Synhibin: a new calcium-dependent membrane-binding protein that inhibits synexin-induced chromaffin granule aggregation and fusion

We report the isolation and purification of synhibin, a new M_r 68000 protein, which inhibits synexin. Synexin mediates Ca²⁺-dependent chromaffin granule aggregation and fusion, processes perhaps important during exocytosis. Our data indicate that synhibin action involves competition with synexin for a site on the chromaffin granule membrane involved in membrane contact. Synhibin may thus be an important intracellular regulator of synexin action during secretion.     

The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding

Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca²⁺-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion.     

Ca-regulated secretory granule exocytosis in pancreatic and parotid acinar cells

Protein secretion from acinar cells of the pancreas and parotid glands is controlled by G-protein coupled receptor activation and generation of the cellular messengers Ca²⁺, diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca²⁺. However, in addition to diverse signaling pathways, acinar cells have large ∼1 μm diameter SGs (∼30 fold larger diameter than synaptic vesicles), respond to stimulation at slower rates (seconds versus milliseconds), demonstrate significant constitutive secretion, and in isolated acini, undergo sequential compound SG–SG exocytosis at the apical membrane. Exocytosis proceeds as an initial rapid phase that peaks and declines over 3 min followed by a prolonged phase that decays to near basal levels over 20–30 min. Studies indicate the early phase is triggered by Ca²⁺ and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca²⁺-sensing protein synatotagmin 1 (syt1) and the accessory protein complexin 2. The molecular details for regulation of VAMP8-mediated SG exocytosis and the prolonged phase of secretion are still emerging. Here we review the known regulatory molecules that impact the sequential exocytic process of SG tethering, docking, priming and fusion in acinar cells.     

Fusion of human erythrocytes induced by uranyl acetate and rare earth metals

Incubation of human erythrocytes with either uranyl ions (UO₂²⁺) or rare earth metals (La³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺ and Yb³⁺) at 37°C for 30–45 min resulted in the fusion of erythrocytes. Redistribution of membrane-associated particles was observed using colloidal-iron charge labelling and freeze-fracture electron microscopy. The fusion of erythrocytes induced by these agents, unlike Ca²⁺, did not exhibit the absolute requirement for phosphate. Moreover, agglutination and fusion by these agents was observed in neuraminidase-treated erythrocytes in contrast to Ca²⁺- and phosphate-induced fusion. Inhibitors of intrinsic transglutaminase activity partially inhibited (35–45%) the fusion induced by UO₂²⁺ suggesting that cross-linking of membrane proteins results in protein-free areas of lipid where fusion may be initiated.     

Arabidopsis Synaptotagmin SYT1, a Type I Signal-anchor Protein,Requires Tandem C2 Domains for Delivery to the Plasma Membrane

The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C₂A and C₂B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C₂A-C₂B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C₂B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins.     

Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

Ca²⁺-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca²⁺ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca²⁺ sensitivity of both CV-plasma membrane and CV–CV fusion. If Sr²⁺, a weak Ca²⁺ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca²⁺, suggesting that IA acts at the Ca²⁺-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency.     

Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca²⁺-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca²⁺-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.     

Munc18a Does Not Alter Fusion Rates Mediated by Neuronal SNAREs,Synaptotagmin, and Complexin

Sec1/Munc18 (SM) proteins are essential for membrane trafficking, but their molecular mechanism remains unclear. Using a single vesicle-vesicle content-mixing assay with reconstituted neuronal SNAREs, synaptotagmin-1, and complexin-1, we show that the neuronal SM protein Munc18a/nSec1 has no effect on the intrinsic kinetics of both spontaneous fusion and Ca²⁺-triggered fusion between vesicles that mimic synaptic vesicles and the plasma membrane. However, wild type Munc18a reduced vesicle association ∼50% when the vesicles bearing the t-SNAREs syntaxin-1A and SNAP-25 were preincubated with Munc18 for 30 min. Single molecule experiments with labeled SNAP-25 indicate that the reduction of vesicle association is a consequence of sequestration of syntaxin-1A by Munc18a and subsequent release of SNAP-25 (i.e. Munc18a captures syntaxin-1A via its high affinity interaction). Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association. In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions. Our results suggest that Munc18a primarily acts at the prefusion stage.     

