Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles

Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca²⁺-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells. We found that down-regulation of both Syt I and Syt IX resulted in a significant loss of Ca²⁺-dependent LDCV exocytosis. Moreover, our results suggest Syt I and Syt IX play redundant role in controlling the choice of fusion modes. Down-regulation of both Syt I and Syt IX renders more fusion in the kiss-and-run mode. We conclude that Syt I and Syt IX function redundantly in Ca²⁺-sensing and fusion pore dilation on LDCVs in PC12 cells.     

Widespread Dysregulation of Peptide Hormone Release in Mice Lacking Adaptor Protein AP-3

The regulated secretion of peptide hormones, neural peptides and many growth factors depends on their sorting into large dense core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used an RNAi screen to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and in particular, LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal rather than biosynthetic pathway. We therefore used mocha mice to determine whether loss of AP-3 also dysregulates peptide release in vivo. We find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble cargo and LDCV membrane proteins, reducing the response to stimulation. We also observe increased basal release of both insulin and glucagon from pancreatic islet cells of mocha mice, suggesting a global disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but the analysis of mice lacking each of these isoforms individually and together shows that loss of both is required to reproduce the effect of the mocha mutation on the regulated pathway. In addition, we show that loss of the related adaptor protein AP-1 has a similar effect on regulated secretion but exacerbates the effect of AP-3 RNAi, suggesting distinct roles for the two adaptors in the regulated secretory pathway.     

以消散场成像和单微粒跟踪技术揭示GLUT4囊泡和分泌囊泡的不同动态过程

葡萄糖转运子蛋白4(glucose transporter 4,GLUT4)在维持体内葡萄糖动态平衡的过程中起着至关重要的作用。GLUT4贮存囊泡(GLUT4 storage vesicle,GSV)和神经内分泌细胞中的分泌囊泡含有许多相同的蛋白。研究证明这些蛋白调节了分泌囊泡的胞内转运过程,但是GLUT4囊泡和分泌囊泡是否具有相同的胞内动态过程还未阐明。文章以3T3-L1纤维原细胞中的GSV和神经内分泌细胞PC12细胞中的分泌囊泡:致密核心大囊泡(large dense core vesicle,LDCV)为研究对象,使用消散场显微成像技术和单微粒跟踪技术直观观察了活体细胞内单个GSV和LDCV的三维运动轨迹。通过以适当方程拟合单个囊泡的均方位移曲线,发现两种囊泡都具有三种运动模式。定量分析显示作自由扩散运动和方向性扩散运动的GSV数量明显多于LDCV。对比GSV和LDCV的三维扩散系数,发现GSV的扩散系数中值为7.2×10-4μm2/s,而LDCV的扩散系数中值仅为1.94×10-4μm2/s。这一结果说明GSV的活动性远大于LDCV,提示GSV的胞内转运过程涉及不同的分子机制。     

Sorting of vesicular monoamine transporter 2 to the regulated secretory pathway confers the somatodendritic exocytosis of monoamines

The release of monoamine neurotransmitters from cell bodies and dendrites has an important role in behavior, but the mechanism (vesicular or non vesicular) has remained unclear. Because the location of vesicular monoamine transporter 2 (VMAT2) defines the secretory vesicles capable of monoamine release, we have studied its trafficking to assess the potential for monoamine release by exocytosis. In neuroendocrine PC12 cells, VMAT2 localizes exclusively to large dense-core vesicles (LDCVs), and we now show that cytoplasmic signals target VMAT2 directly to LDCVs within the biosynthetic pathway. In neurons, VMAT2 localizes to a population of vesicles that we now find undergo regulated exocytosis in dendrites. Although hippocampal neurons do not express typical LDCV proteins, transfected chromogranins A, B, and brain-derived neurotrophic factor (BDNF) colocalize with VMAT2. VMAT2 thus defines a population of secretory vesicles that mediate the activity-dependent somatodendritic release of multiple retrograde signals involved in synaptic function, growth, and plasticity.     

The calcium-sensing protein synaptotagmin 7 is expressed on different endosomal compartments in endocrine,neuroendocrine cells or neurons but not on large dense core vesicles

Synaptotagmin (syt) isoforms function as calcium sensor in post-Golgi transport although the precise transport step and compartment(s) concerned are still not fully resolved. As syt7 has been proposed to operate in lysosomal exocytosis and in exocytosis of large dense core vesicles (LDCVs), we have addressed the distribution of endogenous syt7 in insulin-secreting cells. These cells express different syt7 isoforms comparable to neurons. According to subcellular fractionation and quantitative confocal immunocytochemistry, syt7 is not found on LDCVs or on synaptic-like microvesicles but colocalizes with Rab7 on endosomes and to structures near to or at the plasma membrane. Similarly, endogenous syt7 was absent from LDCVs in pheochromocytoma PC12 cells. In contrast, syt7 localised to lysosomes in both, PC12 cells and hippocampal neurons. In conclusion, endogenous syt7 shows a wider distribution than previously reported but does not qualify as vesicular calcium sensor in SLMV or LDCV exocytosis according to its localisation.     

