Dense-core Vesicle Exocytosis Research Articles

The role of myosin V in exocytosis and synaptic plasticity

In neuroscience, myosin V motor proteins have attracted attention since they are highly expressed in brain, and absence of myosin Va in man leads to a severe neurological disease called Griscelli syndrome. While in some cells myosin V is described to act as a vesicle transport motor, an additional role in exocytosis has emerged recently. In neurons, myosin V has been linked to exocytosis of secretory vesicles and recycling endosomes. Through these functions, it is implied in regulating important brain functions including the release of neuropeptides by exocytosis of large dense-core vesicles and the insertion of neurotransmitter receptors into post-synaptic membranes. This review focuses on the role of myosin V in (i) axonal transport and stimulated exocytosis of large dense-core vesicles to regulate the secretion of neuroactive substances, (ii) tethering of the endoplasmic reticulum at cerebellar synapses to permit long-term depression, (iii) recycling of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors at hippocampal synapses during long-term potentiation, and (iv) recycling of nicotinic acetylcholine receptors at the neuromuscular junction. Myosin V is thus discussed as an important modulator of synaptic plasticity.

Phosphoinositides Function Asymmetrically for Membrane Fusion, Promoting Tethering and 3Q-SNARE Subcomplex Assembly

Phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are essential for rapid SNARE-dependent fusion of yeast vacuoles and other organelles. These phosphoinositides also regulate the fusion of reconstituted proteoliposomes. The reconstituted reaction allows separate analysis of phosphoinositide-responsive subreactions: fusion with SNAREs alone, with the addition of the HOPS tethering factor, and with the further addition of the SNARE complex disassembly chaperones Sec17p and Sec18p. Using assays of membrane tethering, trans-SNARE pairing, and lipid mixing, we found that PI(3)P and PI(4,5)P(2) have distinct functions that are asymmetric with respect to R-SNARE (Nyv1p) and the 3Q-SNAREs (Vam3p, Vti1p, and Vam7p). Fusion reactions with the Q-SNAREs and R-SNARE on separate membranes showed that PI(3)P has two distinct functions. PI(3)P on Q-SNARE proteoliposomes promoted Vam7p binding and association with the other two Q-SNAREs. PI(3)P on R-SNARE proteoliposomes was recognized by the PX domain of Vam7p on Q-SNARE proteoliposomes to promote tethering, although this function could be supplanted by the tethering activity of HOPS. PI(4,5)P(2) stimulated fusion when it was on R-SNARE proteoliposomes, apposed to Q-SNARE proteoliposomes bearing PI(3)P. These functions are essential for the phosphoinositide-dependent synergy between HOPS and Sec17p/Sec18p in promoting rapid fusion.

Versatile roles for myosin Va in dense core vesicle biogenesis and function

The motor protein myosin Va is involved in multiple successive steps in the development of dense-core vesicles, such as in the membrane remodelling during their maturation, their transport along actin filaments and the regulation of their exocytosis. In the present paper, we summarize the current knowledge on the roles of myosin Va in the different steps of dense-core vesicle biogenesis and exocytosis, and compare findings obtained from different cell types and experimental systems.

Effects of Rab3 and Rab27 on Exocytotic Activity and Docking in Mouse Chromaffin Cells

The small GTPases Rab3A and Rab27A are endogenously expressed and specifically localized to dense-core vesicles in PC12 cells and are implicated in the late steps of dense-core vesicle exocytosis. What remains unknown is the individual and collective impact of these proteins on the size of the releasable pools and the rate at which vesicles are primed into these pools. Chromaffin cells isolated from Rab3A-/-, Rab27-/-, and double knock-out mice were used to examine the density of docked vesicles, granule mobility at the membrane, and exocytotic activity. Docked granules were visualized using electron micrographs and quantified based on their placement with respect to the plasma membrane. Mobility of granules was examined using total internal reflection fluorescence (TIRF) microscopy with virus-infected chromaffin cells expressing NPY-cherry. Changes in membrane capacitance were recorded from chromaffin cells in adrenal slices using patch clamp electrophysiology, providing data reflecting fusion kinetics and the refilling of releasable pools. The present study has identified the contributions of Rab3 and Rab27 to release readiness of dense-core vesicles in primary mouse chromaffin cells.

