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Address correspondence to H. Kasai, Dept. of Cell Physiology, National Institute for Physiological Sciences, Graduate University of Advanced Studies, Myodaiji, Okazaki 444-8585, Japan. Tel.: 81-564-55-7831. Fax: 81-564-53-7341. email: hkasai{at}nips.ac.jp
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Abstract |
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Key Words: diabetes; two-photon imaging; secretion; SNARE; pancreatic islet
Abbreviations used in this paper: AM, acetoxymethyl ester; AU, arbitrary unit; [Ca2+]i, cytosolic-free Ca2+ concentration; NP-EGTA, o-nitrophenylEGTA; ROI, region of interest; SNAP25, synaptosome-associated protein of 25 kD; SRB, sulforhodamine B.
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Introduction |
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The dynamics of sequential exocytosis in pancreatic acini have been revealed by two-photon excitation microscopy (Nemoto et al., 2001). In this approach, the secretory glands are placed in a solution containing a polar fluorescent tracer, and the individual exocytic events are detected by the appearance of -shaped fluorescent profiles. We have found that the inner filter effect of bright fluid-phase tracers with large extinction coefficients, such as sulforhodamine B (SRB; Takahashi et al., 2002), is absent in two-photon excitation imaging, so that we can use high concentrations of polar tracers to image individual exocytic events. Sequential exocytosis, which proceeds deep within the cytoplasm, is apparent as beadlike strands of exocytosed vesicles. The rapid appearance of a large fluorescent profile corresponding to multigranular exocytosis was rarely detected in pancreatic acini (Nemoto et al., 2001). Two-photon excitation imaging with an extracellular dye is thus a reliable method with which to quantify the two types of compound exocytosis in intact tissue.
This method has previously been applied to pancreatic islets to monitor the exocytosis of individual insulin granules (Takahashi et al., 2002). We have now systematically investigated the possible occurrence of sequential and multigranular exocytosis in ß cells of intact islets as well as performed real-time measurements of the redistribution of a SNARE protein (Wei et al., 2000), SNAP25 (synaptosome-associated protein of 25 kD), during individual sequential exocytic events, taking advantage of the simultaneous multicolor imaging facility of the two-photon microscope (Nemoto et al., 2001; Takahashi et al., 2002).
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Results |
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Effects of cAMP, PMA, and temperature on sequential exocytosis
Exposure of islets either to 2 µM forskolin, which increases the intracellular concentration of cAMP, or to 400 nM PMA, which activates protein kinase C, in the presence of 20 mM glucose resulted in a two- to fourfold increase in the frequency of insulin exocytosis compared with that apparent in the presence of 20 mM glucose alone (Table I). However, the proportion of sequential exocytic events was unaffected by either of these agents (Table I). The contribution of sequential exocytosis to total exocytosis also did not differ between islets stimulated with 20 mM glucose and those stimulated either with acetylcholine or with a high concentration of K+ (Table I).
Although sequential exocytosis is frequent in the exocrine pancreas even at room temperature (Table I), we tested the possibility that its frequency might be increased in ß cells at higher temperatures (3335°C). Although the frequency of glucose-induced exocytosis was approximately doubled by increasing the temperature, the proportion of sequential exocytic events was not significantly altered (P > 0.1, Table I). Indeed, the contribution of sequential exocytosis was not significantly increased even in the presence of 2 µM forskolin or 400 nM PMA at 3335°C (Table I).
Quantitation of multigranular exocytosis
To evaluate the possible occurrence of multigranular exocytosis in ß cells, we determined the distribution of the fluorescence intensity of individual exocytic events (Fig. 2 A). The distribution was skewed, with an excess of large components. However, the diameters of granules estimated from the fluorescence intensities (see Materials and methods; Fig. 2 B) fit well with a Gaussian distribution (Fig. 2 B, red line), which is consistent with previous ultrastructural data (Dean, 1973) and can account for the skewed distribution in the volume of granules (Fig. 2 A, red line; Bekkers et al., 1990). The distributions of fluorescence intensity and granule diameter were not affected by exposure of islets to forskolin (Fig. 2) or PMA (not depicted) in the presence of 20 mM glucose. The intensity distribution was also similar at 3335°C (unpublished data). Thus, these data indicate the occurrence of little if any multigranular exocytosis in ß cells.
