[Frontiers in Bioscience 16, 1197-1210, January 1, 2011]
Regulation of resident and newcomer insulin granules by calcium and SNARE proteins
Kyota Aoyagi, Mica Ohara-Imaizumi, Shinya Nagamatsu
Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, 181-8611, Japan
TABLE OF CONTENTS
1. ABSTRACT
Insulin, stored in large dense core granules, is biphasically exocytosed by glucose stimulation in pancreatic beta-cells. Several molecules, such as SNARE proteins, and Ca2+ ion are involved in the regulation of insulin exocytosis. Indeed, studies using gene targeting mice revealed critical roles of SNARE proteins and their accessory proteins, which may be associated with diabetes mellitus. In particular, the total internal reflection fluorescent (TIRF) imaging technique shed new light on the molecular mechanism of the insulin exocytotic process. In this review we discuss the mechanism of insulin exocytosis mainly from a point of view of imaging techniques.
2. INTRODUCTION
Insulin, a peptide hormone, is released from pancreatic beta-cells of the islets of Langerhans. Secreted insulin accelerates glucose uptake into target tissues and also suppresses glucose output from the liver. Therefore, insulin negatively regulates the concentration of the blood glucose. Because a dysfunction or insufficiency of the insulin secretory response is commonly observed in various forms of type 1 and type 2 diabetes mellitus (1-3), elucidation of the molecular mechanism underlying insulin secretion is quite important. The regulated exocytosis consists of several elementary stages (4-6). Understanding the insulin exocytotic process has recently much progressed, though the molecular mechanism of insulin exocytosis is still unclear. Here, we discuss the molecular mechanism of insulin secretion in pancreatic beta-cells and recent results mainly using TIRF microscopy.
3. OVERVIEW OF INSULIN SECRETION
3.1. Functional coupling between glucose stimulation and insulin secretion
Insulin secretion is coupled to the electrical activity elicited by the elevations of the blood glucose level (Figure 1). When the concentration of extracellular glucose is increased, glucose enters the beta-cell through high-capacity glucose transporter type 2 (GLUT2) in rodents (7) and subsequent metabolic breakdown of the sugar results in an elevation of the cytosolic ATP/ADP ratio (8, 9). This increased ATP/ADP ratio in turn induces the closure of ATP-sensitive K+ channels (KATP channels), leading to the depolarization of the membrane potential (10). The membrane depolarization induces the opening of voltage-gated Ca2+ channels to increase the intracellular calcium concentration ((Ca2+)i) (11). In response to the (Ca2+)i elevation, insulin-containing granules (insulin granules) fuse with the plasma membrane resulting in the release of insulin into the extracellular space. A rise in (Ca2+)i, however, is not likely be the only regulator of exocytosis (12). G protein (13, 14), long-chain fatty acyl moieties (15, 16), and protein kinases (17, 18) also contribute to the induction of exocytosis. Because of space limitation, we do not discuss the mechanism of insulin exocytosis induced by those factors.
3.2. Biphasic insulin secretion
A physiological feature of insulin secretion is that fuel secretagogue, glucose, induces a biphasic pattern of insulin secretion. Dr. Grodsky clearly demonstrated the characteristics of the glucose-induced insulin secretion by in vitro perfusion of rat pancreas (19) as follows (Figure 2): 1) the initial response is a transient but rapid elevation of insulin release (first phase), which terminates in 5 to 10 min; 2) this is followed by a progressibly increasing second phase (prominent in rat, but low or flat in mice) (20). However, interestingly, non-metabolizable stimulation, such as high KCl, evoked transient insulin secretion very similar to the glucose-induced first phase, but cannot induce sustained second phase (12). The different secretory patterns evoked by metabolizable and non-metabolizable stimuli suggest that distinct mechanisms underlie the first and second phase secretion.
3.3. Exocytotic process and SNARE functions
In neurons and neuroendocrine cells, neurotransmitters and hormones are secreted by a sequential process, exocytosis. A large number of studies using these secretory cells revealed that the process leading to Ca2+-dependent secretion consists of some elementary steps:; 1) translocation of secretory vesicles from inside the cell to the plasma membrane region, 2) docking of vesicles to specific sites on the plasma membrane, 3) ATP- and temperature-dependent priming of vesicles to acquire the competency for the subsequent fusion step, and 4) fusing of the vesicular and plasma membranes to release the vesicular contents into the extracellular space (4-6). It is widely accepted that Ca2+-dependent secretion is accomplished by conducting this series of elementary steps on the plasma membrane.
This Ca2+-dependent exocytosis is mediated by a group of proteins, known as "Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins" such as syntaxin, synaptosomal-associated protein of 25 kDa (SNAP-25), and vesicle associated membrane protein-2 (VAMP2)/synaptobrevin. Syntaxin and SNAP-25 localized in the plasma membrane and VAMP2 on secretory vesicles associate to form a ternary core complex, and the formation of this SNARE complex is thought to force the secretory vesicle membrane and the plasma membrane into close proximity, initiating membrane fusion (4, 5, 21). Indeed, SNARE proteins reconstituted into distinct artificial liposomes were demonstrated to mediate membrane fusion (22). The functional importance of SNARE proteins on Ca2+-dependent exocytosis has been established using tetanus and botulinum neurotoxins. These neurotoxins specifically cleave SNARE proteins and severely suppress the Ca2+-dependent neurotransmitter release (23). Furthermore, the genetic ablation of SNARE proteins abolished electrical depolarization-induced exocytosis in neurons and chromaffin cells (24-27).
The mechanism of insulin secretion in beta-cells shows great similarity to that of Ca2+-dependent secretion in neurons and neuroendocrine cells. In beta-cells, a subset of insulin granules is morphologically docked to the plasma membrane (28, 29) and ATP- and temperature-dependent steps prior to Ca2+-dependent fusion are shown to be essential for electrical depolarization-induced insulin secretion (30-32). The expression of SNARE proteins in beta-cells has also been reported (30, 33-38). In permeabilized insulin-secreting cells, tetanus and botulinum neurotoxins severely inhibited Ca2+-dependent insulin secretion (30, 34, 35), and inhibition of the SNARE function by specific antibodies or synthetic peptides also markedly suppressed Ca2+-dependent exocytosis (39, 40). In addition, the expression of syntaxin and SNAP-25 were reduced in diabetic Goto-Kakizaki rat in which glucose-induced insulin secretion was severely impaired. The insufficiency of insulin secretion in response to glucose stimulation was significantly recovered by exogenously expressed syntaxin and SNAP-25 (41-43), indicating the functional linkage between insulin secretion and SNARE proteins.
It is of note that a substantial amount of insulin was released even when the SNARE function was inhibited in beta-cells. Furthermore, in non-permeabilized living beta-cells, inhibition of the SNARE functions caused only mild suppression of Ca2+-dependent insulin secretion (44, 45), and transient expression of botulinum neurotoxin almost completely inhibited KCl-evoked insulin secretion but a large amount of glucose-induced insulin secretion was preserved (45). These results suggest the possibility that pancreatic beta-cells have an alternative secretory pathway that is botulinum neurotoxin-insensitive and involved in the second phase secretion. Although numerous findings on the insulin secretory mechanism have been drawn from a large number of studies based on the similarity of the secretory response of pancreatic beta-cells to that of other secretory systems, it should be important to elucidate the mechanism specific for the characteristic biphasic insulin secretion in beta-cells.
3.4. Current model for biphasic insulin secretion
As mentioned above, SNARE proteins appear to play a key role in insulin exocytosis, but, it is unknown how these proteins function in biphasic insulin release. Thus, the mechanism underlying the glucose-induced biphasic insulin release is still largely unclear. Rorsman and his colleagues proposed a model for the biphasic insulin secretion in which insulin granules can be divided into two functionally distinct pools with different releasability (46, 47). Membrane capacitance measurements revealed that the elevation of (Ca2+)i induced a burst of the exocytotic response, and subsequently the rate of exocytosis gradually dropped and eventually reached a lower steady-state level (31, 48, 49). It is currently accepted that the initial drop of the exocytotic rate reflects the depletion of insulin granules which can immediately undergo exocytosis in response to the (Ca2+)i elevation, and the pool of these release-competent insulin granules is referred to as the "readily releasable pool (RRP)". A series of electrophysiological techniques revealed that the RRP consists of 20 - 100 granules in single beta-cells (31, 49, 50, 51). Given that a single beta-cell contains ~10,000 insulin granules (28, 29), most insulin granules are incompetent for Ca2+-dependent exocytosis and consequently are assumed to reside in a "reserve pool". Because the number of insulin granules in the RRP is not sufficient to support a sustained exocytotic response, the RRP should be refilled, and the reserve pool is thought to be the source of insulin granules that are replenished into the RRP. The ATP and temperature-dependent reactions were shown to be required for the sustained exocytotic response evoked by electrical depolarization (31, 32), indicating that insulin granules in the pre-primed and fusion-incompetent state(s) should undergo priming steps for replenishing the RRP. Therefore, the steady-state level of exocytosis detected by capacitance measurements would reflect the rate at which the RRP is replenished by insulin granules recruited from the reserve pool.