Loose coupling between calcium channels and sites of exocytosis in chromaffin cells

Calcium microdomains generated by tight clusters of calcium channels regulate fusion of small vesicles at the synaptic terminal and have also been suggested to trigger exocytosis of large dense-core vesicles from neuroendocrine cells. To test this idea, we have compared sites of exocytosis and the spatial distribution of calcium channels in chromaffin cells. Fusion of individual vesicles was visualized using interference reflection microscopy and the submembranous calcium signal was assessed using total internal reflection fluorescence microscopy. Depolarization triggered a burst of exocytosis from up to seven sites in a membrane area of 11 microm(2), but these sites did not colocalize with calcium microdomains. Instead, calcium influx occurred in large patches (averaging 34 microm(2)) containing a mixture of P/Q- and N-type channels. About 20% of fusion events occurred outside calcium channel patches. Further, the delay between the onset of stimulation and a burst of exocytosis was prolonged for several seconds by increasing the concentration of the slow calcium chelator EGTA from 1.5 to 5 mM. These results demonstrate that while calcium channels and release sites tend to congregate in specialized regions of the surface membrane, these have dimensions of several micrometres. The dominant calcium signal regulating release in chromaffin cells is generated by the cooperative action of many channels operating over distances of many micrometres rather than discrete clusters of calcium channels generating localized microdomains.

Age-dependent preferential dense-core vesicle exocytosis in neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein.

Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K(+)] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

A database search of the Paramecium genome reveals 34 genes related to Ca(2+)-release channels of the inositol-1,4,5-trisphosphate (IP(3)) or ryanodine receptor type (IP(3)R, RyR). Phylogenetic analyses show that these Ca(2+) release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP(3)Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP(3)Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca(2+) stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP(3) channel, a group II member (P. tetraurelia IP(3)R(N)-1), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons. Knockdown of P. tetraurelia IP(3)R(N) affects trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca(2+) from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca(2+) as signaling molecule.

Dominant role of lipid rafts L‐type calcium channel in activity‐dependent potentiation of large dense‐core vesicle exocytosis

Calcium influx triggers exocytosis by promoting vesicle fusion with the plasma membrane. However, different subtypes of voltage-gated calcium channel (VGCC) have distinct roles in exocytosis. We previously reported that repetitive stimulation induces activity-dependent potentiation (ADP) which represents the increase of neurotransmitter release. Here, we show that L-type VGCC have a dominant role in ADP of large dense-core vesicle (LDCV) exocytosis. Repetitive stimulation activating VGCC can induce ADP, whereas activation of bradykinin (BK) G protein-coupled receptors or purinergic P2X cation channels can not. L-type VGCC has the dominant role in ADP of LDCV exocytosis by regulating Protein Kinase C (PKC)-epsilon translocation and phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a target molecule of PKC-epsilon. We provide evidence that L-type VGCC, PKC-epsilon, and MARCKS, but not Q-type VGCC, are selectively located in lipid rafts. Also, PKC-epsilon translocation induced by L-type VGCC activation occurs in lipid rafts. Disruption of lipid rafts abolishes ADP of LDCV exocytosis and changes the fusion pore kinetics without affecting the first stimulation-induced exocytosis, showing that lipid rafts are involved in the potentiation process. Taken together, we suggest that L-type VGCC in lipid rafts selectively mediates ADP of LDCV exocytosis by regulating PKC-epsilon translocation and MARCKS phosphorylation.

Synaptotagmin‐7 is a principal Ca2+ sensor for Ca2+‐induced glucagon exocytosis in pancreas

Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

Imaging of evoked dense-core-vesicle exocytosis in hippocampal neurons reveals long latencies and kiss-and-run fusion events

Evoked neuropeptide secretion in the central nervous system occurs slowly, but the basis for slow release is not fully understood. Whereas exocytosis of single synaptic vesicles in neurons and of dense-core vesicles (DCVs) in endocrine cells have been directly visualized, single DCV exocytic events in neurons of the central nervous system have not been previously studied. We imaged DCV exocytosis in primary cultured hippocampal neurons using fluorescent propeptide cargo and total internal reflectance fluorescence microscopy. The majority of Ca(2+)-triggered exocytic events occurred from immobile plasma-membrane-proximal DCVs in the cell soma, whereas there were few events in the neurites. Strikingly, DCVs in the cell soma exhibited 50-fold greater release probabilities than those in neurites. Latencies to depolarization-evoked fusion for DCVs were surprisingly long, occurring with an average time constant (tau) of 16 seconds for DCVs in the soma and even longer for DCVs in neurites. All of the single DCV release events exhibited rapid fusion-pore openings and closures, the kinetics of which were highly dependent upon Ca(2+) levels. These ;kiss-and-run' events were associated with limited cargo secretion. Thus, the slow evoked release of neuropeptides could be attributed to very prolonged latencies from stimulation to fusion and transient fusion-pore openings that might limit cargo secretion.

