Docking is not a prerequisite but a temporal constraint for fusion of secretory granules

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic β cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.     

Loss of Granuphilin and Loss of Syntaxin-1A Cause Differential Effects on Insulin Granule Docking and Fusion

The Rab27 effector granuphilin/Slp4 is essential for the stable attachment (docking) of secretory granules to the plasma membrane, and it also inhibits subsequent fusion. Granuphilin is thought to mediate these processes through interactions with Rab27 on the granule membrane and with syntaxin-1a on the plasma membrane and its binding partner Munc18-1. Consistent with this hypothesis, both syntaxin-1a- and Munc18-1-deficient secretory cells, as well as granuphilin null cells, have been observed to have a deficit of docked granules. However, to date there has been no direct comparative analysis of the docking defects in those mutant cells. In this study, we morphometrically compared granule-docking states between granuphilin null and syntaxin-1a null pancreatic β cells derived from mice having the same genetic background. We found that loss of syntaxin-1a does not cause a significant granule-docking defect, in contrast to granuphilin deficiency. Furthermore, we newly generated granuphilin/syntaxin-1a double knock-out mice, characterized their phenotypes, and found that the double mutant mice represent a phenocopy of granuphilin null mice and do not represent phenotypes of syntaxin-1a null mice, including their granule-docking behavior. Because granuphilin binds to syntaxin-2 and syntaxin-3 as well as syntaxin-1a, it likely mediates granule docking through interactions with those multiple syntaxins on the plasma membrane.     

Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic beta cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic alpha cells, in contrast to another effector, granuphilin, in beta cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca(2+) concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca(2+)-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca(2+)-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.     

Munc 18-1 and granuphilin collaborate during insulin granule exocytosis

Munc 18-1 is a member of the Sec/Munc family of syntaxin-binding proteins known to bind to the plasma membrane Q-SNARE syntaxin1 and whose precise role in regulated exocytosis remains controversial. Here, we show that Munc 18-1 plays a positive role in regulated insulin secretion from pancreatic beta cells. Munc 18-1 depletion caused a loss in the secretory capacity of both transiently transfected INS 1E cells and a stable clone with tetracycline-regulated Munc 18-1 RNA interference. In addition, Munc 18-1-depleted cells exhibited defective docking of insulin granules to the plasma membrane and accumulated insulin in the trans Golgi network. Furthermore, glucose stimulation after Munc 18-1 depletion resulted in the rapid formation of autophagosomes. In contrast, overexpression of Munc 18-1 had no effect on insulin secretion. Although there was no detectable interaction between Munc 18-1 and Munc-18-interacting protein 1 or calcium/calmodulin-dependent serine protein kinase, Munc 18-1 associated with the granular protein granuphilin. This association was regulated by glucose and was required for the specific interaction of insulin granules with syntaxin1. We conclude that Munc 18-1 and granuphilin collaborate in the docking of insulin granules to the plasma membrane in an initial fusion-incompetent state, with Munc 18-1 subsequently playing a positive role in a later stage of insulin granule exocytosis.     

Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a

Secretory vesicle exocytosis is a highly regulated process involving vesicle targeting, priming, and membrane fusion. Rabs and SNAREs play a central role in executing these processes. We have shown recently that Rab27a and its effector, granuphilin, are involved in the exocytosis of insulin-containing secretory granules through a direct interaction with the plasma membrane syntaxin 1a in pancreatic beta cells. Here, we demonstrate that fluorescence-labeled insulin granules are peripherally accumulated in cells overexpressing granuphilin. The peripheral location of granules is well overlapped with both localizations of granuphilin and syntaxin 1a. The plasma membrane targeting of secretory granules is promoted by wild-type granuphilin but not by granuphilin mutants that are defective in binding to either Rab27a or syntaxin 1a. Granuphilin directly binds to the H3 domain of syntaxin 1a containing its SNARE motif. Moreover, introduction of the H3 domain into beta cells induces a dissociation of the native granuphilin-syntaxin complex and a marked reduction of newly docked granules. These results indicate that granuphilin plays a role in tethering insulin granules to the plasma membrane by an interaction with both Rab27a and syntaxin 1a. The complex formation of these three proteins may contribute to the specificity of the targeting process during the exocytosis of insulin granules.     

Granuphilin modulates the exocytosis of secretory granules through interaction with syntaxin 1a

The molecular mechanism for the regulated exocytosis of dense-core granules in endocrine cells remains relatively uncharacterized compared to that of synaptic vesicles in neurons. A novel set of Rab and its effector, Rab27a/granuphilin, which is localized on insulin granules in pancreatic beta cells, was recently identified. Here we demonstrate that granuphilin directly binds to syntaxin 1a on the plasma membrane, and this interaction is regulated by Rab27a. Granuphilin shows affinity to syntaxin 1a with a closed conformation but not to mutant syntaxin 1a, which adopts an open conformation constitutively. Overexpression of granuphilin significantly enhances basal insulin secretion but profoundly inhibits high K(+)-induced insulin secretion. The effect of granuphilin on insulin secretion was impaired by its mutation that disrupts the binding to either Rab27a or syntaxin 1a. Thus, granuphilin is the first regulator in the exocytotic pathway that functions by directly connecting two critical vesicle transport proteins, Rab and SNARE.     

ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic beta cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic beta cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic beta cells.     

The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules

Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K(+)-induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.     

Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy: distinct behavior of granule motion in biphasic insulin release.

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 beta 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.     

Transduction of MIN6 beta cells with TAT-syntaxin SNARE motif inhibits insulin exocytosis in biphasic insulin release in a distinct mechanism analyzed by evanescent wave microscopy

To investigate the in vivo interaction of syntaxin-mediated soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) assembly and insulin exocytosis in biphasic release, we examined the dynamics of insulin granule motion such as docking and fusion with the plasma membrane when the syntaxin SNARE motif (H3 domain) was transduced into living MIN6 beta cells. TAT-H3, produced by fusion of the protein transduction domain of human immunodeficiency virus-1 TAT to the syntaxin-H3 domain, was rapidly transduced into the subplasmalemmal region in living MIN6 cells. Immunoblotting analysis followed by immunoprecipitation on TAT-H3-treated MIN6 cells showed that TAT-H3 binds SNAP-25 and VAMP-2 in vivo. Transduction of MIN6 cells with TAT-H3 caused a decrease in both the first and second phase of insulin release. We therefore quantitatively analyzed approaching, docking, and fusing of green fluorescent protein-labeled single insulin granules in TAT-H3-transduced MIN6 cells by evanescent wave microscopy. Under high glucose stimulation, TAT-H3 treatment not only reduced the fusion events from previously docked granules for the first 120 s (first phase of release) but also strongly inhibited the docking and fusion from newly recruited insulin granules after this point (second phase of release). During the second phase of release we observed a marked reduction in the accumulation of newly docked insulin granules; subsequently, fusion events were significantly decreased. TAT-H3 treatment by itself, however, did not alter the number of previously docked granules without stimulation. We conclude that introduction of the H3 domain into MIN6 cells inhibits biphasic insulin release by two mechanisms. 1) In the first phase of insulin release, the H3 domain interferes with previously docked granules to be fused, and 2) in the second phase of insulin release reduced fusion events result from a marked decline of newly docked granules. Thus, syntaxin-mediated SNARE assembly modulates insulin exocytosis in biphasic insulin release in a distinct way.     

Insulin exocytotic mechanism by imaging technique

Insulin is stored in pancreatic beta cell granules, and released biphasically by the exocytotic mechanism induced by nutrient glucose. Insulin exocytosis must be critically regulated to finely control body glucose homeostasis because insulin is the only hormone that can promptly reduce the blood glucose level. Recent advanced techniques in molecular biology and electrophysiology revealed the molecular mechanism of insulin release in the process from glucose entry to increased [Ca(2+)](i). However, the insulin exocytotic process such as translocation, docking and fusion of insulin granules was largely unknown. In order to reveal the molecular mechanism of this process, we utilized a newly innovated imaging technique, TIRF imaging system. Here we review recent results of our studies into docking and fusion of insulin granules analyzed by TIRF system.     

Site of docking and fusion of insulin secretory granules in live MIN6 beta cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy

To determine the site of insulin exocytosis in the pancreatic beta 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 anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 beta 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 synaptosomal-associated 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-beta-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 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.     

Interaction of syntaxin with alpha-fodrin, a major component of the submembranous cytoskeleton

The soluble N-ethyl maleimide-sensitive factor attachment protein receptor machinery is involved in membrane docking and fusion. In this machinery, the syntaxin family is a central coordinator and participates in multiple protein-protein interactions. In this study we have shown that alpha-fodrin, nonerythroid spectrin, is a new binding partner of the syntaxin family. alpha-Fodrin bound to syntaxin-1a, -3, and -4, all of which are localized on the plasma membrane. Syntaxin-3 interacted with alpha-fodrin in dose-dependent and saturable manners but not with alpha-spectrin, erythroid spectrin. Syntaxin-3 interacted with alpha-fodrin through its C-terminal coiled-coil region. Binding of Munc18 or SNAP-25 to syntaxin-1a inhibited the interaction of alpha-fodrin with syntaxin-1a. Available evidence indicates that alpha-fodrin is implicated in exocytosis, but a precise mode of action of alpha-fodrin in exocytosis remains unclear. Our results suggest that alpha-fodrin regulates exocytosis through the interaction with members of the syntaxin family.     

Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis

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 beta 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(-/-) beta 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.     

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION*

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075–11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.     

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.     

Insulin stimulation of GLUT4 exocytosis, but not its inhibition of endocytosis, is dependent on RabGAP AS160

Insulin maintains whole body blood glucose homeostasis, in part, by regulating the amount of the GLUT4 glucose transporter on the cell surface of fat and muscle cells. Insulin induces the redistribution of GLUT4 from intracellular compartments to the plasma membrane, by stimulating a large increase in exocytosis and a smaller inhibition of endocytosis. A considerable amount is known about the molecular events of insulin signaling and the complex itinerary of GLUT4 trafficking, but less is known about how insulin signaling is transmitted to GLUT4 trafficking. Here, we show that the AS160 RabGAP, a substrate of Akt, is required for insulin stimulation of GLUT4 exocytosis. A dominant-inhibitory mutant of AS160 blocks insulin stimulation of exocytosis at a step before the fusion of GLUT4-containing vesicles with the plasma membrane. This mutant, however, does not block insulin-induced inhibition of GLUT4 endocytosis. These data support a model in which insulin signaling to the exocytosis machinery (AS160 dependent) is distinct from its signaling to the internalization machinery (AS160 independent).