The observation of the biphasic exocytotic response detected by capacitance measurements was extended to glucose-induced insulin secretion by Rorsman's group (46, 47, 52, 53). They proposed that the insulin granules in the RRP would be responsible for the glucose-induced first phase and that the sustained insulin secretion during the second phase would correspond to the rate of replenishment of insulin granules from the reserve pool to the RRP. The estimated number of granules in the RRP was very close to that of insulin granules released during the glucose-induced first phase (47, 54), suggesting that the insulin granules in the RRP would be responsible for the first phase insulin secretion. Meanwhile, although there is no experimental evidence showing that ATP- and temperature-dependent priming steps are required for the glucose-induced second phase insulin secretion in beta-cells, there is a consensus that recruitment of insulin granules from the reserve pool to the RRP should be required for the second phase insulin secretion because the number of insulin granules in the RRP in the non-stimulated condition cannot support the biphasic insulin secretion lasting for a few hours (55). Although the model provided by Rorsman's group seems to fit very well with the feature of biphasic insulin secretion, this model is entirely based on the hypothesis that all insulin granules should be released from the RRP. However, several independent studies on neurons recently raised a question about this hypothesis by demonstrating that not only the RRP but also another pool provided secretory vesicles for exocytosis (56-58). Indeed, there had been no direct observation of real-time insulin granule motion in glucose-stimulated beta-cells to ascertain whether all insulin granules exhibited the same dynamic behavior prior to fusion. However, a recent advance in technology, total internal reflection fluorescent (TIRF) imaging system, allowed us to directly observe the dynamic motion of single insulin granules undergoing insulin exocytosis.
4. IMAGING ANALYSIS OF INSULIN SECRETION
4.1. TIRF microscopy
TIRF microscopy is a technique that specifically illuminates fluorophores within a closely restricted layer just adjacent to the interface at which total internal reflection occurs (59, 60). When angled light strikes the interface and the light undergoes total internal reflection, some of the light penetrates the interface as an electromagnetic field called "evanescent wave". Because the evanescent wave vanishes exponentially with distance from the interface, the penetration depth of the evanescent field is usually less than 100 nm. Therefore, when cells are grown on a coverslip, a very thin layer adjacent to the coverslip can be specifically illuminated by the evanescent wave formed by the total internal reflection occurring at the interface between the coverslip and extracellular medium. In pioneering work by Daniel Axelrod, this technique was first applied to the study of live cells, primarily to investigate cell surface adhesions (61). Then, several studies of protein dynamics, topography of cell-substrate contacts, endocytosis and exocytosis using this technique were reported (62-67).
4.2. Application of TIRF microscopy to imaging analysis of the dynamics of secretory granules
Because the exocytotic process occurs in the vicinity of the plasma membrane, TIRF microscopy is a powerful tool to analyze the exocytotic process of individual insulin granules in living pancreatic beta-cells. To observe the insulin secretory process under TIRF microscopy, insulin granules should be labeled with fluorescent probes. Previously, a weak base fluorescent dye, acridine orange, was used for labeling granules because the inside of most vesicles is acidic (68). However, acridine orange accumulates not only in insulin granules but also other secretory vesicles such as GABA-containing small synaptic-like vesicles. Thus, the expression of GFP-tagged insulin would be best to specifically analyze the motion of insulin granules. GFP-tagged neuropeptide Y and phogrin were also used to label insulin granules (69-72), but their expression patterns were not completely identical (71). Therefore, we made a recombinant adenovirus encoding human preproinsulin tagged with GFP, which is localized in insulin secretory granules (73), allowing us to analyze the real-time motion of insulin granules.
4.3. Docked granules detected by electron and TIRF microscopies
When pancreatic beta-cells expressing insulin-GFP are imaged by TIRF microscopy, punctate fluorescent spots can be observed (74, 75). These punctate signals most likely correspond to docked insulin granules because of the following supporting evidence. First, the intensity of the evanescent field exponentially declines with the distance from the coverslip. Thus, the intensity of excitation light at 100 nm away from the coverslip declines by 89% in our TIRF microscopy setting (73-75). Second, the number of fluorescent spots observed under TIRF microscopy is similar to that of insulin granules morphologically docked on the plasma membrane detected by electron microscopy. Under TIRF microscopy, 253.3 + 10.2 per 200 um2 punctate signals were detected (75). Using the morphometoric data obtained by electron microscopy (29), this value can be converted to 692 fluorescent spots per single beta-cell. Because two studies using electron microscopy reported that 600 - 700 insulin granules morphologically docked on the plasma membrane (28, 29), our result obtained by TIRF microscopy is very similar to those from electron microscopy. Finally, in pancreatic beta-cells prepared from syntaxin1A-/- mice or diabetic Goto-Kakizaki rats, the number of punctate signals detected by TIRF microscopy was severely reduced. The electron microscopic analyses of these animals also revealed the dramatic reduction of morphologically docked insulin granules (41, 74, 75). Taken together, insulin GFP signals observed under the TIRF microscopy are highly likely to correspond to insulin granules morphologically docked on the plasma membrane detected by electron microscopy.
4.4. Analysis of biphasic insulin secretion by TIRF microscopy
TIRF microscopy revealed that glucose stimulation induced interesting exocytotic responses that originated from two distinct types of insulin granules with completely different behaviors prior to fusion; "previously docked granules" that are visible before the onset of stimulation under TIRF microscopy, and "newcomer granules" that cannot be detected by TIRF microscopy or are dimly visible before stimulation (Figure 3). It should be noted that in primary cultured pancreatic beta-cells, newcomer granules fuse immediately after they reach the plasma membrane. The time from landing to fusion is less than 50 ms (74, 75). More interestingly, these two types of fusion events reveal different temporal patterns. Fusions originating from previously docked granules are detected preferentially during the first phase, whereas fusion events during the second phase mostly arose from newcomer granules (41, 74, 75).
Some recent studies demonstrated that glucose stimulation evoked pulsatile insulin secretion not only in isolated pancreatic islets but also at the single beta-cell level (76-78). However, many groups could not always detect the pulsatile response (75, 79-83). Chow and his colleagues observed the glucose-induced pulsatile insulin secretion under TIRF microscopy but they did not address the granule behavior prior to the fusion (77). Thus, which types of insulin granules are involved in the pulsatile secretion currently remains unclear.
4.5. Relationship between SNARE proteins and biphasic insulin release
We examined the relationship between syntaxin and the biphasic insulin release using TIRF microscopy. Because syntaxin was shown to play a crucial role in secretory granule docking and subsequent fusion steps in the exocytotic process (84-86), we performed image analysis to reveal the relationship between syntaxin localization on the plasma membrane and the dynamic motion of insulin granules using dual-color TIRF microscopy. It was reported that syntaxin was not homogenously distributed but was enriched in distinct sites and formed numerous clusters in an artificially isolated plasma membrane (87-89). The clustered distribution of syntaxin together with its functional importance in docking and the subsequent exocytotic process strongly implicated the dynamic interaction between syntaxin clusters and the exocytotic sites of insulin granules. Using the protein transduction domain of HIV-1 TAT protein, which has been shown to efficiently cross biological membranes (90-91), syntaxin clusters in living beta-cells and insulinoma MIN6 cells were visualized by fluorescently labeled antibodies (75, 93). Syntaxin clusters colocalized with a substantial number of docked insulin granules detected by TIRF microscopy, and high KCl stimulation evoked the exocytosis of insulin granules docked on the syntaxin clusters in living MIN6 cells (93). Glucose stimulation also induced the exocytosis of insulin granules docked on the syntaxin clusters in living pancreatic beta-cells (75). We also found that ELKS, a protein localized in presynaptic active zones (94, 95) and involved in membrane trafficking (95, 96), also exhibited the clustered localization in the plasma membrane, and a substantial fraction of the exocytotic responses was observed on the ELKS clusters (97). These results prompted us to further explore the physiological role of SNARE proteins using syntaxin1A-/- pancreatic beta-cells and beta-cells treated with interlukin-1beta to reduce the amount of SNAP-25 (75, 98).
TIRF microscopy revealed that genetic ablation of syntaxin1A severely reduced the number of docked insulin granules and also that a reduction of SNAP-25 by interleukin-1beta diminished the docked insulin granules (75, 98). These results clearly indicate that syntaxin and SNAP-25 are key components for the docking of insulin granules. In line with these results, a series of recent studies in chromaffin cells demonstrated that syntaxin-SNAP-25 complex is essential for the docking of secretory granules (84, 86). Specific cleavage of syntaxin by botulinum neurotoxin resulted in the loss of docked secretory granules (85), and an equivalent docking defect was observed in SNAP-25-/- chromaffin cells (84). Therefore, these reports based on electron microscopic analysis strongly support our TIRF microscopy results. Furthermore, concomitantly with the reduced number of docked insulin granules, genetic ablation of syntaxin1A and reduction of the amount of SNAP-25 by interleukin-1beta treatment selectively disturbed the glucose-induced exocytotic responses originating from previously docked granules during the first phase (75, 98). These results clearly demonstrated the functional linkage between docked insulin granules and the first phase insulin secretion.