Molecular mechanism of attachment process of dense-core vesicles to the plasma membrane in neuroendocrine cells

The molecular mechanisms by which large dense-core vesicles tethering, docking, priming, and exocytosis of their various cargoes from neuroendocrine cells have been the subject of intense debate during the past few years. Recent studies have suggested that the monomeric GTPase Rab27 subfamily and its cell-type- or tissue-type-specific Rab27-binding protein(s) (also called “Rab27 effector(s)”) are present on the dense-core vesicle membrane and might regulate the tethering, docking, priming, and exocytosis of hormones. Rab27 effector proteins, synaptotagmin- like proteins (Slps) and rabphilin, consist of a Rab27-binding domain and Ca 2+- and phospholipids-binding tandem C2 domains on their N- and C-terminal, respectively. Biochemical and live cell imaging analysis have revealed that Rab27 and its effectors make complexes before binding to the plasma membrane targeting partners, such as Munc18-1/syntaxin1-a complex or SNAP-25. These complexes positively or negatively regulate the tethering and/or docking (i.e. attachment) process of exocytosis. Here I discuss the data showing the molecular mechanisms of how Rab27 and its effectors regulate the dense-core vesicle tethering and/or docking to the plasma membrane in neuroendocrine cells.

Role of the Vesicular Chloride Transporter ClC-3 in Neuroendocrine Tissue

ClC-3 is an intracellular chloride transport protein known to reside on endosomes and synaptic vesicles. The endogenous protein has been notoriously difficult to detect in immunohistological experiments because of the lack of reliable antibodies. Using newly generated antibodies, we now examine its expression pattern at the cellular and subcellular level. In all tissues examined, immunostaining indicated that ClC-3 is a vesicular protein, with a prominent expression in endocrine cells like adrenal chromaffin cells and pancreatic islet cells. In line with a possible function of ClC-3 in regulating vesicle trafficking or exocytosis in those secretory cells, capacitance measurements and amperometry indicated that exocytosis of large dense-core vesicles (LDCVs) was decreased in chromaffin cells from ClC-3 knock-out mice. However, immunohistochemistry complemented with subcellular fractionation showed that ClC-3 is not detectable on LDCVs of endocrine cells, but localizes to endosomes and synaptic-like microvesicles in both adrenal chromaffin and pancreatic beta cells. This observation points to an indirect influence of ClC-3 on LDCV exocytosis in chromaffin cells, possibly by affecting an intracellular trafficking step.

Molecular identification of a SNAP-25-like SNARE protein in Paramecium.

Using database searches of the completed Paramecium tetraurelia macronuclear genome with the metazoan SNAP-25 homologues, we identified a single 21-kDa Qb/c-SNARE in this ciliated protozoan, named P. tetraurelia SNAP (PtSNAP), containing the characteristic dual heptad repeat SNARE motifs of SNAP-25. The presence of only a single Qb/c class SNARE in P. tetraurelia is surprising in view of the multiple genome duplications and the high number of SNAREs found in other classes of this organism. As inferred from the subcellular localization of a green fluorescent protein (GFP) fusion construct, the protein is localized on a variety of intracellular membranes, and there is a large soluble pool of PtSNAP. Similarly, the PtSNAP that is detected with a specific antibody in fixed cells is associated with a number of intracellular membrane structures, including food vacuoles, the contractile vacuole system, and the sites of constitutive endo- and exocytosis. Surprisingly, using gene silencing, we could not assign a role to PtSNAP in the stimulated exocytosis of dense core vesicles (trichocysts), but we found an increased number of food vacuoles in PtSNAP-silenced cells. In conclusion, we identify PtSNAP as a Paramecium homologue of metazoan SNAP-25 that shows several divergent features, like resistance to cleavage by botulinum neurotoxins.

VAMP2 interacts directly with the N terminus of Kv2.1 to enhance channel inactivation

Recently, we demonstrated that the Kv2.1 channel plays a role in regulated exocytosis of dense-core vesicles (DCVs) through direct interaction of its C terminus with syntaxin 1A, a plasma membrane soluble NSF attachment receptor (SNARE) component. We report here that Kv2.1 interacts with VAMP2, the vesicular SNARE partner that is also present at high concentration in neuronal plasma membrane. This is the first report of VAMP2 interaction with an ion channel. The interaction was demonstrated in brain membranes and characterized using electrophysiological and biochemical analyses in Xenopus oocytes combined with an in vitro binding analysis and protein modeling. Comparative study performed with wild-type and mutant Kv2.1, wild-type Kv1.5, and chimeric Kv1.5N/Kv2.1 channels revealed that VAMP2 enhanced the inactivation of Kv2.1, but not of Kv1.5, via direct interaction with the T1 domain of the N terminus of Kv2.1. Given the proposed role for surface VAMP2 in the regulation of the vesicle cycle and the important role for the sustained Kv2.1 current in the regulation of dendritic calcium entry during high-frequency stimulation, the interaction of VAMP2 with Kv2.1 N terminus may contribute, alongside with the interaction of syntaxin with Kv2.1 C terminus, to the activity dependence of DCV release.

