The molecular mechanisms underlying "kiss and run" or "cavicapture" exocytosis of dense core secretory vesicles are presently unclear. Although dynamin-1 has previously been implicated in the recapture process in neurons, the recruitment of this fission protein to a single exocytosing vesicle has not been examined in real time during peptide release from pancreatic ␤-cells. Imaged simultaneously in clonal insulin-secreting cells by dual color total internal reflection fluorescence microscopy, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y and green fluorescent protein (GFP)-tagged synaptotagmin-1 or synaptobrevin-2 rapidly diffused from sites of exocytosis, whereas the vesicle membrane protein phogrin and tissue plasminogen activator (tPA) were retained, consistent with fusion pore closure. Vesicle recovery frequently involved the recruitment of enhanced GFP-tagged dynamin-1, and GTPase-defective dynamin-1(K44E) increased the dwell time of tPA-mRFP at the plasma membrane. By contrast, recruitment of GFP chimeras of clathrin, epsin, and amphiphysin was not observed. Expression of dynamin-1(K535A), mutated in the pleckstrin homology domain, caused the apparent full fusion of vesicles, as reported by the additional release of tPA-mRFP (15-nm diameter) and enhanced GFP-tagged phogrin. We conclude that re-uptake of vesicles after peptide release by cavicapture corresponds to a novel form of endocytosis in which dynamin-1 stabilizes and eventually closes the fusion pore, with no requirement for "classical" endocytosis for retreat from the plasma membrane.

AMP-activated protein kinase (AMPK) has recently been implicated in the control of preproinsulin gene expression in pancreatic islet β-cells [da Silva Xavier, Leclerc, Salt, Doiron, Hardie, Kahn and Rutter (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4023–4028]. Using pharmacological and molecular strategies to regulate AMPK activity in rat islets and clonal MIN6 β-cells, we show here that the effects of AMPK are exerted largely upstream of insulin release. Thus forced increases in AMPK activity achieved pharmacologically with 5-amino-4-imidazolecarboxamide riboside (AICAR), or by adenoviral overexpression of a truncated, constitutively active form of the enzyme (AMPKα1.T172D), blocked glucose-stimulated insulin secretion. In MIN6 cells, activation of AMPK suppressed glucose metabolism, as assessed by changes in total, cytosolic or mitochondrial [ATP] and NAD(P)H, and reduced increases in intracellular [Ca2+] caused by either glucose or tolbutamide. By contrast, inactivation of AMPK by expression of a dominant-negative form of the enzyme mutated in the catalytic site (AMPKα1.D157A) did not affect glucose-stimulated increases in [ATP], NAD(P)H or intracellular [Ca2+], but led to the unregulated release of insulin. These results indicate that inhibition of AMPK by glucose is essential for the activation of insulin secretion by the sugar, and may contribute to the transcriptional stimulation of the preproinsulin gene. Modulation of AMPK activity in the β-cell may thus represent a novel therapeutic strategy for the treatment of type 2 diabetes mellitus.

Exocytotic release of neuropeptides and hormones is generally believed to involve the complete merger of the secretory vesicle with the plasma membrane. However, recent data have suggested that "kiss-and-run" mechanisms may also play a role. Here, we have examined the dynamics of exocytosis in pancreatic MIN6 beta cells by imaging lumen- (neuropeptide Y/pH-insensitive yellow fluorescent protein; NPY.Venus) or vesicle membrane-targeted fluorescent probes (synaptobrevin-2/enhanced green fluorescent protein; synapto.pHluorin, or phosphatase on the granule of insulinoma-enhanced green fluorescent protein, phogrin.EGFP) by evanescent wave microscopy. Unexpectedly, NPY.Venus release events occurred much less frequently (13%-40% maximal rate) than those of synapto.pHluorin, even though the latter molecule, but not phogrin.EGFP, usually diffused away from the site of fusion. Thus, the majority of exocytosis occurs in these cells by kiss-and-run events that involve either the release of small molecules only, small molecules and selected membrane proteins, or all soluble cargoes ("pure," "mixed," and "full" kiss-and-run, respectively). Changes in the activity of synaptotagmin IV, achieved here by overexpression of the wild-type protein, may allow different stimuli to alter the ratio of these events, and thus the release of selected vesicle cargoes.

Recent studies have suggested that two small GTPases, Rab3A and Rab27A, play a key role in the late steps of dense-core vesicle exocytosis in endocrine cells; however, neither the precise mechanisms by which these two GTPases regulate dense-core vesicle exocytosis nor the functional relationship between them is clear. In this study, we expressed a number of different Rab proteins, from Rab1 to Rab41 in PC12 cells and systematically screened them for those that are specifically localized on dense-core vesicles. We found that four Rabs (Rab3A, Rab27A, Rab33A, Rab37) are predominantly targeted to dense-core vesicles in PC12 cells, and that three of them (Rab3A, Rab27A, Rab33A) are endogenously expressed on dense-core vesicles. We further investigated the effect of silencing each Rab with specific small interfering RNA on vesicle dynamics by total internal reflection fluorescence microscopy in a single PC12 cell. Silencing either Rab3A or Rab27A in PC12 cells significantly decreased the number of dense-core vesicles docked to the plasma membrane without altering the kinetics of individual exocytotic events, whereas silencing of Rab33A had no effect at all. Simultaneous silencing of Rab3A and Rab27A caused a significantly greater decrease in number of vesicles docked to the plasma membrane. Our findings indicate that Rab3A and Rab27A cooperatively regulate docking step(s) of dense-core vesicles to the plasma membrane.