Several lines of evidences also support the idea that docked insulin granules would be involved in the first phase insulin secretion. According to the model for biphasic insulin secretion proposed by Rorsman's group, the RRP should consist of a subset of docked insulin granules that have experienced priming steps. Therefore, part of the docked insulin granules should be responsible for the first phase insulin secretion. In good agreement with this model, an ultrastructural study revealed that 600 - 700 insulin granules are morphologically docked at the plasma membrane in single beta-cell (28, 29), and the first phase insulin secretion mimicked by high KCl stimulation was accompanied by a significant reduction of the number of docked insulin granules (28). Second, Sharp and his colleagues used the molecular interaction between VAMP2 and syntaxin to estimate the amount of docked insulin granules (99). The amount of syntaxin-VAMP2 complex was decreased during the first 10 min after the onset of glucose stimulation (corresponding to the first phase), suggesting that molecularly docked insulin granules were responsible for the first phase insulin secretion. Third, electrophysiological experiments showed that ~50 insulin granules were estimated to functionally interact with L-type voltage-gated Ca2+ channels to constitute the RRP (50). These independent results obtained from ultrastructural, molecular, electrophysiological, and biochemical approaches clearly demonstrated that a subset of docked insulin granules constitute the RRP and is, at least partly, responsible for the glucose-stimulated first phase insulin secretion.
4.6. Second phase insulin release and fusions from newcomer granules
Although new technologies revealed that SNARE proteins are involved in the fusion from previously docked granules during the first phase insulin release, the molecules related to the exocytosis of newcomer granules during the second phase are not well understood yet. Interestingly, we found several differences between fusions from newcomers and those from previously docked granules.
First, fusions arising from newcomer granules were detectable in syntaxin1A-/- beta-cells. This was consistent with the perifusion experiments showing that genetic ablation of syntaxin1A impaired the first phase but not the second phase insulin secretion. This result indicates that fusions from newcomer granules are preserved in syntaxin1A-/- cells, because the second phase insulin release mostly consisted of fusions from newcomer granules. In addition, fusions from newcomer granules were detected outside of syntaxin1 clusters on the plasma membrane (75). These results suggest that fusions from newcomer granules were 1) independent of syntaxin1A and 2) involved in the second phase insulin secretion. Consistent with these results, a previous study showed that botulinum neurotoxin, which specifically cleaves several syntaxin isoforms, inhibited 95% of KCl-induced insulin secretion, but only 25% of glucose-induced secretion (45). In addition, the reduction of syntaxin1 and SNAP-25 in cultured beta-cells prepared from diabetic GK rats affected the fusions originating from previously docked granules but not those from newcomer granules. Furthermore, newcomer fusions were not sensitive to the reduction of SNAP-25 and VAMP2 by interleukin-1beta treatment. Thus, syntaxin1A, SNAP-25 and VAMP2 may not be involved in newcomer fusion. Indeed, SNARE isoform-independent exocytosis has been reported (25, 27). In VAMP2 or SNAP25 deleted neurons, evoked neurotransmitter release was completely abolished but spontaneous release was preserved. In addition, several independent studies in neurons recently demonstrated that secretory vesicles involved in evoked neurotransmitter release and spontaneous release belonged to the distinct vesicle pools (56-58). If this is the case, what molecules are involved in newcomer fusion? What mechanisms work in the fusion process of newcomers? The mechanism of exocytosis for previously docked granules appears to be different from that of newcomer granules. Thus, it would be possible that different SNARE protein isoforms are involved in these two pathways. Alternatively, the conformation of proteins on these granules might be different because the acidification of the inside of granules was reported to be important for the sustained exocytotic response in electrophysiologically stimulated beta-cells (100), and the granular acidification may affect the conformation of proteins on the granules (101). Further studies are required to identify the molecules regulating the dynamics of newcomer granules.
4.7. Different characteristics of Ca2+ sensing in previously docked and newcomer granules
Recently, we reported that the Ca2+ sensitivities of fusions originating from previously docked and newcomer granules are different (102). A rapid and marked elevation in the subplasma membrane Ca2+ concentration ((Ca2+)PM) caused fusions mostly from previously docked granules, whereas a slow and sustained elevation in (Ca2+)PM induced fusions from newcomer granules. These results were in good agreement with a recent kinetic model study, describing that the Ca2+ concentrations determine the fusion events of previously docked granules and those of newcomer granules (103). In addition, L-type voltage gated Ca2+ channels were tightly associated with rapid exocytosis and the first phase insulin secretion (104), whereas R-type Ca2+ channels regulated the second phase insulin secretion (105). These studies suggested that the Ca2+ ion has different impacts on the first and second phase secretion. Therefore, it is conceivable that there are different Ca2+-sensing mechanisms controlling fusions from previously docked versus newcomer granules. Synaptotagmins are candidates for Ca2+ sensors of membrane fusion, in concert with SNARE complex. In primary beta-cells, synaptotagmin 5, 7, and 9 are expressed (106-109), but it still remains unclear whether these synaptotagmins regulate the glucose-induced first or second phase insulin secretion. It is also possible that Ca2+ may control the recruitment of insulin granules during the second phase via miosyn Va (110, 111).
4.8. Regulation of second phase insulin release by phosphatidylinositol 3-kinase
Phosphatidylinositol 3-kinase (PI3K), a key component that transmits the insulin signal to downstream effectors (112, 113), is reported to negatively regulate the glucose-induced second phase insulin secretion (114-117). Although class 1B PI3K, a minor component of PI3K in pancreatic beta-cells, was reported to positively modulate the KCl-stimulated insulin secretion in beta-cells (118), its involvement in the glucose-induced biphasic insulin secretion is unknown. On the other hand, the genetic ablation of p85a, a major regulatory subunit of PI3K in beta-cells, resulted in the enhancement of insulin secretion preferentially during the second phase (114). A highly selective inhibitor for PI3K, wortmannin, was also well-known to enhance the insulin secretion (115, 116) without affecting the glucose-induced first phase release (117). Because PI3K regulates the trafficking of intracellular vesicles and exocytosis in many cell types (119, 120), PI3K and its downstream effectors should be involved in the regulation of the dynamics and exocytosis of newcomer granules, and consequently the second phase insulin secretion.
The experiments shown in Figure 4 were performed to examine whether the insulin-PI3K pathway could regulate fusions from newcomer granules. Here, insulin granules in MIN6 cells were labeled by insulin-GFP and observed under TIRF microscopy. Stimulation with 22 mM glucose applied at 0 min induced the biphasic exocytotic responses as we reported previously (75). When MIN6 cells were pre-treated with 0.1 uM wortmannin for 15 min, the number of exocytotic responses from newcomer granules during the second phase but not that of the first phase was markedly increased (Figure 4). The effect of wortmannin on newcomer granules during the second phase was specific because over-expression of a dominant negative form of p85a induced the same effect on newcomer granule fusion (unpublished data). These results, together with previous reports, clearly indicate that downstream effector(s) of PI3K would regulate the dynamics of newcomer granules and the second phase insulin secretion. Although more than 100 molecules are reported to be activated downstream of PI3K, Akt, a Ser/Thr kinase activated downstream of PI3K, would be of particular interest because Akt phosphorylates and regulates several molecules that are involved in insulin secretion including Rac1 (121), Pak1 (122), cystein string protein (CSP) (123), and AS160 (124, 125).
4.9. Akt substrates, possible regulator of second phase insulin secretion
Cdc42, a small GTPase protein, was recently reported to selectively and positively regulate the second phase insulin secretion (126). Because the positive effect of Cdc42 on insulin secretion was dependent on its downstream effectors, Pak1 and Rac1, it is reasonable to assume that the second phase insulin secretion would be regulated by Pak1 and Rac1. Although the role of Rac1 and Pak1 in the biphasic insulin secretion is still unclear, Li et. al. reported that expression of a dominant-negative form of Rac1 caused the inhibition of glucose-induced, but not KCl-induced, insulin secretion indicating the involvement of Rac1 in the second phase release (127). In addition, the phosphorylation at Ser71 of Rac1 by Akt was reported to inhibit the activation of Rac1 (121) suggesting that the inhibition of PI3K should activate Rac1 and enhance the second phase insulin secretion. Akt also phosphorylates and modestly activates Pak1, a Ser/Thr kinase, but the role of the activated Pak1 on the biphasic insulin secretion still remains to be elucidated.