Munc18-1 promotes large dense-core vesicle docking.

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.     

Regulation of atrial natriuretic peptide secretion by a novel Ras-like protein

Atrial cardiomyocytes, neurons, and endocrine tissues secrete neurotransmitters and peptide hormones via large dense-core vesicles (LDCVs). We describe a new member of the Ras family of G-proteins, named RRP17, which is expressed specifically in cardiomyocytes, neurons, and the pancreas. RRP17 interacts with Ca(2+)-activated protein for secretion-1 (CAPS1), one of only a few proteins known to be associated exclusively with LDCV exocytosis. Ectopic expression of RRP17 in cardiomyocytes enhances secretion of atrial natriuretic peptide (ANP), a regulator of blood pressure and natriuresis. Conversely, genetic deletion of RRP17 in mice results in dysmorphic LDCVs, impaired ANP secretion, and hypertension. These findings identify RRP17 as a component of the cellular machinery involved in regulated secretion within the heart and potential mediator of the endocrine influence of the heart on other tissues.     

The vesicular monoamine transporter 2 contains trafficking signals in both its N-glycosylation and C-terminal domains

The vesicular acetylcholine transporter (VAChT) and the vesicular monoamine transporter (VMAT) belong to the same transporter family that packages acetylcholine into synaptic vesicles (SVs) and biogenic amines into large dense core vesicles (LDCVs) and/or SVs, respectively. These transporters share similarities in sequence and structure with their N- and C-terminal domains located in the cytoplasm. When expressed in PC12 cells, VMAT2 localizes to LDCV, whereas VAChT is found mainly on synaptic-like microvesicles. Previous studies have shown that the cytoplasmic C-terminal domain of VAChT contains signals targeting this transporter to SVs. However, the targeting signals for VMAT have not been completely elucidated. To identify signals targeting VMAT2 to LDCV, the subcellular localization of VMAT2-VAChT chimeras was analyzed in PC12 cells. Chimeras having either the N-terminal region through transmembrane domain 2 of VMAT2 or the C-terminal domain of VMAT2 do not traffic to LDCV efficiently. In contrast, chimeras having both of these regions, or the luminal glycosylated loop in conjunction with transmembrane domains 1 and 2 and the C-terminal domain of VMAT2, traffic to LDCV. Treatment of PC12 cells with 1-deoxymannojirimycin, a specific alpha-mannosidase I inhibitor, causes VMAT2 to localize to synaptic-like microvesicles. The results indicate that both mature N-linked glycosylation and the C-terminus are important for proper trafficking of VMAT2 and that the locations of trafficking signals in VMAT2 and VAChT are surprisingly different.     

Two distinct secretory vesicle�Cpriming steps in adrenal chromaffin cells

Priming of large dense-core vesicles (LDCVs) is a Ca²⁺-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca²⁺ concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.     

Distinct Subcellular Distribution of δ-Opioid Receptor Fused with Various Tags in PC12 Cells

In small dorsal root ganglion neurons, δ-opioid receptors (DORs) have been found to be mainly distributed in the cytoplasm and often associated with the membrane of large dense-core vesicles (LDCVs) that contain neuropeptides. To study the distribution of DORs under various physiological or pharmacological conditions, the receptors fused with different tags are constructed, transfected into cells or animals, and examined with microscopy. In this study, we show that DOR with different tags have distinct patterns of subcellular distribution in neuroendocrine cells, PC12 cells. Both immunostaining and vesicle fraction analysis showed that the native DORs expressed in PC12 cells were mainly associated with LDCVs. In transfected PC12 cells, DOR tagged with Myc or hemagglutinin exhibited LDCV localization. However, DOR fused with GFP at N- or C-terminus was found to be mainly localized on the cell surface, and mediated the function of DOR agonist. Therefore, the distribution of DOR fused with GFP differs from the native DORs. These results suggest that the subcellular distribution of the receptor could be better presented by the fused tag with smaller molecular size. Special issue article in honor of Dr. Ji-Sheng Han.     