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca²⁺. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10^?7 M and 10^?4 M Ca²⁺, was specific for Ca²⁺, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca²⁺ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca²⁺ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca²⁺ concentrations we have not found a major influence of Ca²⁺ asymmetry on fusion. Incorporation of the ganglioside GM₃, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca²⁺-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca²⁺ ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6 · 10}{}^{\mathrm{?6}}\text{M}$) and low affinity sites ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.2 · 10}{}^{\mathrm{?4}}\text{M}$).     

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca²⁺-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca²⁺-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca²⁺-sensitive regulatory pathway for zymogen granule exocytosis.     

Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells

Synaptotagmin VII (Syt VII), which has a higher Ca²⁺ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca²⁺-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca²⁺ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.     

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

In neuroexocytosis, SNAREs and Munc18-1 may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc18-1 stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca²⁺ sensor synaptotagmin-1 (Syt1), providing the structural basis for the conserved function of Sec1/Munc18 proteins in exocytosis. However, when full-length Syt1 is present, no enhancement of SNARE zippering and no acceleration of Ca²⁺-triggered content mixing by Munc18-1 are observed. Thus, our results show that Syt1 acts as an antagonist for Munc18-1 in SNARE zippering and fusion pore opening. Although the Sec1/Munc18 family may serve as part of the fusion machinery in other exocytotic pathways, Munc18-1 may have evolved to play a different role, such as regulating syntaxin-1a in neuroexocytosis.     

Identifying the targets of the amplifying pathway for insulin secretion in pancreatic beta-cells by kinetic modeling of granule exocytosis

A kinetic model for insulin secretion in pancreatic β-cells is adapted from a model for fast exocytosis in chromaffin cells. The fusion of primed granules with the plasma membrane is assumed to occur only in the “microdomain” near voltage-sensitive L-type Ca²⁺-channels, where [Ca²⁺] can reach micromolar levels. In contrast, resupply and priming of granules are assumed to depend on the cytosolic [Ca²⁺]. Adding a two-compartment model to handle the temporal distribution of Ca²⁺ between the microdomain and the cytosol, we obtain a unified model that can generate both the fast granule fusion and the slow insulin secretion found experimentally in response to a step of membrane potential. The model can simulate the potentiation induced in islets by preincubation with glucose and the reduction in second-phase insulin secretion induced by blocking R-type Ca²⁺-channels (Ca_V2.3). The model indicates that increased second-phase insulin secretion induced by the amplifying signal is controlled by the “resupply” step of the exocytosis cascade. In contrast, enhancement of priming is a good candidate for amplification of first-phase secretion by glucose, cyclic adenosine 3′:5′-cyclic monophosphate, and protein kinase C. Finally, insulin secretion is enhanced when the amplifying signal oscillates in phase with the triggering Ca²⁺-signal.     

Plasma membrane calcium pump activity is affected by the membrane protein concentration: Evidence for the involvement of the actin cytoskeleton

Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca²⁺ from the cell. Specific Ca²⁺-ATPase activity of erythrocyte membranes increased steeply up to 1.5-5 times when the membrane protein concentration decreased from 50 μg/ml to 1 μg/ml. The activation by dilution was also observed for ATP-dependent Ca²⁺ uptake into vesicles from Sf9 cells over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca²⁺ or Ca²⁺-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.     

Calcium-dependent Regulation of SNARE-mediated Membrane Fusion by Calmodulin

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca²⁺ sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca²⁺-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca²⁺/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K_D = 500 nm) and syntaxin 1 (K_D = 2 μm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca²⁺ sensors act antagonistically in SNARE-mediated fusion.     

Fusion of isolated synaptic vesicles as a model of the terminal stage of regulated exocytosis