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Redistribution of SNAP25 associated with sequential exocytosis
It has been proposed that sequential exocytosis is facilitated by the lateral diffusion of target SNARE proteins from the plasma membrane into the membrane of the fused granule (Nemoto et al., 2001). To test this hypothesis, we transfected islets with an adenoviral vector encoding an ECFP-SNAP25 fusion protein (Fig. 4 B). The ECFP-SNAP25 protein was localized predominantly to the plasma membrane, which is consistent with the localization of the endogenous SNARE protein (Jacobsson et al., 1994). Expression of ECFP-SNAP25 did not affect either the frequency of exocytic events induced by 20 mM glucose in the presence of 2 µM forskolin (26.1 ± 7.7 events cell1 min1, n = 7) or the proportion of sequential exocytosis (1.3%, n = 312, P > 0.1).
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We quantified the increase in ECFP-SNAP25 fluorescence apparent 520 s after the onset of the SRB signal for sequential exocytic events, solitary fusion events, and arbitrary regions of the plasma membrane without exocytosis, respectively (Fig. 5 A). The distributions of the increases in ECFP-SNAP25 fluorescence (Fig. 5 A, inset) differed significantly between sequential and solitary exocytic events (Smirnov test, P < 0.01) and between solitary events and arbitrary regions (P < 0.05). No increase in fluorescence larger than 15 arbitrary units (AU) was detected in arbitrary regions (Fig. 5 A). If we set the threshold level for significant redistribution at 15 AU, the redistribution was significant in 54% of sequential exocytic events (n = 36) but in only 5% of solitary exocytic events (n = 354; 2 test, P < 0.01). The mean fluorescence increase for the events with significant redistribution was larger in sequential exocytic events (mean ± SD = 25.6 ± 10.7 AU, n = 14, P < 0.01) than in solitary exocytic events (20.8 ± 6.3 AU, n = 18). The mean baseline fluorescence was 20.6 ± 12.5 AU (n = 17).
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Effects of cyclodextrin treatment
To investigate whether or not cholesterol-dependent lipid rafts were involved in regulating lateral diffusion of t-SNARE (Chamberlain et al., 2001), we treated islet preparations with methyl-ß-cyclodextrin (15 mM in Sol A, 3060 min). Such treatment was known to deplete cholesterol selectively from membrane (<40%), leaving phospholipid unaffected (Lang et al., 2001), and to induce dispersion of the clusters of the intrinsic plasma membrane-associated SNAREs in PC12 cells (Lang et al., 2001). When we stimulated cyclodextrin-treated islets with 20 mM glucose and 2 µM forskolin, the frequency of insulin exocytosis was reduced to 20% (4.8 events cell1 min1, n = 4, Table I), as reported in PC12 cells (Lang et al., 2001). However, the proportion of sequential exocytosis was increased by 3.7-fold (Table I, 8.9%, n = 416). In addition, significant redistribution of ECFP-SNAP25 fluorescence (threshold level at 15 AU, Fig. 4 I) was more frequently observed by 2.7-fold (14.9%, n = 201) than in untreated control cases (5.8%, n = 361).
Treatment with methyl-ß-cyclodextrin also reduced the delay between the onset of exocytosis and that of the ECFP-SNAP25 signal from 8.2 ± 7.2 s (n =12) to 2.4 ± 2.5 s (n = 19, P < 0.01) in solitary events, and from 6.3 ± 6.1 s (n = 19) to 3.5 ± 2.3 s (n = 4, P > 0.05) in sequential events. The time during which SNAP-25 signal reaches the maximal values was not affected by cyclodextrin (unpublished data). The mean fluorescence increase for the events with significant redistribution was not significantly different from control in solitary events (21.8 ± 8.1 AU, n = 30, P > 0.05), and in sequential events (26.5 ± 16.9 AU, n = 7, P > 0.05).