CSP is a presynaptic protein involved in neurotransmitter release. Several studies revealed that CSP is associated with the membrane of secretory vesicles and regulated various aspects of exocytosis: Ca2+-influx, docking, and Ca2+-dependent fusion steps (128, 129). The expression of CSP in beta-cells was reported, and the involvement of CSP in Ca2+-dependent insulin exocytosis was demonstrated (130, 131). Because CSP was shown to interact with syntaxin and synaptotagmin through its N-terminal region, the regulation of exocytosis by CSP was at least partially mediated by these molecular interactions (132, 133). Interestingly, Akt and PKA phosphorylate the same residue, Ser10 (123, 134), and this phosphorylation reduces the binding affinity of CSP to syntaxin and synaptotagmin (123, 132). Thus, the inhibition of PI3K would be expected to reinforce the interaction of CSP with syntaxin and synaptotagmin resulting in the enhancement of insulin secretion.
AS160 was initially identified as an Akt substrate in 3T3-L1 cells (124). Insulin stimulation induced the phosphorylation of five residues (Ser318, Ser570, Ser588, Thr642, Thr751) in AS160, and the phosphorylation of these residues was essential for insulin-dependent exocytosis of GLUT4- containing granules in 3T3-L1 cells (125). Recently, Bouzakri et. al. reported that AS160 regulated glucose-stimulated insulin secretion in beta-cells (135). They showed that AS160 was also phosphorylated by Akt in response to PI3K activation, and depletion of AS160 by siRNA suppressed the glucose-induced insulin secretion. Interestingly, in contrast to the glucose-induced insulin secretion, the basal insulin release was enhanced in AS160 depleted MIN6 cells and beta-cells. Because fusion events can be spontaneously observed in a non-stimulated condition and TIRF microscopy revealed that most spontaneous fusion events arose from newcomer granules (75), it might be interesting to hypothesize that AS160 has dual effects on the exocytosis of insulin granules: a positive effect for fusions from previously docked granules but a negative effect on fusions from newcomer granules.
5. PROPOSED MODEL FOR BIPHASIC INSULIN SECRETION
From our observations and supporting evidences described above, we propose a model for the dynamics of insulin granules during the glucose-induced biphasic secretion, in which two distinct pathways for exocytosis are involved (Figure 5). The glucose-induced first phase insulin secretion consists of fusions mostly originating from previously docked granules, which is also supplemented by those from newcomer granules. The proportion of newcomer fusions during the first phase seems to depend on the experimental conditions (41, 75, 94, 136). On the other hand, in our model, mainly based on evidence from direct imaging techniques, the second phase secretion is mostly derived from newcomer granules which directly fuse with the plasma membrane without passing through the RRP. Besides fusions originating from newcomer granules during the second phase, insulin granules are newly recruited to the docked granule pool. However, only a few of the newly recruited insulin granules are released (74) probably because they are not primed.
To validate our model, the molecular mechanism underlying the dynamics of newcomer granules should be elucidated to identify the specific molecules involved in the second phase insulin secretion. As discussed above, newcomer granules translocate from the inside pool to the plasma membrane in response to stimulation, but we did not have any result concerning the translocation process of newcomer granules; what molecule regulates the dynamic motion of newcomer granules and triggers the translocation, and how do they translocate to the plasma membrane? To answer these questions, the analysis of intracellular insulin granules is becoming more important to shed new light on the molecular mechanisms of the second phase insulin secretion.
6. ACKNOWLEDGEMENT
We are grateful to Mr. Brent Bell (Department of Respiratory and Infectious Diseases, Tohoku University Graduate School of Medicine) for reading this manuscript. This work was supported by the following resources: KAKENHI (C-20570189, 21113523, B-20390260), Sumitomo Foundation, Astellas Foundation for Research on Metabolic Disorders, Research Foundation for Opto-Science and Technology, Kyorin Univ. Sch. Med. Collaboration Project, NOVARTIS Foundation (Japan) for the Promotion of Science.
7. REFERRENCE
1. Polonsky KS: Lilly Lecture 1994. The beta-cell in diabetes: from molecular genetics to clinical research. Diabetes 44, 705-717 (1995)
10.2337/diabetes.44.6.705
http://dx.doi.org/10.2337/diabetes.44.6.705
2. Taylor SI, Accili D, Imai Y: Insulin resistance or insulin deficiency. Which is the primary cause of NIDDM? Diabetes 43, 735-740 (1994)
3. Turner RC, Hattersley AT, Shaw JT, Levy JC: Type II diabetes: clinical aspects of molecular biological studies. Diabetes 44, 1-10 (1995)
10.2337/diabetes.44.1.1
http://dx.doi.org/10.2337/diabetes.44.1.1
4. Sudhof TC: The synaptic vesicle cycle. Annu Rev Neurosci 27, 509-547 (2004)
10.1146/annurev.neuro.26.041002.131412
http://dx.doi.org/10.1146/annurev.neuro.26.041002.131412
5. Jahn R, Lang T, Sudhof TC: Membrane fusion. Cell 112, 519-533 (2003)
10.1016/S0092-8674(03)00112-0
http://dx.doi.org/10.1016/S0092-8674(03)00112-0
6. Klenchin VA, Martin TF: Priming in exocytosis: attaining fusion-competence after vesicle docking. Biochimie 82, 399-407 (2000)
10.1016/S0300-9084(00)00208-X
http://dx.doi.org/10.1016/S0300-9084(00)00208-X
7. Johnson JH, Newgard CB, Milburn JL, Lodish HF, Thorens B: The high Km glucose transporter of islets of Langerhans is functionally similar to the low affinity transporter of liver and has an identical primary sequence. J Biol Chem 265, 6548-6551 (1990)
8. Ghosh A, Ronner P, Cheong E, Khalid P, Matschinsky FM: The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta-cells in the isolated perfused rat pancreas. J Biol Chem 266, 22887-22892 (1991)
9. Detimary P, Jonas JC, Henquin JC: Possible links between glucose-induced changes in the energy state of pancreatic B cells and insulin release. Unmasking by decreasing a stable pool of adenine nucleotides in mouse islets. J Clin Invest 96, 1738-1745 (1995)
10.1172/JCI118219
http://dx.doi.org/10.1172/JCI118219
10. Ashcroft FM: K(ATP) channels and insulin secretion: a key role in health and disease. Biochem Soc Trans 34, 243-246 (2006)
10.1042/BST20060243
http://dx.doi.org/10.1042/BST20060243
11. Rorsman P: The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint. Diabetologia 40, 487-495 (1997)
10.1007/s001250050706
http://dx.doi.org/10.1007/s001250050706
12. Gembal M, Gilon P, Henquin JC: Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells. J Clin Invest 89, 1288-1295 (1992)
10.1172/JCI115714
http://dx.doi.org/10.1172/JCI115714
13. Li G, Regazzi R, Balch WE, Wollheim CB: Stimulation of insulin release from permeabilized HIT-T15 cells by a synthetic peptide corresponding to the effector domain of the small GTP-binding protein rab3. FEBS Lett 327, 145-149 (1993)
10.1016/0014-5793(93)80159-R
http://dx.doi.org/10.1016/0014-5793(93)80159-R
14. Baffy G, Yang L, Wolf BA, Williamson JR: G-protein specificity in signaling pathways that mobilize calcium in insulin-secreting beta-TC3 cells. Diabetes 42, 1878-1882 (1993)
10.2337/diabetes.42.12.1878
http://dx.doi.org/10.2337/diabetes.42.12.1878