Short elements with charged amino acids form clusters to sort protachykinin into large dense-core vesicles

The sorting of neuropeptide tachykinins into large dense-core vesicles (LDCVs) is a key step in their regulated secretion from neurons. However, the sorting mechanism for protachykinin has not yet to be clearly resolved. In this study, we report that the clustered short elements with charged amino acids regulate the efficiency of protachykinin sorting into LDCVs. A truncation experiment showed that the propeptide and the mature peptide-containing sequence of protachykinin were sorted into LDCVs. These two regions exhibit a polarized distribution of charged amino acids. The LDCV localization of the propeptide was gradually decreased with an increasing number of neutral amino acids. Furthermore, the short element with four to five amino acids containing two charged residues was found to be a basic unit for LDCV sorting that enables regulated secretion. In the native propeptide sequence, these charged short elements were clustered to enhance the intermolecular aggregation by electrostatic interaction and produce a gradual and additive effect on LDCV sorting. The optimal conditions for intermolecular aggregation of protachykinin were at millimolar Ca(2+) concentrations and pH 5.5-6.0. These results demonstrate that the charged short elements are clustered such that they serve as aggregative signals and regulate the efficiency of protachykinin sorting into LDCVs. These findings reveal a novel mechanism for the sorting of neuropeptides into a regulated secretory pathway.     

Transport of receptors, receptor signaling complexes and ion channels via neuropeptide-secretory vesicles

Stimulus-induced exocytosis of large dense-core vesicles (LDCVs) leads to discharge of neuropeptides and fusion of LDCV membranes with the plasma membrane. However, the contribution of LDCVs to the properties of the neuronal membrane remains largely unclear. The present study found that LDCVs were associated with multiple receptors, channels and signaling molecules, suggesting that neuronal sensitivity is modulated by an LDCV-mediated mechanism. Liquid chromatography-mass spectrometry combined with immunoblotting of subcellular fractions identified 298 proteins in LDCV membranes purified from the dorsal spinal cord, including G-protein-coupled receptors, G-proteins and other signaling molecules, ion channels and trafficking-related proteins. Morphological assays showed that δ-opioid receptor 1 (DOR1), β2 adrenergic receptor (AR), G(αi2), voltage-gated calcium channel α2δ1 subunit and P2X purinoceptor 2 were localized in substance P (SP)-positive LDCVs in small-diameter dorsal root ganglion neurons, whereas β1 AR, Wnt receptor frizzled 8 and dishevelled 1 were present in SP-negative LDCVs. Furthermore, DOR1/G(αi2)/G(β1γ5)/phospholipase C β2 complexes were associated with LDCVs. Blockade of the DOR1/G(αi2) interaction largely abolished the LDCV localization of G(αi2) and impaired stimulation-induced surface expression of G(αi2). Thus, LDCVs serve as carriers of receptors, ion channels and preassembled receptor signaling complexes, enabling a rapid, activity-dependent modulation of neuronal sensitivity.     

CAPS1 regulates catecholamine loading of large dense-core vesicles

CAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs.     

EGFP-Tagged Vasopressin Precursor Protein Sorting Into Large Dense Core Vesicles and Secretion From PC12 Cells

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary.2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques.3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.     

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH- terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.     

Corelease and differential exit via the fusion pore of GABA, serotonin, and ATP from LDCV in rat pancreatic beta cells

The release of gamma-aminobutyric acid (GABA) and ATP from rat beta cells was monitored using an electrophysiological assay based on overexpression GABA(A) or P2X2 receptor ion channels. Exocytosis of LDCVs, detected by carbon fiber amperometry of serotonin, correlated strongly (approximately 80%) with ATP release. The increase in membrane capacitance per ATP release event was 3.4 fF, close to the expected capacitance of an individual LDCV with a diameter of 0.3 microm. ATP and GABA were coreleased with serotonin with the same probability. Immunogold electron microscopy revealed that approximately 15% of the LDCVs contain GABA. Prespike "pedestals," reflecting exit of granule constituents via the fusion pore, were less frequently observed for ATP than for serotonin or GABA and the relative amplitude (amplitude of foot compared to spike) was smaller: in some cases the ATP-dependent pedestal was missing entirely. An inward tonic current, not dependent on glucose and inhibited by the GABA(A) receptor antagonist SR95531, was observed in beta cells in clusters of islet cells. Noise analysis indicated that it was due to the activity of individual channels with a conductance of 30 pS, the same as expected for individual GABA(A) Cl- channels with the ionic gradients used. We conclude that (a) LDCVs accumulate ATP and serotonin; (b) regulated release of GABA can be accounted for by exocytosis of a subset of insulin-containing LDCVs; (c) the fusion pore of LDCVs exhibits selectivity and compounds are differentially released depending on their chemical properties (including size); and (d) a glucose-independent nonvesicular form of GABA release exists in beta cells.     