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Discussion |
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Our data support the hypothesis that redistribution of a target SNARE protein from the plasma membrane into the membrane of a fused granule after opening of the fusion pore plays an important role in sequential exocytosis in ß cells, as has been proposed for pancreatic acinar cells (Nemoto et al., 2001). Thus, sequential exocytosis in ß cells was preferentially associated with a redistribution of ECFP-SNAP25 apparent after opening of the fusion pore. Furthermore, the cyclodextrin treatment, which enhanced redistribution of SNAP25 by 2.6-fold, increased the proportion of sequential exocytosis by 3.7-fold. The observation that 46% of sequential exocytic events were not associated with such redistribution might be attributable to the small signal/noise ratio of ECFP-SNAP25 fluorescence or to the presence of endogenous SNAP25. Conversely, the redistribution of ECFP-SNAP25 was associated with only 5% of solitary exocytic events, and the extent of the redistribution in these instances was smaller than that apparent for sequential events. This result suggests that sequential exocytosis is regulated by the redistribution of SNAP25 in a concentration-dependent manner. Infrequency (5%) or inhibition of the lateral diffusion of SNAP25 might be the major factor limiting the frequency of sequential exocytosis in pancreatic ß cells. In mast cells, where massive sequential exocytosis occurs (Alvarez de Toledo and Fernandez, 1990), substantial relocation of SNAP23 was found after exocytosis (Guo et al., 1998). Such relocation might, at least partly, reflect redistribution of SNAP23 into granules as in ß cells.
We found that the depletion of cholesterol by methyl-ß-cyclodextrin from the plasma membrane increased the frequency of redistribution of ECFP-SNAP25 and shortened the latency between the onset of primary exocytosis and that of redistribution of ECFP-SNAP25. Cholesterol binds directly to syntaxin 1A (Lang et al., 2001), and SNAP25 forms a heterodimer with syntaxin 1A (Chamberlain et al., 2001). Therefore, the depletion of cholesterol may facilitate the diffusion of SNAP25 along the plasma membrane and thereby enhances redistribution of SNAP25 into fused granules. Alternatively, cholesterol depletion may alter properties of the fusion pore so that diffusion of SNAP25 along fusion pore is facilitated. The more frequent and rapid redistribution of SNAP25 can account for the increase in the proportion of sequential exocytosis, but only to 8.9%, and the major molecular mechanisms of the inhibition of sequential exocytosis remain to be clarified.
The suppression of sequential exocytosis in ß cells may play an important role in the maintenance of blood glucose concentration. First, the rate of insulin exocytosis triggered by glucose was relatively low (11 events cell1 min1) and the time course of secretion is relatively long, persisting for up to several minutes or hours. Thus, the suppression of sequential exocytosis prevents massive mobilization of insulin granules that would result in hypersecretion and in depletion of insulin. Second, the rarity of sequential exocytosis obliges ß cells to transport insulin granules to the cell surface for exocytosis (Hisatomi et al., 1996; Rorsman and Renström, 2003). Such transportation, which is energy dependent, might act as a fuel sensor for insulin granule exocytosis (Takahashi et al., 1999; Kasai et al., 2002; Rorsman and Renström, 2003). Thus, the suppression of sequential exocytosis in ß cells may be an important aspect of the secretory physiology of these cells.