15. Ashcroft FM, Proks P, Smith PA, Ammala C, Bokvist K, Rorsman P: Stimulus-secretion coupling in pancreatic beta cells. J Cell Biochem 55 Suppl, 54-65 (1994)
10.1002/jcb.240550007
http://dx.doi.org/10.1002/jcb.240550007
16. Prentki M, Corkey BE: Are the beta-cell signaling molecules malonyl-CoA and cystolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM? Diabetes 45, 273-283 (1996)
10.2337/diabetes.45.3.273
http://dx.doi.org/10.2337/diabetes.45.3.273
17. Sorenson RL, Brelje TC, Roth C: Effect of tyrosine kinase inhibitors on islets of Langerhans: evidence for tyrosine kinases in the regulation of insulin secretion. Endocrinology 134, 1975-1978 (1994)
10.1210/en.134.4.1975
http://dx.doi.org/10.1210/en.134.4.1975
18. Ratcliff H, Jones PM: Effects of okadaic acid on insulin secretion from rat islets of Langerhans. Biochim Biophys Acta 1175, 188-191 (1993)
10.1016/0167-4889(93)90022-H
http://dx.doi.org/10.1016/0167-4889(93)90022-H
19. O'Connor MD, Landahl H, Grodsky GM: Comparison of storage- and signal-limited models of pancreatic insulin secretion. Am J Physiol 238, R378-R389 (1980)
20. Ma YH, Wang J, Rodd GG, Bolaffi JL, Grodsky GM: Differences in insulin secretion between the rat and mouse: role of cAMP. Eur J Endocrinol 132, 370-376 (1995)
10.1530/eje.0.1320370
http://dx.doi.org/10.1530/eje.0.1320370
21. Sudhof TC, Rothman JE: Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474-477 (2009)
10.1126/science.1161748
http://dx.doi.org/10.1126/science.1161748
22. Sollner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE: A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75, 409-418 (1993)
23. Humeau Y, Doussau F, Grant NJ, Poulain B: How botulinum and tetanus neurotoxins block neurotransmitter release. Biochimie 82, 427-446 (2000)
10.1016/S0300-9084(00)00216-9
http://dx.doi.org/10.1016/S0300-9084(00)00216-9
24. Sorensen JB, Nagy G, Varoqueaux F, Nehring RB, Brose N, Wilson MC, Neher E: Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23. Cell 114, 75-86 (2003)
10.1016/S0092-8674(03)00477-X
http://dx.doi.org/10.1016/S0092-8674(03)00477-X
25. Schoch S, Deak F, Konigstorfer A, Mozhayeva M, Sara Y, Sudhof TC, Kavalali ET: SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science 294, 1117-1122 (2001)
10.1126/science.1064335
http://dx.doi.org/10.1126/science.1064335
26. Borisovska M, Zhao Y, Tsytsyura Y, Glyvuk N, Takamori S, Matti U, Rettig J, Sudhof T, Bruns D: v-SNAREs control exocytosis of vesicles from priming to fusion. EMBO J 24, 2114-2126 (2005)
10.1038/sj.emboj.7600696
http://dx.doi.org/10.1038/sj.emboj.7600696
27. Washbourne P, Thompson PM, Carta M, Costa ET, Mathews JR, Lopez-Benditó G, Molnar Z, Becher MW, Valenzuela CF, Partridge LD, Wilson MC: Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat Neurosci 5, 19-26 (2002)
10.1038/nn783
http://dx.doi.org/10.1038/nn783
28. Olofsson CS, Gopel SO, Barg S, Galvanovskis J, Ma X, Salehi A, Rorsman P, Eliasson L: Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells. Pflugers Arch 444, 43-51 (2002)
10.1007/s00424-002-0781-5
http://dx.doi.org/10.1007/s00424-002-0781-5
29. Straub SG, Shanmugam G, Sharp GW: Stimulation of insulin release by glucose is associated with an increase in the number of docked granules in the beta-cells of rat pancreatic islets. Diabetes 53, 3179-3183 (2004)
10.2337/diabetes.53.12.3179
http://dx.doi.org/10.2337/diabetes.53.12.3179
30. Regazzi R, Wollheim CB, Lang J, Theler JM, Rossetto O, Montecucco C, Sadoul K, Weller U, Palmer M, Thorens B: VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca2+-but not for GTP gamma S-induced insulin secretion. EMBO J 14, 2723-2730 (1995)
31. Eliasson L, Renstom E, Ding WG, Proks P, Rorsman P: Rapid ATP-dependent priming of secretory granules precedes Ca2+-induced exocytosis in mouse pancreatic B-cells. J Physiol 503, 399-412 (1997)
10.1111/j.1469-7793.1997.399bh.x
http://dx.doi.org/10.1111/j.1469-7793.1997.399bh.x
32. Renstom E, Eliasson L, Bokvist K, Rorsman P: Cooling inhibits exocytosis in single mouse pancreatic B-cells by suppression of granule mobilization. J Physiol 494, 41-52 (1996)
33. Nagamatsu S, Fujiwara T, Nakamichi Y, Watanabe T, Katahira H, Sawa H, Akagawa K: Expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells. Syntaxin 1A, but not 1B, plays a negative role in regulatory insulin release pathway. J Biol Chem 271, 1160-1165 (1996)
10.1074/jbc.271.2.1160
http://dx.doi.org/10.1074/jbc.271.2.1160
34. Sadoul K, Lang J, Montecucco C, Weller U, Regazzi R, Catsicas S, Wollheim CB, Halban PA: SNAP-25 is expressed in islets of Langerhans and is involved in insulin release. J Cell Biol 128, 1019-1028 (1995)
10.1083/jcb.128.6.1019
http://dx.doi.org/10.1083/jcb.128.6.1019
35. Sadoul K, Berger A, Niemann H, Weller U, Roche PA, Klip A, Trimble WS, Regazzi R, Catsicas S, Halban PA: SNAP-23 is not cleaved by botulinum neurotoxin E and can replace SNAP-25 in the process of insulin secretion. J Biol Chem 272, 33023-33027 (1997)
10.1074/jbc.272.52.33023
http://dx.doi.org/10.1074/jbc.272.52.33023
36. Jacobsson G, Bean AJ, Scheller RH, Juntti-Berggren L, Deeney JT, Berggren PO, Meister B: Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells. Proc Natl Acad Soc U S A 91, 12487-12491 (1994)
10.1073/pnas.91.26.12487
http://dx.doi.org/10.1073/pnas.91.26.12487
37. Wheeler MB, Sheu L, Ghai M, Bouquillon A, Grondin G, Weller U, Beaudoin AR, Bennett MK, Trimble WS, Gaisano HY: Characterization of SNARE protein expression in beta cell lines and pancreatic islets. Endocrinology 137, 1340-1348 (1996)
10.1210/en.137.4.1340
http://dx.doi.org/10.1210/en.137.4.1340
38. Nagamatsu S, Sawa H, Nakamichi Y, Kondo Y, Matsushima S, Watanabe T: Non-functional role of syntaxin 2 in insulin exocytosis by pancreatic beta cells. Cell Biochem Funct 15, 237-242 (1997)
10.1002/(SICI)1099-0844(199712)15:4<237::AID-CBF746>3.0.CO;2-U
http://dx.doi.org/10.1002/(SICI)1099-0844(199712)15:4<237::AID-CBF746>3.0.CO;2-U
39. Martin F, Salinas E, Vazquez J, Soria B, Reig JA: Inhibition of insulin release by synthetic peptides shows that the H3 region at the C-terminal domain of syntaxin-1 is crucial for Ca2+- but not for guanosine 5'-(gamma-thio)triphosphate-induced secretion. Biochem J 320, 201-205 (1996)
40. Martín F, Moya F, Gutierrez LM, Reig JA, Soria B: Role of syntaxin in mouse pancreatic beta cells. Diabetologia 38, 860-863 (1995)
10.1007/s001250050364
http://dx.doi.org/10.1007/s001250050364
41. Kawai J, Ohara-Imaizumi M, Nakamichi Y, Okamura T, Akimoto Y, Matsushima S, Aoyagi K, Kawakami H, Watanabe T, Watada H, Kawamori R, Nagamatsu S: Insulin exocytosis in Goto-Kakizaki rat beta-cells subjected to long-term glinide or sulfonylurea treatment. Biochem J 412, 93-101 (2008)
10.1042/BJ20071282
http://dx.doi.org/10.1042/BJ20071282
42. Ohara-Imaizumi M, Nishiwaki C, Nakamichi Y, Kikuta T, Nagai S, Nagamatsu S: Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analysed by total internal reflection fluorescence microscopy. Diabetologia 47, 2200-2207 (2004)
10.1007/s00125-004-1579-0
http://dx.doi.org/10.1007/s00125-004-1579-0
43. Nagamatsu S, Nakamichi Y, Yamamura C, Matsushima S, Watanabe T, Ozawa S, Furukawa H, Ishida H: Decreased expression of t-SNARE, syntaxin 1, and SNAP-25 in pancreatic beta-cells is involved in impaired insulin secretion from diabetic GK rat islets: restoration of decreased t-SNARE proteins improves impaired insulin secretion. Diabetes 48, 2367-2373 (1999)
10.2337/diabetes.48.12.2367
http://dx.doi.org/10.2337/diabetes.48.12.2367