Synaptotagmin IV regulates dense core vesicle (DCV) release in LβT2 cells

Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca 2+ sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear. The function of Syt IV is of particular interest because it is rapidly up-regulated by chronic depolarization and seizures. Using RNAi-mediated gene silencing, we have explored the role of Syt I and IV on secretion in a pituitary gonadotrope cell line. Downregulation of Syt IV clearly reduced Ca 2+-triggered exocytosis of dense core vesicles (DCVs) in LβT2 cells. Syt I silencing, however, had no effect on vesicular release.

PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells

Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca(2+)-dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides.

CAPS1 and CAPS2 Regulate Stability and Recruitment of Insulin Granules in Mouse Pancreatic β Cells

CAPS1 and CAPS2 regulate dense-core vesicle release of transmitters and hormones in neuroendocrine cells, but their precise roles in the secretory process remain enigmatic. Here we show that CAPS2(-/-) and CAPS1(+/-);CAPS2(-/-) mice, despite having increased insulin sensitivity, are glucose intolerant and that this effect is attributable to a marked reduction of glucose-induced insulin secretion. This correlates with diminished Ca(2+)-dependent exocytosis, a reduction in the size of the morphologically docked pool, a decrease in the readily releasable pool of secretory vesicles, slowed granule priming, and suppression of second-phase (but not first-phase) insulin secretion. In beta cells of CAPS1(+/-);CAPS2(-/-) mice, the lowered insulin content and granule numbers were associated with an increase in lysosome numbers and lysosomal enzyme activity. We conclude that although CAPS proteins are not required for Ca(2+)-dependent exocytosis to proceed, they exert a modulatory effect on insulin granule priming, exocytosis, and stability.

Rabphilin directly interacts with SNAP-25 and regulates the docking step of dense-core vesicle exocytosis in PC12 cells

Rabphilin directly interacts with SNAP-25 and regulates the docking step of dense-core vesicle exocytosis in PC12 cells

Increased plasma membrane localization of O‐glycosylation‐deficient mutant of synaptotagmin I in PC12 cells

Synaptotagmin I (Syt I) is a Ca2+-binding protein on synaptic vesicles and presumably functions as a Ca2+ sensor for neurotransmitter release. Native Syt I protein in neuroendocrine PC12 cells undergoes several posttranslational modifications, such as O-glycosylation, N-glycosylation, and fatty acylation, and the latter two modifications have been shown to be required for the proper function of murine Syt I in PC12 cells. However, nothing is known about the physiological significance of the O-glycosylation of Syt I in dense-core vesicle exocytosis in PC12 cells. In this study, we created an O-glycosylation-deficient mutant (named TA = T15A/T16A) and an N-glycosylation-deficient mutant of Syt I (named T26A) and investigated their subcellular distribution in Syt I-deficient PC12 cells, where other Syt isoforms (e.g., IV and IX) and other membrane trafficking proteins (e.g., Rab27A, SNAP-25, syntaxin-1, and VAMP-2) are normally expressed. We found that some cells expressing high level of recombinant wild-type (WT) Syt I protein show mistargeting of Syt I(WT) protein to the plasma membrane, whereas most of the cells show normal dense-core vesicle localization of Syt I(WT) protein. Similar mistargeting was also observed in cells expressing high levels of the Syt I(T26A) and Syt I(TA) mutants, but the mistargeting of the Syt I(TA) mutant to the plasma membrane was much more evident than with the Syt I(WT) or (T26A) mutant. The results indicate that O-glycosylation, not N-glycosylation, is partially involved in efficient targeting of Syt I protein to dense-core vesicles in PC12 cells.

Mechanisms of exocytosis

Catecholamines and peptides secreted from dense-core vesicles (DCVs) of adrenal chromaffin cells regulate a wide variety of physiological processes. For instance, the release of noradrenaline and adrenaline plays a key role in regulating heart rate and blood pressure. Thus understanding the mechanisms of secretory processes of DCVs is crucial for understanding the basis of diseases such as hypertension. DCVs undergo several stages of secretory processing before they are exocytosed. These include docking, priming and triggering of membrane fusion/exocytosis. Molecular studies of DCV exocytosis have identified many proteins critically involved in DCV secretion. These proteins include SNARE proteins, Munc18-1, phosphatidylinositol transfer protein, type I phosphatidylinositol-4-phosphate-5-kinases, NSF, Munc13, CAPS1, synaptotagmins, RalA/RalB GTPases and exocyst proteins. In this article, I will discuss the functions of these proteins within the context of the stages (i.e. docking, priming and triggering of membrane fusion/exocytosis) in DCV secretion.