Priming in exocytosis: attaining fusion-competence after vesicle docking

Membrane contact established by tethering or docking mechanisms is not a sufficient condition for membrane fusion. In neural and neuroendocrine cells, only a small fraction of secretory vesicles docked at the plasma membrane are fusion-competent and undergo rapid ATP-independent fusion in response to Ca(2+) elevations. Additional biochemical events termed 'priming' are essential to render vesicles competent for Ca(2+)-triggered fusion. The priming of vesicles is ATP-dependent and a number of ATP-dependent priming reactions have been characterized in permeable neuroendocrine cells. These involve NSF-mediated priming of SNARE protein complexes, the ATP-dependent synthesis of phosphoinositides, and protein kinase-mediated protein phosphorylation. In addition, munc13 is an important protein involved in priming synaptic vesicles. An emphasis in this review is on recent work indicating that priming events identified in the pathways of regulated exocytosis share many features with pre-fusion processes characterized in constitutive fusion pathways.     

Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming

Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles.     

CAPS-1 and CAPS-2 are essential synaptic vesicle priming proteins

Before transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca(2+) levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca(2+) triggered release of transmitters.     

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.     

分泌囊泡启动步骤和相关调节蛋白研究进展

囊泡启动是细胞调节性分泌中非常关键的一个步骤,囊泡启动后才具备与膜融合的能力.囊泡的启动过程需要SNARE复合体的形成和许多其它蛋白如Muncl3、RIM、CAPS等的参与,但不同类型的分泌囊泡其关键的启动因子并不相同.本文主要从分泌囊泡的启动过程和不同囊泡所特异的启动因子着手,综述了囊泡转运过程中启动步骤的最新研究进展.     

Modulating vesicle priming reveals that vesicle immobilization is necessary but not sufficient for fusion-competence

In neurons and neuroendocrine cells, docked vesicles need to undergo priming to become fusion competent. Priming is a multi-step process that was shown to be associated with vesicle immobilization. However, it is not known whether vesicle immobilization is sufficient to acquire complete fusion competence. To extend our understanding of the physical manifestation of vesicle priming, we took advantage of tomosyn, a SNARE-related protein that specifically inhibits vesicle priming, and measured its effect on vesicle dynamics in live chromaffin cells using total internal reflection fluorescence microscopy. We show here that while in control cells vesicles undergo immobilization before fusion, vesicle immobilization is attenuated in tomosyn overexpressing cells. This in turn increases the turnover rate of vesicles near the membrane and attenuates the fusion of newcomer vesicles. Moreover, the release probability of immobile vesicles in tomosyn cells is significantly reduced, suggesting that immobilization is an early and necessary step in priming but is insufficient, as further molecular processes are needed to acquire complete fusion competence. Using tomosyn as a molecular tool we provide a mechanistic link between functional docking and priming and suggest that functional docking is the first step in vesicle priming, followed by molecular modifications that do not translate into changes in vesicle mobility.     

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

The Ca²⁺-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.     

Vesicle pools, docking, priming, and release

The release of neurotransmitter from synaptic vesicles represents the final event by which presynapses send their chemical signal to the receiving postsynapses. Prior to fusion, synaptic vesicles undergo a series of maturation events, most notably the membrane-delimited docking and priming steps. Physiological and optical experiments with high-time resolution have allowed the distinction of vesicles in different maturation states with respect to fusion, the so-called vesicle pools. In this review, we define the various vesicle pools and discuss pathways leading into and out of these pools. We also provide an overview of an array of proteins that have been identified or are speculated to play a role in the transition between the various vesicle pools.Work in the lab of the authors is supported by grants from the Deutsche Forschungsgemeinschaft and the European Community and by local funding (HOMFOR).     

Identification of the minimal protein domain required for priming activity of Munc13-1

Most nerve cells communicate with each other through synaptic transmission at chemical synapses. The regulated exocytosis of neurotransmitters, hormones, and peptides occurs at specialized membrane areas through Ca2+-triggered fusion of secretory vesicles with the plasma membrane . Prior to fusion, vesicles are docked at the plasma membrane and must then be rendered fusion-competent through a process called priming. The molecular mechanism underlying this priming process is most likely the formation of the SNARE complex consisting of Syntaxin 1, SNAP-25, and Synaptobrevin 2. Members of the Munc13 protein family consisting of Munc13-1, -2, -3, and -4 were found to be absolutely required for this priming process . In the present study, we identified the minimal Munc13-1 domain that is responsible for its priming activity. Using Munc13-1 deletion constructs in an electrophysiological gain-of-function assay of chromaffin-granule secretion, we show that priming activity is mediated by the C-terminal residues 1100-1735 of Munc13-1, which contains both Munc13-homology domains and the C-terminal C2 domain. Priming by Munc13-1 appears to require its interaction with Syntaxin 1 because point mutants that do not bind Syntaxin 1 do not prime chromaffin granules.     

Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells

In chromaffin cells the number of large dense-core vesicles (LDCVs) which can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, we overexpressed Munc13-1 in chromaffin cells and stimulated secretion by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, were increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, was increased by the same factor. The response to a second flash, however, was greatly reduced, indicating a depletion of release-competent vesicles. Since there was no apparent change in the number of docked vesicles, we conclude that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles.     

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca²⁺-triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca²⁺-triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.     

Synaptic ribbons: machines for priming vesicle release?

Synaptic ribbons are specialized organelles that hold vesicles close to the active zone of sensory synapses, but their function is mysterious. Acute disruption of the ribbon complex using light has now revealed that it has a role in priming synaptic vesicles for fusion.     

A minimal domain responsible for Munc13 activity

Munc13 proteins are essential in neurotransmitter release, controlling the priming of synaptic vesicles to a release-ready state. The sequences responsible for this priming activity are unknown. Here we identify a large alpha-helical domain of mammalian Munc13-1 that is autonomously folded and is sufficient to rescue the total arrest in neurotransmitter release observed in hippocampal neurons lacking Munc13s.     

The structure and function of ‘active zone material’ at synapses

The docking of synaptic vesicles on the presynaptic membrane and their priming for fusion with it to mediate synaptic transmission of nerve impulses typically occur at structurally specialized regions on the membrane called active zones. Stable components of active zones include aggregates of macromolecules, ‘active zone material’ (AZM), attached to the presynaptic membrane, and aggregates of Ca²⁺-channels in the membrane, through which Ca²⁺ enters the cytosol to trigger impulse-evoked vesicle fusion with the presynaptic membrane by interacting with Ca²⁺-sensors on the vesicles. This laboratory has used electron tomography to study, at macromolecular spatial resolution, the structure and function of AZM at the simply arranged active zones of axon terminals at frog neuromuscular junctions. The results support the conclusion that AZM directs the docking and priming of synaptic vesicles and essential positioning of Ca²⁺-channels relative to the vesicles'' Ca²⁺-sensors. Here we review the findings and comment on their applicability to understanding mechanisms of docking, priming and Ca²⁺-triggering at other synapses, where the arrangement of active zone components differs.     

UNC-13 interaction with syntaxin is required for synaptic transmission

Neurotransmitter secretion at synapses is controlled by several processes-morphological docking of vesicles at release sites, priming of docked vesicles to make them fusion competent, and calcium-dependent fusion of vesicles with the plasma membrane . In worms, flies, and mice, mutants lacking UNC-13 have defects in vesicle priming . Current models propose that UNC-13 primes vesicles by stabilizing Syntaxin's "open" conformation by directly interacting with its amino-terminal regulatory domain . However, the functional significance of the UNC-13/Syntaxin interaction has not been tested directly. A truncated protein containing the Munc homology domains (MHD1 and MHD2) and the carboxy-terminal C2 domain partially rescued both the behavioral and secretion defects of unc-13 mutants in C. elegans. A double mutation in MHD2 (F1000A/K1002A) disrupts the UNC-13/Syntaxin interaction. The rate of endogenous synaptic events and the amplitude of nerve-evoked excitatory post-synaptic currents (EPSCs) were both significantly reduced in UNC-13S(F1000A/K1002A). However, the pool of primed (i.e., fusion-competent) vesicles was normal. These results suggest that the UNC-13/Syntaxin interaction is conserved in C. elegans and that, contrary to current models, the UNC-13/Syntaxin interaction is required for nerve-evoked vesicle fusion rather than synaptic-vesicle priming. Thus, UNC-13 may regulate multiple steps of the synaptic-vesicle cycle.     

SNARE protein recycling by αSNAP and βSNAP supports synaptic vesicle priming

Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.     

Actin is not an essential component in the mechanism of calcium-triggered vesicle fusion

Actin has been suggested as an essential component in the membrane fusion stage of exocytosis. In some model systems disruption of the actin filament network associated with exocytotic membranes results in a decrease in secretion. Here we analyze the fast Ca2+-triggered membrane fusion steps of regulated exocytosis using a stage-specific preparation of native secretory vesicles (SV) to directly test whether actin plays an essential role in this mechanism. Although present on secretory vesicles, selective pharmacological inhibition of actin did not affect the Ca2+-sensitivity, extent, or kinetics of membrane fusion, nor did the addition of exogenous actin or an anti-actin antibody. There was also no discernable affect on inter-vesicle contact (docking). Overall, the results do not support a direct role for actin in the fast, Ca2+-triggered steps of regulated membrane fusion. It would appear that actin acts elsewhere within the exocytotic cycle.     

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.     

The Role of Phospholipase D in Regulated Exocytosis

There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca²⁺-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca²⁺ sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.     

CAPS in search of a lost function

Ca2+-dependent activator protein for secretion (CAPS) is an evolutionarily conserved secretory protein that was previously thought to mediate Ca2+-triggered fusion of dense-core vesicles. In an elegant study of CAPS1-deficient mice, Speidel et al. (this issue of Neuron) now show that CAPS function may have been misunderstood. CAPS appears to act upstream of fusion in the biogenesis or maintenance of mature secretory vesicles, raising the possibility of a completely new type of function for an essential component of the secretory machinery.