44. Vikman J, Ma X, Hockerman GH, Rorsman P, Eliasson L: Antibody inhibition of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 reduces rapid exocytosis in insulin-secreting cells. J Mol Endocrinol 36, 503-515 (2006)
10.1677/jme.1.01978
http://dx.doi.org/10.1677/jme.1.01978
45. Land J, Zhang H, Vaidyanathan VV, Sadoul K, Niemann H, Wollheim CB: Transient expression of botulinum neurotoxin C1 light chain differentially inhibits calcium and glucose induced insulin secretion in clonal beta-cells. FEBS Lett 419, 13-17 (1997)
10.1016/S0014-5793(97)01411-7
http://dx.doi.org/10.1016/S0014-5793(97)01411-7
46. Rorsman P, Eliasson L, Renstom E, Gromada J, Barg S, Gopel S: The Cell Physiology of Biphasic Insulin Secretion. News Physiol Sci 15, 72-77 (2000)
47. Eliasson L, Abdulkader F, Braun M, Galvanovskis J, Hoppa MB, Rorsman P: Novel aspects of the molecular mechanisms controlling insulin secretion. J Physiol 586, 3313-3324 (2008)
10.1113/jphysiol.2008.155317
http://dx.doi.org/10.1113/jphysiol.2008.155317
48. Eliasson L, Proks P, Ammala C, Ashcroft FM, Bokvist K, Renstom E, Rorsman P, Smith PA: Endocytosis of secretory granules in mouse pancreatic beta-cells evoked by transient elevation of cytosolic calcium. J Physiol 493, 755-767 (1996)
49. Renstom E, Eliasson L, Rorsman P: Protein kinase A-dependent and -independent stimulation of exocytosis by cAMP in mouse pancreatic B-cells. J Physiol 502, 105-118 (1997)
10.1111/j.1469-7793.1997.105bl.x
http://dx.doi.org/10.1111/j.1469-7793.1997.105bl.x
50. Barg S, Eliasson L, Renstom E, Rorsman P: A subset of 50 secretory granules in close contact with L-type Ca2+ channels accounts for first-phase insulin secretion in mouse beta-cells. Diabetes 511, S74-S82 (2002)
10.2337/diabetes.51.2007.S74
http://dx.doi.org/10.2337/diabetes.51.2007.S74
51. Gromada J, Hoy M, Renstom E, Bokvist K, Eliasson L, Gopel S, Rorsman P: CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells. J Physiol 518, 745-759 (1999)
10.1111/j.1469-7793.1999.0745p.x
http://dx.doi.org/10.1111/j.1469-7793.1999.0745p.x
52. Rorsman P, Renstom E: Insulin granule dynamics in pancreatic beta cells. Diabetologia 46, 1029-1045 (2003)
10.1007/s00125-003-1153-1
http://dx.doi.org/10.1007/s00125-003-1153-1
53. Barg S, Lindqvist A, Obermüller S: Granule docking and cargo release in pancreatic beta-cells. Biochem Soc Trans 36, 294-299 (2008)
10.1042/BST0360294
http://dx.doi.org/10.1042/BST0360294
54. Straub SG, Sharp GW: Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes Metab Res Rev 18, 451-463 (2002)
10.1002/dmrr.329
http://dx.doi.org/10.1002/dmrr.329
55. Bratanova-Tochkova TK, Cheng H, Daniel S, Gunawardana S, Liu YJ, Mulvaney-Musa J, Schermerhorn T, Straub SG, Yajima H, Sharp GW: Triggering and augmentation mechanisms, granule pools, and biphasic insulin secretion. Diabetes 511, S83-S90 (2002)
10.2337/diabetes.51.2007.S83
http://dx.doi.org/10.2337/diabetes.51.2007.S83
56. Fredj NB, Burrone J: A resting pool of vesicles is responsible for spontaneous vesicle fusion at the synapse. Nat Neurosci 12, 751-758 (2009)
10.1038/nn.2317
http://dx.doi.org/10.1038/nn.2317
57. Mathew SS, Pozzo-Miller L, Hablitz JJ: Kainate modulates presynaptic GABA release from two vesicle pools. J Neurosci 28, 725-731 (2008)
10.1523/JNEUROSCI.3625-07.2008
http://dx.doi.org/10.1523/JNEUROSCI.3625-07.2008
58. Sara Y, Virmani T, Deak F, Liu X, Kavalali ET: An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron 45, 563-573 (2005)
10.1016/j.neuron.2004.12.056
http://dx.doi.org/10.1016/j.neuron.2004.12.056
59. Axerlod D: Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764-774 (2001)
10.1034/j.1600-0854.2001.21104.x
http://dx.doi.org/10.1034/j.1600-0854.2001.21104.x
60. Steyer JA, Almers W: A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol 2, 268-275 (2001)
10.1038/35067069
http://dx.doi.org/10.1038/35067069
61. Axelrod D: Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89, 141-145 (1981)
10.1083/jcb.89.1.141
http://dx.doi.org/10.1083/jcb.89.1.141
62. Truskey GA, Burmeister JS, Grapa E, Reichert WM: Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances. J Cell Sci 103, 491-499 (1992)
63. Vale RD, Funatsu T, Pierce DW, Romberg L, Harada Y, Yanagida T: Direct observation of single kinesin molecules moving along microtubules. Nature 380, 451-453 (1996)
10.1038/380451a0
http://dx.doi.org/10.1038/380451a0
64. Sund SE, Axelrod D: Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching. Biophys J 79, 1655-1669 (2000)
10.1016/S0006-3495(00)76415-0
http://dx.doi.org/10.1016/S0006-3495(00)76415-0
65. Dickson RM, Norris DJ, Tzeng YL, Moerner WE: Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels. Science 274, 966-969 (1996)
10.1126/science.274.5289.966
http://dx.doi.org/10.1126/science.274.5289.966
66. Oheim M, Loerke D, Stühmer W, Chow RH: The last few milliseconds in the life of a secretory granule. Docking, dynamics and fusion visualized by total internal reflection fluorescence microscopy (TIRFM). Eur Biophys J 27, 83-98 (1998)
10.1007/s002490050114
http://dx.doi.org/10.1007/s002490050114
67. Sako Y, Minoghchi S, Yanagida T: Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol 2, 168-172 (2000)
10.1038/35004044
http://dx.doi.org/10.1038/35004044
68. Tsuboi T, Zhao C, Terakawa S, Rutter GA: Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event. Curr Biol 10, 1307-1310 (2000)
10.1016/S0960-9822(00)00756-9
http://dx.doi.org/10.1016/S0960-9822(00)00756-9
69. Pouli AE, Emmanouilidou E, Zhao C, Wasmeier C, Hutton JC, Rutter GA: Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaera. Biochem J 333, 193-199 (1998)
70. Tsuboi T, Rutter GA: Multiple forms of "kiss-and-run" exocytosis revealed by evanescent wave microscopy. Curr Biol 13, 563-567 (2003)
10.1016/S0960-9822(03)00176-3
http://dx.doi.org/10.1016/S0960-9822(03)00176-3
71. Tsuboi T, McMahon HT, Rutter GA: Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells. J Biol Chem 279, 47115-47124 (2004)
10.1074/jbc.M408179200
http://dx.doi.org/10.1074/jbc.M408179200
72. Gridley S, Lane WS, Garner CW, Lienhard GE: Novel insulin-elicited phosphoproteins in adipocytes. Cell Signal 17, 59-66 (2005)
10.1016/j.cellsig.2004.05.013
http://dx.doi.org/10.1016/j.cellsig.2004.05.013
73. Ohara-Imaizumi M, Nakamichi Y, Tanaka T, Ishida H, Nagamatsu S: Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy: distinct behavior of granule motion in biphasic insulin release. J Biol chem. 277, 3805-3808 (2001)
10.1074/jbc.C100712200
http://dx.doi.org/10.1074/jbc.C100712200
74. Ohara-Imaizumi M, Nishiwaki C, Kikuta T, Nagai S, Nakamichi Y, Nagamatsu S: TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells. Biochem J 381, 13-18 (2004)
10.1042/BJ20040434
http://dx.doi.org/10.1042/BJ20040434
75. Ohara-Imaizumi M, Fujiwara T, Nakamichi Y, Okamura T, Akimoto Y, Kawai J, Matsushima S, Kawakami H, Watanabe T, Akagawa K, Nagamatsu S: Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis. J Cell Biol 177, 695-705 (2007)
10.1083/jcb.200608132
http://dx.doi.org/10.1083/jcb.200608132
76. Gilon P, Shepherd RM, Henquin JC: Oscillations of secretion driven by oscillations of cytoplasmic Ca2+ as evidences in single pancreatic islets. J Biol Chem 268, 22265-22268 (1993)
77. Michael DJ, Xiong W, Geng X, Drain P, Chow RH: Human insulin vesicle dynamics during pulsatile secretion. Diabetes 56, 1277-1288 (2007)
10.2337/db06-0367
http://dx.doi.org/10.2337/db06-0367
78. Dyachok O, Idevall-Hagren O, Sagetorp J, Tian G, Wuttke A, Arrieumerlou C, Akusjärvi G, Gylfe E, Tengholm A: Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion. Cell Metab 8, 26-37 (2008)