In the study of membrane fusion, which is the terminal stage of exocytosis, we used a simplified model consisting of homotypic membranes of isolated synaptic vesicles (SV) obtained from the synaptosomal fraction of rat brain tissue. It was shown that fusion of SV develops in the presence of cytoplasmic proteins and 10^–7 to 10^–5 M Ca²⁺ ions. This conclusion was made based on changes in the intensity of fluorescence of a probe, R18. Calcium ions were found to be the most effective activators of the membrane fusion when the effects of bivalent cations, Ca²⁺, Sr²⁺, and Ba²⁺, were compared. ATP induced membrane fusion both in the presence and in the absence of Ca²⁺, and the effects of ATP and Ca²⁺ were additive. These findings allow us to believe that there are factors in the system containing SV and soluble proteins of synaptosomes, which initiate fusion of the membranes under the influence of not only Ca²⁺ but also ATP. The intensity of Ca²⁺-dependent fusion of SV dropped after trypsin treatment, i.e., proteolysis resulted in modulation of the sensitivity of vesicular proteins and/or a change in their capability of evoking membrane fusion. Monoclonal antibodies against synaptotagmin and synaptobrevin inhibited fusion of SV, but only partly. Our results support the concept that Ca²⁺-regulated membrane fusion is possible without the involvement of the entire SNARE complex.Neirofiziologiya/Neurophysiology, Vol. 36, No. 4, pp. 272–280, July–August, 2004.This revised version was published online in April 2005 with a corrected cover date.     

Assay of dense-core vesicle exocytosis using permeabilized PC12 cells

Exocytosis plays an essential role in fundamental cellular events by secreting neurotransmitters, hormones, and cytokines. Although the minimal molecular components termed SNARE that govern membrane fusion have been identified, the precise mechanisms behind the finely-tuned regulation of exocytosis executed by many molecules in addition to the actions of SNARE remain to be fully identified. Here, we evaluated a model system for assaying catecholamine secretion from permeabilized rat pheochromocytoma PC12 cells, in which the structural integrity required was preserved adequately. Among several chemical reagents used for the cell permeabilization and freezing-thawing procedures, the treatment of cells with digitonin at concentrations of 7.5–15 μM was most suitable for the secretion assay, as it was considered to cause mild disruption of the plasma membrane, enabling free access to small molecules such as Ca²⁺ and ATP to the minimal membrane fusion machinery. No additional cytosolic proteins were required to reconstitute the secretion. In this assay model, ATP was necessary to maintain the priming state before Ca²⁺-triggered exocytosis but was not required for the Ca²⁺-triggered membrane fusion process itself. The present study provides a useful cell model for exploring novel molecules that may be implicated in exocytosis such as those playing regulatory roles in addition to the “minimal membrane fusion machinery for exocytosis”, which does not require any additional special apparatus.     

ALG-2 and peflin regulate COPII targeting and secretion in response to calcium signaling

ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca²⁺ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca²⁺ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by ∼50%. We found that at steady-state Ca²⁺, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca²⁺ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration—phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca²⁺-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca²⁺ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca²⁺ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.     

Polyamines Biological modulators of membrane fusion

The effect of polyamines on the kinetics of Ca²⁺- and Mg²⁺-mediated membrane fusion was studied by following the intermixing of the contents of vesicles composed of phosphatidate/phosphatidylserine/ phosphatidylethanolamine/cholesterol (1:2:3:2). Addition of polyamines at specific concentration ranging from 40 to 400 μM promoted aggregation of the vesicles. In addition, low levels of spermine (50–100 μM) enhanced both Ca²⁺ - and Mg²⁺-mediated fusion. The initial fusion rate of this membrane system increased more than 200-fold when fusion was initiated by Ca²⁺ after 5 min pre-incubation of vesicles with 50 μM spermine. These results indicate that in addition to their other known effects on cellular metabolism, polyamines may be involved in modulating intracellular membrane fusion.     

Synaptotagmin IV Acts as a Multi-Functional Regulator of Ca<Superscript>2+</Superscript>-Dependent Exocytosis

In response to stimuli, secretary cells secrete a variety of signaling molecules packed in vesicles (e.g., neurotransmitters and peptide hormones) into the extracellular space by exocytosis. The vesicle secretion is often triggered by calcium ion (Ca²⁺) entered into secretary cells and achieved by the fusion of secretory vesicles with the plasma membrane. Recent accumulating evidence has indicated that members of the synaptotagmin (Syt) family play a major role in Ca²⁺-dependent exocytosis, and Syt I, in particular, is now widely accepted as the major Ca²⁺-sensor for synchronous neurotransmitter release. Involvement of other Syt isoforms in Ca²⁺-dependent exocytotic events other than neurotransmitter release has also been reported, and the Syt IV isoform is of particular interest, because Syt IV has several unique features not found in Syt I (e.g., immediate early gene product induced by deporalization and postsynaptic localization). In this article, we summarize the literature on the multi-functional role of Syt IV in Ca²⁺-dependent exocytosis.