Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuropeptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH 4Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.PC-12 cells ͉ evanescent-field microscopy ͉ endocytosis ͉ kiss and run

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic β cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A−/−) mice. Synt1A−/− β cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

In vertebrates, beta cells are aggregated in the form of pancreatic islets. Within these islets, communication between beta cells inhibits basal insulin secretion and enhances glucose-stimulated insulin secretion, thus contributing to glucose homeostasis during fasting and feeding. In the search for the underlying molecular mechanism, we have discovered that beta cells communicate via ephrin-As and EphAs. We provide evidence that ephrin-A5 is required for glucose-stimulated insulin secretion. We further show that EphA-ephrin-A-mediated beta cell communication is bidirectional: EphA forward signaling inhibits insulin secretion, whereas ephrin-A reverse signaling stimulates insulin secretion. EphA forward signaling is downregulated in response to glucose, which indicates that, under basal conditions, beta cells use EphA forward signaling to suppress insulin secretion and that, under stimulatory conditions, they shift to ephrin-A reverse signaling to enhance insulin secretion. Thus, we explain how beta cell communication in pancreatic islets conversely affects basal and glucose-stimulated insulin secretion to improve glucose homeostasis.

We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic beta-cells, from normal and diabetic Goto-Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary beta-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from 'newcomers'. In diabetic GK rat beta-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat beta-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat beta-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat beta-cells.

The monomeric small GTPase Rab27a is specifically localized on both secretory granules and lysosome-related organelles. Although natural mutations of the Rab27a gene in human Griscelli syndrome and in ashen mice cause partial albinism and immunodeficiency reflecting the dysfunction of lysosome-related organelles, phenotypes resulting from the defective exocytosis of secretory granules have not been reported. To explore the roles of Rab27a in secretory granules, we analyzed insulin secretion profiles in ashen mice. Ashen mice showed glucose intolerance after a glucose load without signs of insulin resistance in peripheral tissues or insulin deficiency in the pancreas. Insulin secretion from isolated islets was decreased specifically in response to high glucose concentrations but not other nonphysiological secretagogues such as high K + concentrations, forskolin, or phorbol ester. Neither the intracellular Ca 2+ concentration nor the dynamics of fusion pore opening after glucose stimulation were altered. There were, however, marked reductions in the exocytosis from insulin granules predocked on the plasma membrane and in the replenishment of docked granules during glucose stimulation. These results provide the first genetic evidence to our knowledge for the role of Rab27a in the exocytosis of secretory granules and suggest that the Rab27a/effector system mediates glucose-specific signals for the exocytosis of insulin granules in pancreatic β cells.

To study insulin exocytosis by monitoring the single insulin secretory granule motion, evanescent wave microscopy was used to quantitatively analyze the final stage of insulin exocytosis with biphasic release. Green fluorescent protein-tagged insulin transfected in MIN6 ␤ cells was packed in insulin secretory granules, which appeared to preferentially dock to the plasma membrane. Upon fusion evoked by secretagogues, evanescent wave microscopy revealed that fluorescence of green fluorescent protein-tagged insulin brightened, spread (within 300 ms), and then vanished. Under KCl stimulation, which represents the 1st phase of release, the successive fusion events were seen mostly from previously docked granules for the first minute, followed by the recruitment of new granules to the plasmalemmal docking sites. Stimulation with glucose, in contrast, caused the fusion events from previously docked granules for the first 120 s, thereafter a continuous fusion (2nd phase of release) was observed over 10 min mostly from newly recruited granules that progressively accumulated on the plasma membrane. Thus, our data revealed the distinct behavior of the insulin granule motion during the 1st and 2nd phase of release.

In preparation for the metabolic demands of pregnancy, β cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased β cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a −/− mice exhibited impaired glucose tolerance despite normally increased β cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in β cells, which increased Ca 2+ uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the β cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy. P regnancy places unique demands on the metabolism of the mother. As the pregnancy progresses and the nutrient requirements of the fetus increase, rising levels of placental hormones reduce maternal insulin sensitivity, thereby maintaining the maternal/fetal gradient of glucose and the flow of nutrients to the fetus. The mother balances the resulting increase in insulin demand with structural and functional changes in the islets that generate increased and hyperdynamic insulin secretion. β cell numbers increase, the threshold for glucose decreases, and glucose-stimulated insulin secretion (GSIS) increases (1-3). Failure to reach this balance of insulin demand with insulin production results in gestational diabetes (4).However, the changes in the maternal islets are not simply a response to increased insulin demand, as they precede the development of insulin resistance. Instead, these changes correlate more closely with levels of circulating maternal lactogens (prolactin and placental lactogen) that signal through the prolactin receptor on the β cell (5-9). Downstream of the prolactin receptor, multiple pathway components have been identified that contribute to the maternal increase in β cell mass (10-16), but not the changes in GSIS.In response to the lactogen signaling during pregnancy, levels of both isoforms of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin (5-hydroxytryptamine; 5-HT), rise dramatically in the islet (13,17,18). Islet serotonin acts in an autocrine/paracrine manner through the Gα q -coupled serotonin receptor 5-HT2b receptor (Htr2b) to increase β cell proliferation and mass at midgestation and through Gα i -coupled 5-HT1d receptor (Htr1d) to reduce β cell mass at the end of gestation (13). These dynamic changes in 5-HT receptor (Htr) expression can explain the shifts in β cell proliferation during pregnancy.

To determine the site of insulin exocytosis in the pancreatic ␤ cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled antisyntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 ␤ cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomalassociated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mM KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-␤-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 ␤ cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.Insulin is stored in large dense core vesicles in pancreatic ␤ cells and is released by exocytosis when glucose levels rise (1). We (2) and others (3, 4) have demonstrated previously that soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) 1 are expressed in pancreatic ␤ cells and play an important role in the insulin exocytotic process (2, 5, 6). However, it is not known how t-SNAREs are structurally organized and distributed in the pancreatic ␤ cell plasma membrane and how they spatially interact with the insulin granule during docking and fusion. A recent study described the distribution and spatial organization of t-SNAREs using the procedure of plasma membrane sheets derived from PC12 cells (7, 8), but technical difficulties have prevented such a study in pancreatic ␤ cells.Recently, we developed an approach using a green fluorescent protein (GFP)-tagged insulin granule system combined with total internal reflection microscopy (TIRFM) (9). Using this system, we were able to observe the motion of single insulin granules such as in docking and fusion in the exocytotic process during physiological stimulation. TIRF illuminates fluorophores close to the plasma membrane (within ϳ100 nm) (10), allowing us to observe with high resolution not only the single insulin granules approaching, docking, and fusing with the plasma membrane but also...