10.1016/j.cmet.2008.06.003
http://dx.doi.org/10.1016/j.cmet.2008.06.003
79. Oh E, Thurmond DC: Munc18c depletion selectively impairs the sustained phase of insulin release. Diabetes 58, 1165-1174 (2009)
10.2337/db08-1059
http://dx.doi.org/10.2337/db08-1059
80. Fujimoto W, Miki T, Ogura T, Zhang M, Seino Y, Satin LS, Nakaya H, Seino S: Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice. Diabetologia 52, 863-872 (2009)
10.1007/s00125-009-1306-y
http://dx.doi.org/10.1007/s00125-009-1306-y
81. Wijesekara N, Dai FF, Hardy AB, Giglou PR, Bhattacharjee A, Koshkin V, Chimienti F, Gaisano HY, Rutter GA, Wheeler MB: Beta cell-specific Znt8 deletion in mice causes marked defects in insulin processing, crystallization and secretion. Diabetologia 53, 1656-1668 (2010)
10.1007/s00125-010-1733-9
http://dx.doi.org/10.1007/s00125-010-1733-9
82. Lopez JA, Kwan EP, Xie L, He Y, James DE, Gaisano HY: The RalA GTPase is a central regulator of insulin exocytosis from pancreatic islet beta cells. J Biol Chem 283, 17939-17945 (2008)
10.1074/jbc.M800321200
http://dx.doi.org/10.1074/jbc.M800321200
83. Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T: Granuphilin molecularly dockes insulin granules to the fusion machinery. J Cell Biol 171, 99-109 (2005)
10.1083/jcb.200505179
http://dx.doi.org/10.1083/jcb.200505179
84. de Wit H, Walter AM, Milosevic I, Gulyas-Kovacs A, Riedel D, Sørensen JB, Verhage M: Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes. Cell 138, 935-946 (2009)
10.1016/j.cell.2009.07.027
http://dx.doi.org/10.1016/j.cell.2009.07.027
85. de Wit H, Cornelisse LN, Toonen RF, Verhage M: Docking of secretory vesicles is syntaxin dependent. PLoS One 1, e126 (2006)
10.1371/journal.pone.0000126
http://dx.doi.org/10.1371/journal.pone.0000126
86. Verhage M, Sørensen JB: Vesicle docking in regulated exocytosis. Traffic 9, 1414-1424 (2008)
10.1111/j.1600-0854.2008.00759.x
http://dx.doi.org/10.1111/j.1600-0854.2008.00759.x
87. Lang T, Bruns D, Wenzel D, Riedel D, Holroyd P, Thiele C, Jahn R: SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis. EMBO J 20, 2202-2213 (2001)
10.1093/emboj/20.9.2202
http://dx.doi.org/10.1093/emboj/20.9.2202
88. Lang T, Margittai M, Holzler H, Jahn R: SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs. J Cell Biol 158, 751-760 (2002)
10.1083/jcb.200203088
http://dx.doi.org/10.1083/jcb.200203088
89. Aoyagi K, Sugaya T, Umeda M, Yamamoto S, Terakawa S, Takahashi M: The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters. J Biol Chem 280, 17346-17352 (2005)
10.1074/jbc.M413307200
http://dx.doi.org/10.1074/jbc.M413307200
90. Stein S, Weiss A, Adermann K, Lazarovici P, Hochman J, Wellhoner H: A disulfide conjugate between anti-tetanus antibodies and HIV (37-72)Tat neutralizes tetanus toxin inside chromaffin cells. FEBS Lett 458, 383-386 (1996)
10.1016/S0014-5793(99)01186-2
http://dx.doi.org/10.1016/S0014-5793(99)01186-2
91. Steffen W: Tat-mediated delivery of antibodies into cultured cells. Methods Mol Biol 161, 141-148 (2001)
10.1385/1-59259-051-9:141
http://dx.doi.org/10.1385/1-59259-051-9:141
92. Niesner U, Halin C, Lozzi L, Günthert M, Neri P, Wunderli-Allenspach H, Zardi L, Neri D: Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides. Bioconjug Chem 13, 729-736 (2002)
10.1021/bc025517+
http://dx.doi.org/10.1021/bc025517+
93. Ohara-Imaizumi M, Nishiwaki C, Kikuta T, Kumakura K, Nakamichi Y, Nagamatsu S: 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. J Biol Chem 279, 8403-8408 (2004)
10.1074/jbc.M308954200
http://dx.doi.org/10.1074/jbc.M308954200
94. Deguchi-Tawarada M, Inoue E, Takao-Rikitsu E, Inoue M, Ohtsuka T, Takai Y: CAST2: identification and characterization of a protein structurally related to the presynaptic cytomatrix protein CAST. Genes Cells 9, 15-23 (2004)
10.1111/j.1356-9597.2004.00697.x
http://dx.doi.org/10.1111/j.1356-9597.2004.00697.x
95. Wang Y, Liu X, Biederer T, Sudhof TC: A family of RIM-binding proteins regulated by alternative splicing: Implications for the genesis of synaptic active zones. Proc Natl Acad Sci U S A 99, 14464-14469 (2002)
10.1073/pnas.182532999
http://dx.doi.org/10.1073/pnas.182532999
96. Monier S, Jollivet F, Janoueix-Lerosey I, Johannes L, Goud B: Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport. Traffic 3, 289-297 (2002)
10.1034/j.1600-0854.2002.030406.x
http://dx.doi.org/10.1034/j.1600-0854.2002.030406.x
97. Ohara-Imaizumi M, Ohtsuka T, Matsushima S, Akimoto Y, Nishiwaki C, Nakamichi Y, Kikuta T, Nagai S, Kawakami H, Watanabe T, Nagamatsu S: 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. Mol Biol Cell 16, 3289-3300 (2005)
10.1091/mbc.E04-09-0816
http://dx.doi.org/10.1091/mbc.E04-09-0816
98. Ohara-Imaizumi M, Cardozo AK, Kikuta T, Eizirik DL, Nagamatsu S: The cytokine interleukin-1beta reduces the docking and fusion of insulin granules in pancreatic beta-cells, preferentially decreasing the first phase of exocytosis. J Biol Chem 279, 41271-41274 (2004)
10.1074/jbc.C400360200
http://dx.doi.org/10.1074/jbc.C400360200
99. Daniel S, Noda M, Straub SG, Sharp GW: Identification of the docked granule pool responsible for the first phase of glucose-stimulated insulin secretion. Diabetes 48, 1686-1690 (1999)
10.2337/diabetes.48.9.1686
http://dx.doi.org/10.2337/diabetes.48.9.1686
100. Barg S, Huang P, Eliasson L, Nelson DJ, Obermuller S, Rorsman P, Thevenod F, Renstom E: Priming of insulin granules for exocytosis by granular Cl- uptake and acidification. J. Cell Sci. 114, 2145-2154 (2001)
101. Barg S: Mechanisms of exocytosis in insulin-secreting B-cells and glucagon-secreting A-cells. Pharmacol. Toxicol. 92, 3-13 (2003)
10.1034/j.1600-0773.2003.920102.x
http://dx.doi.org/10.1034/j.1600-0773.2003.920102.x
102. Ohara-Imaizumi M, Aoyagi K, Nakamichi Y, Nishiwaki C, Sakurai T, Nagamatsu S: Pattern of rise in subplasma membrane Ca2+ concentration determines type of fusing insulin granules in pancreatic beta cells. Biochem Biophys Res Commun 385, 291-295 (2009)
10.1016/j.bbrc.2009.04.155
http://dx.doi.org/10.1016/j.bbrc.2009.04.155
103. Pedersen MG, Sherman A: Newcomer insulin secretory granules as a highly calcium-sensitive pool. Proc Natl Acad Sci U S A 106, 7432-7436 (2009)
10.1073/pnas.0901202106
http://dx.doi.org/10.1073/pnas.0901202106
104. Schulla V, Renstom E, Feil R, Feil S, Franklin I, Gjinovci A, Jing XJ, Laux D, Lundquist I, Magnuson MA, Obermuller S, Olofsson CS, Salehi A, Wendt A, Klugbauer N, Wollheim CB, Rorsman P, Hofmann F: Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice. EMBO J 22, 3844-3854 (2003)
10.1093/emboj/cdg389
http://dx.doi.org/10.1093/emboj/cdg389
105. Jing X, Li DQ, Olofsson CS, Salehi A, Surve VV, Caballero J, Ivarsson R, Lundquist I, Pereverzev A, Schneider T, Rorsman P, Renstom E: CaV2.3 calcium channels control second-phase insulin release. J Clin Invest 115, 146-154 (2005)
10.1172/JCI22518
http://dx.doi.org/10.1172/JCI22518
106. Iezzi M, Eliasson L, Fukuda M, Wollheim CB: Adenovirus-mediated silencing of synaptotagmin 9 inhibits Ca2+-dependent insulin secretion in islets. FEBS Lett 579, 5241-5246 (2005)
10.1016/j.febslet.2005.08.047
http://dx.doi.org/10.1016/j.febslet.2005.08.047
107. Iezzi M, Kouri G, Fukuda M, Wollheim CB: Synaptotagmin V and IX isoforms control Ca2+ -dependent insulin exocytosis. J Cell Sci 117, 3119-3127 (2004)
10.1242/jcs.01179
http://dx.doi.org/10.1242/jcs.01179