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Materials and methods |
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Two-photon excitation imaging
Two-photon excitation imaging of islets was performed with an inverted microscope (model IX70; Olympus) and a laser-scanning microscope (model FluoView; Olympus) equipped with a water-immersion objective lens (UplanApo60xW/IR; numerical aperture, 1.2). The laser power at the specimen was 310 mW, and two-photon excitation was effected at 830 nm, with images acquired every 0.32 s. The fluorescence of SRB, of fura-2 and fura-2FF, and of ECFP was measured at 550650 nm, 400530 nm, and 400490 nm, respectively. In ECFP measurement, laser power and photomultiplier voltage were set to 8 W and 500 V, respectively. 12-bit images were color-coded with autumn color codes of FluoView. All experimental procedures were performed under yellow light illumination (FL40S-Y-F; National) to prevent unintended photolysis of the caged-Ca2+ compound (see the following section). Most experiments were performed at RT (2425°C), although the temperature of the bathing solution was increased to 3335°C with a heater (Daia Medical System) in some experiments.
Exocytic events were counted in a region of interest (ROI) with an area of 3,0005,000 µm2 and were normalized to an area of 2,000 µm2. Given that the thickness of the optical section (T) in our setup is 0.8 µm, the volume of the normalized ROI is 1,600 µm3. We expressed the number of events in the normalized ROI as events per cell, given that the volume of individual ß cells is
1,600 (ß cells have a mean diameter of 14.0 µm and constitute 90% of cells in an islet, or one ß cell/1,596 µm3 of an islet). For example, a frequency of 11.2 (or 45) events per cell per minute can also be expressed as 0.018 (or 0.073) events per square micrometer of plasma membrane per minute (a). In this example, the mean frequency of events (
) within 1 min and a distance of <0.5 µm (2b) is predicted to be between
ab2 and
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(or 0.721.3%) based on Poisson statistics. Peak fluorescence intensities of individual exocytic events were converted to the diameters (2r) of granules with the equation
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Photolysis of a caged-Ca2+ compound
The acetoxymethyl esters (AMs) of the Ca2+ indicators, fura-2 (Molecular Probes) and fura-2FF (Tef Lab), as well as that of the caged-Ca2+ compound NP-EGTA (Molecular Probes) were dissolved in DMSO at a high concentration (210 mM). Islets were loaded with these compounds by incubation for 30 min at 37°C in serum-free DME containing 10 µM fura-2-AM (or fura-2FF-AM), 25 µM NP-EGTA-AM, 0.03% cremphor EL (Molecular Probes), and 0.1% BSA, and were washed with Sol A. Photolysis of NP-EGTA was induced with a brief flash (0.20.5 s) of a mercury lamp (model IX-RFC; Olympus). The resulting increase in [Ca2+]i was monitored by the decrease in fluorescence intensity of fura-2FF (excitation wavelength: 830 nm) and was calculated as
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Adenoviral infection
The mouse cDNA encoding SNAP25b was amplified by PCR with the forward primer 5'-GCGAATTCAATGGCCGAGGACGCAG-3' and the reverse primer 5'-CGGTCGACTTAACCACTTCCCAGCATC-3', which incorporate SalI and EcoRI sites at the 5' and 3' ends of the SNAP25 cDNA sequence, respectively. The PCR product was digested with SalI and EcoRI and inserted into the corresponding sites of the expression vector pECFP-C1 (CLONTECH Laboratories, Inc.), yielding a vector that encodes a fusion protein in which ECFP is attached to the NH2 terminus of SNAP25 (Wei et al., 2000). An adenoviral vector that encodes the ECFP-SNAP25 fusion protein was constructed as described previously (Miyake et al., 1996). Islets were infected with the adenoviral vector (7 x 107 plaque-forming units per milliliter) for 2 h in serum-free culture medium, washed, and incubated for an additional 1018 h before experiments.
Online supplemental material
Online supplemental material shows a video of the real-time image shown in Fig. 1 B. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200312033/DC1.
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Acknowledgments |
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This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Japan Society for the Promotion of Science, as well as by research grants from the Human Frontier Science Program Organization, Yamanouchi Foundation for Research on Metabolic Disorders, and Japan Diabetes Foundation.
Submitted: 3 December 2003
Accepted: 23 March 2004
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