Glucagon-like peptide-1 (GLP-1) is a potent regulator of glucose-stimulated insulin secretion whose mechanisms of action are only partly understood. In the present paper, we show that at low (3mM) glucose concentrations, GLP-1 increases the free intramitochondrial concentrations of both Ca2+ ([Ca2+]m), and ATP ([ATP]m) in clonal MIN6 β-cells. Suggesting that cAMP-mediated release of Ca2+ from intracellular stores is responsible for these effects, increases in [ATP]m that were induced by GLP-1 were completely blocked by the Rp isomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), or by chelation of intracellular Ca2+. Furthermore, inhibition of Ins(1,4,5)P3 (IP3) receptors with xestospongin C, or application of ryanodine, partially inhibited GLP-1-induced [ATP]m increases, and the simultaneous blockade of both IP3 and ryanodine receptors (RyR) completely eliminated the rise in [ATP]m. GLP-1 appeared to prompt Ca2+-induced Ca2+ release through IP3 receptors via a protein kinase A (PKA)-mediated phosphorylation event, since ryanodine-insensitive [ATP]m increases were abrogated with the PKA inhibitor, H89. In contrast, the effects of GLP-1 on RyR-mediated [ATP]m increases were apparently mediated by the cAMP-regulated guanine nucleotide exchange factor cAMP-GEFII, since xestospongin C-insensitive [ATP]m increases were blocked by a dominant-negative form of cAMP-GEFII (G114E,G422D). Taken together, these results demonstrate that GLP-1 potentiates glucose-stimulated insulin release in part via the mobilization of intracellular Ca2+, and the stimulation of mitochondrial ATP synthesis.

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic beta-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative-acting globular tail domain of MyoVa decreased by approximately 50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in beta-cells.

Astrocytes comprise a large population of cells in the brain and are important partners to neighboring neurons, vascular cells, and other glial cells. Astrocytes not only form a scaffold for other cells, but also extend foot processes around the capillaries to maintain the blood–brain barrier. Thus, environmental chemicals that exist in the blood stream could have potentially harmful effects on the physiological function of astrocytes. Although astrocytes are not electrically excitable, they have been shown to function as active participants in the development of neural circuits and synaptic activity. Astrocytes respond to neurotransmitters and contribute to synaptic information processing by releasing chemical transmitters called “gliotransmitters.” State-of-the-art optical imaging techniques enable us to clarify how neurotransmitters elicit the release of various gliotransmitters, including glutamate, D-serine, and ATP. Moreover, recent studies have demonstrated that the disruption of gliotransmission results in neuronal dysfunction and abnormal behaviors in animal models. In this review, we focus on the latest technical approaches to clarify the molecular mechanisms of gliotransmitter exocytosis, and discuss the possibility that exposure to environmental chemicals could alter gliotransmission and cause neurodevelopmental disorders.

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminalRabphilin was originally identified as a specific GTP-Rab3A-binding protein on secretory granules (1, 2) that is involved in the control of regulated secretion, including neurotransmitter release and hormone secretion (3-6). Recent evidence, however, has indicated that rabphilin functions independently of Rab3A. First, rabphilin knock-out animals and Rab3 knock-out animals display distinct phenotypes in terms of neurotransmitter release (7,8). Second, rabphilin promotes dense core vesicle exocytosis by endocrine cells independently of Rab3A (9, 10). Third, we very recently found that an N-terminal Rab-binding domain (RBD) 3 of rabphilin also functions as an effector domain for Rab27A in PC12 cells (11)(12)(13)(14). Although the Rab binding properties of rabphilin have been well documented (1,(11)(12)(13)(14)(15)(16), the function of the C-terminal tandem C2 domains of rabphilin, upon ligand binding during regulated secretion remains largely unknown. Biochemical analysis has indicated that the C2 domains of rabphilin interact with phospholipids in a Ca 2ϩ -dependent manner (17-19), but how the Ca 2ϩ /phospholipid binding to the C2 domains of rabphilin triggers regulated secretion also remains unknown.The functional relationship between Rab27A⅐effector complex and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, fundamental fusion machinery of vesicle exocytosis (20), have recently been reported. Slp4-a/granuphilin-a, another Rab27A effector that functions in certain endocrine cells (21-23) and parotid acinar cells (24), was reported to directly interact with syntaxin IA and/or Munc18-1 (23, 25, 26). In addition, the results of a genetic analysis of Caenorhabditis elegans rabphilin and SNARE (syntaxin, SNAP-25, or VAMP/synaptobrevin) double mutants have suggested that rabphilin modulates SNARE function (8). Thus, it is highly possible that rabphilin physically associates with SNARE proteins itself and/or SNARE-associated proteins but that possibility has never been investigated. In this study we systematically investigated the interaction between rabphilin and SNAREs and SNARE-associated proteins by coimmunoprecipitation assays and found that the C2B domain of rabphilin directly interacts with isolated SNAP-25, a target SNARE localized on the plasma membrane. Because there has never been a detailed description of the function of the C2B domain of rabphilin in the recruitment, docking, priming, and fusion of secretory vesicles in live cells, we also investigated the function of the C2B domain of rabphilin on the motion of a single dense core vesicle during exocytosis in PC12 cells by total internal reflection fluorescence microscopy (TIRF, also called evanescent wave or evanescence microscopy) (27) using vesicletargeted fluorescent proteins (28 -31). Expression of the rabphilin-⌬C2B mutant lacking SNAP-25 binding activity inhibited vesicle docking and markedly decreased the number...