108. Gauthier BR, Duhamel DL, Iezzi M, Theander S, Saltel F, Fukuda M, Wehrle-Haller B, Wollheim CB: Synaptotagmin VII splice variants alpha, beta, and delta are expressed in pancreatic beta-cells and regulate insulin exocytosis. FASEB J 22, 194-206 (2008)
10.1096/fj.07-8333com
http://dx.doi.org/10.1096/fj.07-8333com
109. Gustavsson N, Lao Y, Maximov A, Chuang JC, Kostromina E, Repa JJ, Li C, Radda GK, Sudhof TC, Han W: Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice. Proc Natl Acad Sci U S A 105, 3992-3997 (2008)
10.1073/pnas.0711700105
http://dx.doi.org/10.1073/pnas.0711700105
110. Krementsov DN, Krementsova EB, Trybus KM: Myosin V: regulation by calcium, calmodulin, and the tail domain. J Cell Biol 164, 877-886 (2004)
10.1083/jcb.200310065
http://dx.doi.org/10.1083/jcb.200310065
111. Ivarsson R, Jing X, Waselle L, Regazzi R, Renstom E: Myosin 5a controls insulin granule recruitment during late-phase secretion. Traffic 6, 1027-1035 (2005)
10.1111/j.1600-0854.2005.00342.x
http://dx.doi.org/10.1111/j.1600-0854.2005.00342.x
112. Leibiger IB, Leibiger B, Berggren PO: Insulin feedback action on pancreatic beta-cell function. FEBS Lett 532, 1-6 (2002)
10.1016/S0014-5793(02)03627-X
http://dx.doi.org/10.1016/S0014-5793(02)03627-X
113. Cantley LC: The phosphoinositide 3-kinase pathway. Science 296, 1655-1657 (2002)
10.1126/science.296.5573.1655
http://dx.doi.org/10.1126/science.296.5573.1655
114. Eto K, Yamashita T, Tsubamoto Y, Terauchi Y, Hirose K, Kubota N, Yamashita S, Taka J, Satoh S, Sekihara H, Tobe K, Iino M, Noda M, Kimura S, Kadowaki T: Phosphatidylinositol 3-kinase suppresses glucose-stimulated insulin secretion by affecting post-cytosolic (Ca2+) elevation signals. Diabetes 51, 87-97 (2002)
10.2337/diabetes.51.1.87
http://dx.doi.org/10.2337/diabetes.51.1.87
115. Hagiwara S, Sakurai T, Tashiro F, Hashimoto Y, Matsuda Y, Nonomura Y, Miyazaki J: An inhibitory role for phosphatidylinositol 3-kinase in insulin secretion from pancreatic B cell line MIN6. Biochem Biophys Res Commun 214, 51-59 (1995)
10.1006/bbrc.1995.2255
http://dx.doi.org/10.1006/bbrc.1995.2255
116. Zawalich WS, Zawalich KC: A link between insulin resistance and hyperinsulinemia: inhibitors of phosphatidylinositol 3-kinase augment glucose-induced insulin secretion from islets of lean, but not obese, rats. Endocrinology 141, 3287-3295 (2000)
10.1006/bbrc.1995.2255
http://dx.doi.org/10.1006/bbrc.1995.2255
117. Zawalich WS, Tesz GJ, Zawalich KC: Inhibitors of phosphatidylinositol 3-kinase amplify insulin release from islets of lean but not obese mice. J Endocrinol 174, 247-258 (2002)
10.1677/joe.0.1740247
http://dx.doi.org/10.1677/joe.0.1740247
118. Pigeau GM, Kolic J, Ball BJ, Hoppa MB, Wang YW, Ruckle T, Woo M, Manning Fox JE, MacDonald PE: Insulin granule recruitment and exocytosis is dependent on p110 in insulinoma and human beta-cells. Diabetes 58, 2084-2092 (2009)
10.2337/db08-1371
http://dx.doi.org/10.2337/db08-1371
119. Lindmo K, Stenmark H: Regulation of membrane traffic by phosphoinositide 3-kinases. J Cell Sci 119, 605-614 (2006)
10.1242/jcs.02855
http://dx.doi.org/10.1242/jcs.02855
120. Itakura M, Yamamori S, Kuwahara R, Sekiguchi M, Takahashi M: Two distinct regulatory mechanisms of neurotransmitter release by phosphatidylinositol 3-kinase. J Neurochem 94, 502-509 (2005)
10.1111/j.1471-4159.2005.03242.x
http://dx.doi.org/10.1111/j.1471-4159.2005.03242.x
121. Kwon T, Kwon DY, Chun J, Kim JH, Kang SS: Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1. J Biol Chem 275, 423-428 (2000)
10.1074/jbc.275.1.423
http://dx.doi.org/10.1074/jbc.275.1.423
122. Zhou GL, Zhuo Y, King CC, Fryer BH, Bokoch GM, Field J: Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration. Mol Cell Biol 23, 8058-8069 (2003)
10.1128/MCB.23.22.8058-8069.2003
http://dx.doi.org/10.1128/MCB.23.22.8058-8069.2003
123. Evans GJ, Barclay JW, Prescott GR, Jo SR, Burgoyne RD, Birnbaum MJ, Morgan A: Protein kinase B/Akt is a novel cysteine string protein kinase that regulates exocytosis release kinetics and quantal size. J Biol Chem 281, 1564-1572 (2006)
10.1074/jbc.M503628200
http://dx.doi.org/10.1074/jbc.M503628200
124. Kane S, Sano H, Liu SC, Asara JM, Lane WS, Garner CC, Lienhard GE: A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J Biol Chem 277, 22115-22118 (2002)
10.1074/jbc.C200198200
http://dx.doi.org/10.1074/jbc.C200198200
125. Sano H, Kane S, Sano E, Mîinea CP, Asara JM, Lane WS, Garner CW, Lienhard GE: Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. J Biol Chem 278, 14599-14602 (2003)
10.1074/jbc.C300063200
http://dx.doi.org/10.1074/jbc.C300063200
126. Wang Z, Oh E, Thurmond DC: Glucose-stimulated Cdc42 signaling is essential for the second phase of insulin secretion. J Biol Chem 282, 9536-9546 (2007)
10.1074/jbc.M610553200
http://dx.doi.org/10.1074/jbc.M610553200
127. Li J, Luo R, Kowluru A, Li G: Novel regulation by Rac1 of glucose- and forskolin-induced insulin secretion in INS-1 beta-cells. Am J Physiol Endocrinol Metab 286, E818-E827 (2004)
10.1152/ajpendo.00307.2003
http://dx.doi.org/10.1152/ajpendo.00307.2003
128. Evans GJ, Morgan A, Burgoyne RD: Tying everything together: the multiple roles of cysteine string protein (CSP) in regulated exocytosis. Traffic 4, 653-659 (2003)
10.1034/j.1600-0854.2003.00127.x
http://dx.doi.org/10.1034/j.1600-0854.2003.00127.x
129. Buchner E, Gundersen CB: The DnaJ-like cysteine string protein and exocytotic neurotransmitter release. Trends Neurosci 20, 223-227 (1997)
10.1016/S0166-2236(96)10082-5
http://dx.doi.org/10.1016/S0166-2236(96)10082-5
130. Zhang H, Kelley WL, Chamberlain LH, Burgoyne RD, Wollheim CB, Lang J: Cysteine-string proteins regulate exocytosis of insulin independent from transmembrane ion fluxes. FEBS Lett 437, 267-272 (1998)
10.1016/S0014-5793(98)01233-2
http://dx.doi.org/10.1016/S0014-5793(98)01233-2
131. Brown H, Larsson O, Bränström R, Yang SN, Leibiger B, Leibiger I, Fried G, Moede T, Deeney JT, Brown GR, Jacobsson G, Rhodes CJ, Braun JE, Scheller RH, Corkey BE, Berggren PO, Meister B: Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis. EMBO J 17, 5048-5058 (1998)
10.1093/emboj/17.17.5048
http://dx.doi.org/10.1093/emboj/17.17.5048
132. Evans GJ, Morgan A: Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I. Biochem J 364, 343-347 (2002)
10.1042/BJ20020123
http://dx.doi.org/10.1042/BJ20020123
133. Nie Z, Ranjan R, Wenniger JJ, Hong SN, Bronk P, Zinsmaier KE: Overexpression of cysteine-string proteins in Drosophila reveals interactions with syntaxin. J Neurosci 19, 10270-10279 (1999)
134. Evans GJ, Wilkinson MC, Graham ME, Turner KM, Chamberlain LH, Burgoyne RD, Morgan A: Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis. J Biol Chem 276, 47877-47885 (2001)
10.1074/jbc.M108186200
http://dx.doi.org/10.1074/jbc.M108186200
135. Bouzakri K, Ribaux P, Tomas A, Parnaud G, Rickenbach K, Halban PA: Rab GTPase-activating protein AS160 is a major downstream effector of protein kinase B/Akt signaling in pancreatic beta-cells. Diabetes 57, 1195-1204 (2008)
10.2337/db07-1469
http://dx.doi.org/10.2337/db07-1469
136. Kasai K, Ohara-Imaizumi M, Takahashi N, Mizutani S, Zhao S, Kikuta T, Kasai H, Nagamatsu S, Gomi H, Izumi T: Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation. J Clin Invest 115, 388-396 (2005)
10.1172/JCI22955
http://dx.doi.org/10.1172/JCI22955
Key Words: Pancreatic beta-cell, insulin secretion, TIRF microscopy
Send correspondence to: Shinya Nagamatsu, Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, 181-8611, Japan, Tel : 81-422-47-5511, Fax : 81-422-47-5538, E-mail:shinya@ks.kyorin-u.ac.jp