Morphological docking of secretory vesicles

Calcium-dependent secretion of neurotransmitters and hormones is essential for brain function and neuroendocrine-signaling. Prior to exocytosis, neurotransmitter-containing vesicles dock to the target membrane. In electron micrographs of neurons and neuroendocrine cells, like chromaffin cells many synaptic vesicles (SVs) and large dense-core vesicles (LDCVs) are docked. For many years the molecular identity of the morphologically docked state was unknown. Recently, we resolved the minimal docking machinery in adrenal medullary chromaffin cells using embryonic mouse model systems together with electron-microscopic analyses and also found that docking is controlled by the sub-membrane filamentous (F-)actin. Currently it is unclear if the same docking machinery operates in synapses. Here, I will review our docking assay that led to the identification of the LDCV docking machinery in chromaffin cells and also discuss whether identical docking proteins are required for SV docking in synapses.     

Molecular Diversity in Neurosecretion: Reflections on the Hypothalamo-Neurohypophysial System

1. The diversity of molecules involved in various aspects of neurosecretion, such asproprotein processing, axonal transport of large dense core vesicles (LDCVs), and regulated secretion, is discussed in the context of the hypothalamo-neurohypophysial system (HNS).2. Recent studies have uncovered a family of at least seven processing enzymes known as proprotein convertases (PCs) which are involved in proteolytically cleaving protein precursors at paired basic amino acid motifs to yield biologically active peptides. Three of these, PC1(3), 2, and 5, are found in neurons and are involved in producing regulatedsecretory peptide products.3. The axonal transport of LDCVs occurs on microtubule tracks by still unknown mechanisms. There are over 11 distinct kinesin-related molecules that have now beenidentified as possible microtubule motor candidates.4. Calcium channels in the nervous system are known to be derived from at leastfive -subunit and four -subunit genes with multiple alternatively spliced isoforms in each case. These could account, in part, for the varied calcium currents found in the HNS.5. The large number of proteins and isoforms now demonstrated to be involved inregulated secretion are discussed, with a focus on LDCV compositions and the synaptotag-min gene family.     

Kinesin I and cytoplasmic dynein orchestrate glucose-stimulated insulin-containing vesicle movements in clonal MIN6 beta-cells

Glucose-stimulated mobilization of large dense-core vesicles (LDCVs) to the plasma membrane is essential for sustained insulin secretion. At present, the cytoskeletal structures and molecular motors involved in vesicle trafficking in beta-cells are poorly defined. Here, we describe simultaneous imaging of enhanced green fluorescent protein (EGFP)-tagged LDCVs and microtubules in beta-cells. Microtubules exist as a tangled array, along which vesicles describe complex directional movements. Whilst LDCVs frequently changed direction, implying the involvement of both plus- and minus-end directed motors, inactivation of the minus-end motor, cytoplasmic dynein, inhibited only a small fraction of all vesicle movements which were involved in vesicle recovery after glucose-stimulated exocytosis. By contrast, selective silencing of the plus-end motor, kinesin I, with small interfering RNAs substantially inhibited all vesicle movements. We conclude that the majority of LDCV transport in beta-cells is mediated by kinesin I, whilst dynein probably contributes to the recovery of vesicles after rapid kiss-and-run exocytosis.     

Differential Localization of Vesicular Acetylcholine and Monoamine Transporters in PC12 Cells but Not CHO Cells

Previous studies have indicated that neuro-endocrine cells store monoamines and acetylcholine (ACh) in different secretory vesicles, suggesting that the transport proteins responsible for packaging these neurotransmitters sort to distinct vesicular compartments. Molecular cloning has recently demonstrated that the vesicular transporters for monoamines and ACh show strong sequence similarity, and studies of the vesicular monoamine transporters (VMATs) indicate preferential localization to large dense core vesicles (LDCVs) rather than synaptic-like microvesicles (SLMVs) in rat pheochromocytoma PC12 cells. We now report the localization of the closely related vesicular ACh transporter (VAChT). In PC12 cells, VAChT differs from the VMATs by immunofluorescence and fractionates almost exclusively to SLMVs and endosomes by equilibrium sedimentation. Immunoisolation further demonstrates colocalization with synaptophysin on SLMVs as well as other compartments. However, small amounts of VAChT also occur on LDCVs. Thus, VAChT differs in localization from the VMATs, which sort predominantly to LDCVs. In addition, we demonstrate ACh transport activity in stable PC12 transformants overexpressing VAChT. Since previous work has suggested that VAChT expression confers little if any transport activity in non-neural cells, we also determined its localization in transfected CHO fibroblasts. In CHO cells, VAChT localizes to the same endosomal compartment as the VMATs by immunofluorescence, density gradient fractionation, and immunoisolation with an antibody to the transferrin receptor. We have also detected ACh transport activity in the transfected CHO cells, indicating that localization to SLMVs is not required for function. In summary, VAChT differs in localization from the VMATs in PC12 cells but not CHO cells.