and fertilization envelope elevation occurred within about 1 min after exposure to trypsin, chymotrypsin, or pronase; protease inhibitors prevented these responses. Kallikrein caused cortical granule exocytosis and fertilization envelope elevation, but this response required more time (∼30 min). Exocytosis was also seen in response to a recombinant trypsin, but not to a point-mutated trypsin without proteolytic activity. The extent of exocytosis was similar to that seen at fertilization, as measured by the fluorescent dye FM 1-43. In addition to causing exocytosis, application of trypsin, chymotrypsin, or pronase caused an increase in intracellular free calcium, detected by calcium green dextran, and stimulation of DNA synthesis, detected by incorporation of bromodecxyuridine. Exocytosis also occurred when trypsin or chymotrypsin was applied in artificial sea water in which the free calcium was reduced to a low level (40-70 n M) such that Ca influx would be reduced by >10,000-fold; this indicated that the proteases did not act by damaging the eggs and causing external calcium to leak into the cytoplasm. These findings show that there is an extracellularly exposed protein that when proteolyzed can induce fertilization-like responses; this protein may be a receptor that transduces a signal from the sperm to initiate egg activation at fertilization.

Capturing and quantifying the exocytotic event.

Although exocytosis is now known to be the universal method by which proteins are released from eukaryotic cells, we know surprisingly little of the mechanism by which exocytosis occurs. One reason for this is that it has proved difficult to capture sufficient of these evanescent events to permit their study. The difficulty with which exocytoses can be visualized with standard preparative techniques varies among tissues, but the problem is particularly apparent in the mammalian nervous system. Tannic acid has recently been introduced as an agent by which exocytosed granule cores can be captured and visualized electron-microscopically. Application of tannic acid to the magnocellular neurosecretory system reveals exocytoses from all parts of their terminal arborization within the neural lobe, and also from their dendrites within the hypothalamus. Quantification of the exocytoses in unstimulated tissue and in tissue stimulated by a variety of exogenous and endogenous mechanisms indicates: (a) that exocytosis occurs equally from each unit of membrane of the perivascular nerve endings, and of the axonal swellings that were previously thought to be sites of granule storage, rather than release; (b) that, in the nerve endings, a greater proportion of the stored granules are exocytosed, and thus the endings are specialized for release not by any particular property of their membrane, but by a high surface membrane:volume ratio. Together, the data cast doubt on the hypothesis that exocytosis occurs only at some functionally specialized sites at certain loci in the membrane. Rather, the data favour the hypothesis that magnocellular granules can fuse with any part of the membrane, depending on constraints imposed by the cytoskeleton, and a local increase in cytosolic free calcium level. When applied to hypothalamic central nervous tissue, tannic acid reveals that exocytosis of dense-cored synaptic vesicles occurs preferentially, but not exclusively, at the membrane apposed to the postsynaptic element. However, about half of all exocytoses from synaptic boutons occur at bouton membrane unrelated to the synaptic cleft. In all tissues studied, tannic acid reveals a heterogeneity among secretory cells in the extent of exocytosis that occurs in response to stimulation, and permits an analysis of the degree to which secretion is polarized in any one direction. These results question long-held assumptions concerning the site at which neurones release transmitters and modulators. Tannic acid seems likely to prove a potent tool in the investigation of both the mechanism of exocytosis and the ways in which different types of cells adapt the process to perform their physiol

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils.

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.

Polycation inhibition of exocytosis from sea urchin egg cortex.

The Ca2+-stimulated release of vesicle contents from cortical fragments prepared from sea urchin eggs is an in vitro model for exocytosis. Cortical fragments have been isolated either in suspension (cell surface complex, CSC preparation), or attached to polycation-coated surfaces (cortical lawn, CL preparation). CL, but not CSC, have been reported to undergo a rapid "aging" process whereby they fail to respond to micromolar free Ca2+. Since, in principle, the only difference between the two preparations is the use of polycations in the CL preparation, polycations were suspected of being inhibitory. This hypothesis was tested by evaluating the effects of polycation-containing buffers on the Ca2+ threshold, rate, and extent of exocytosis in CL prepared from the eggs of Strongylocentrotus purpuratus. A sensitive microphotometric assay, based on light scattering by the individual cortical vesicles in the CL, was used to quantitate the exocytotic response. Buffers containing polylysine were found to be potent inhibitors of cortical exocytosis. The Ca2+ threshold of CL that had been treated for 15 min at room temperature with 50 micrograms/ml of polylysine was more than three orders of magnitude greater than that of freshly prepared CL. The other polycations tested (protamine, spermine and neomycin) were also found to be inhibitory, but to a lesser degree than polylysine. Two lines of evidence suggested that the polycations used in the preparation of CL are responsible for the rapid "aging" phenomenon: CSC fragments that had been affixed to polylysine-coated coverslips were shown to acquire "aging" characteristics similar to the CL preparations; control CSC that had been maintained in suspension did not. Radiolabeled poly-L-lysine was shown to dissociate from coated coverslips and redistribute onto CL.

Exocytosis regulates urinary acidification in turtle bladder by rapid insertion of H+ pumps into the luminal membrane.

Urinary acidification by the turtle bladder is due to a H+-ATPase that is located in the luminal membrane. The rate of H+ transport is stimulated by an increase in the ambient CO2. Using the fluorescent dye acridine orange, we showed that the mitochondria-rich cell of this equilibrium contains vesicles whose internal pH is acidic. We measured the pH of these vesicles by using endocytosed fluorescein isothiocyanate-labeled dextran and found it to be near 5.0. The pH increased after treatment with protonophores or metabolic inhibitors, suggesting that it was due to a H+ pump rather than to a Donnan effect. In bladders preloaded with fluorescent dextran, CO2 stimulated exocytosis and H+ transport measured simultaneously in the same bladder. The increase in the H+ current correlated well with the extent of exocytosis, and both were inhibited by pretreatment with colchicine. We conclude that the turtle bladder contains an intracellular reserve of vesicles containing H+ pumps and CO2 stimulates rapid fusion of these vesicles with the luminal membrane with consequent insertion of H+ pumps, thereby stimulating H+ secretion across the whole epithelium.

Biochemical Studies on the Leukocytes in Chediak-Higashi Syndrome

Blood leukocytes from a patient with Chediak-Higashi syndrome (CHS) were compared with normal cells for their capacity of extruding (exocytosis) the lysosomal enzyme myeloperoxidase during phagocytosis or after a treatment with the ionophore A23187 and Ca2+. A decreased rate and extent of exocytosis in phagocytizing CHS cells was observed also with the Ca2+ ionophore. This suggests that a defect in Ca2+ mobilization is not responsible for the impaired secretion of granule content. Isolated granules of CHS cells and of leukocytes were treated with the detergent Triton X-100. Since the solubilization of myeloperoxidase from the CHS granules was much lower than from the normal ones, we suggest that the former organelles have a more resistant membrane.