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Vol. 18, Issue 9, 3502-3511, September 2007
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*Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, United Kingdom; and School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia
Submitted January 12, 2007;
Revised June 12, 2007;
Accepted June 20, 2007
Monitoring Editor: Tom U. Martin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, in granules containing low-molecular-weight neurotransmitters (Spruce et al., 1990; Albillos et al., 1997; Alés et al., 1999; Holroyd et al., 2002) and now in cells with peptide-containing granules (Taraska et al., 2003; Perrais et al., 2004; Tsuboi et al., 2004; Obermuller et al., 2005), evidence increasingly suggests that more complex behavior can occur. Instead of granule collapse, fusion pore opening can be followed by closure. A final closure of the fusion pore is an essential step in the kiss-and-run model of exocytosis (Ceccarelli et al., 1972), where it is the transitional step between exocytosis and the endocytic recovery of the granule (Holroyd et al., 2002; Bauer et al., 2004; Tsuboi et al., 2004).
Although many observations of these fusion pore dynamics are unequivocal, it is still controversial as to how fusion pore closure might be related to, or even regulate, loss of granule content (Bauer et al., 2004; Perrais et al., 2004; Obermuller et al., 2005). Furthermore, although a range of factors, such as dynamin (Graham et al., 2002; Tsuboi et al., 2004), complexin (Archer et al., 2002), calcium (Alés et al., 1999; Haller et al., 2001), and syntaxin (Wang et al., 2003), can affect pore dynamics, the physiological steps that regulate this process are not known.
We have studied exocytosis of zymogen granules in acinar cells of the exocrine pancreas (Palade, 1975). The kinetics of exocytosis are slow (Nemoto et al., 2001; Thorn et al., 2004), and we have proposed that endocytosis may occur by recovery of granule membrane in a piecemeal process (Thorn et al., 2004). Here, we show that the fusion pore formed by the zymogen granule is one of the largest pores identified (Curran et al., 1993; Melikyan et al., 1995) (we estimate a pore diameter of between 29 and 55 nm), and yet it is capable of closing. We provide evidence that this closure is likely a prelude to endocytosis and that it occurs after the majority of granule contents are lost.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), modified to reduce the time in collagenase and to limit mechanical trituration. The resulting preparation was composed mainly of pancreatic lobules and fragments (50–100 cells), which were plated onto poly-L-lysine–coated glass coverslips. In experiments with heavy fluorescein-dextrans, problems with slow dye diffusion led us to modify the above-mentioned protocol to produce smaller tissue fragments by extending the times of trituration and incubating the fragments for 40 min, under constant gentle agitation, in the presence of the dyes and in the presence of 1% soybean trypsin inhibitor before study.
Confocal Imaging
Fixed specimens were imaged using a Zeiss 100M Axioscope confocal laser scanning microscope, with a 63x objective lens (numerical aperture [NA] 1.3). Images were collected with the appropriate filters and captured using the multitrack mode of the microscope to minimize cross-talk to <2%. Fluorescent probes were from Invitrogen (Carlsbad, CA). All other compounds were from Sigma Chemical (Poole, Dorset, United Kingdom) and Sigma (Castle Hill, NSW, Australia). All fixed cell data were obtained from many different tissue fragments from at least two independent preparations.
Assessment of Fusion Pore Dimensions
Lysine-fixable fluorescein-dextran dyes were added to the plated pancreatic fragments. The concentrations, adjusted to take account of the varying molecular weights were as follows: 3-kDa Texas Red-dextran (2 mg/ml), 3-kDa fluorescein-dextran (2 mg/ml), 70-kDa fluorescein-dextran (6.5 mg/ml), 500-kDa fluorescein-dextran (3 mg/ml), and 2000-kDa fluorescein-dextran (8 mg/ml). Cells were stimulated with 2 µM acetylcholine for 5 min at room temperature, and then they were washed and fixed in 4% paraformaldehyde in phosphate-buffered saline for 30 min. The Stokes–Einstein radius for each fluorescein-dextran was calculated from the average molecular mass by using the formula radius = 0.33(molecular mass)0.463 (Venturoli and Rippe, 2005). Diameters are 3 kDa, 2.6 nm; 70 kDa, 11.4 nm; 500 kDa, 28.8 nm; and 2000 kDa, 55.2 nm. The radius of 
-amylase (55 kDa) was calculated as 6.4 nm by using the formula radius = 0.483(molecular mass)0.386.
Protocol for CL-6B Gel Separation
CL-6B beads (Sigma-Aldrich) were washed and packed into a 0.75- x 30-cm column. Dextran blue 2000 kDa was passed through the column to calculate void volume and flow rate (set to 160 µl/min). Fractions were collected every minute into individual wells on a 96-well plate. Dye concentration was determined using absorbance spectroscopy at 590 nm. The column was standardized using apoferritin and thyroglobulin.
The fluorescein-dextrans were diluted in 2 ml PBS, applied to the column, and collected fractions were quantified by fluorescence spectroscopy at 485-nm excitation and 510-nm emission. To purify the fluorescein-dextrans, we collected the appropriate subset of fractions for each of the dyes as shown in the bars above the graph in Figure 2A. These were concentrated in a centrifugal filter with a molecular mass cut-off of 30 kDa (Amicon Ultra; Millipore, Billerica, MA) and resuspended in extracellular solution.
Use of Skeletal Muscle
Mice were humanely killed according to local animal ethics procedures. Extensor digitorum longus muscle was dissected out; small bundles of fibers were separated and then stretched out under oil according to previously published procedures (Launikonis and Stephenson, 2002). Droplets (1 µl) containing the purified fluorescein-dextran dyes or Cascade Blue dye were applied to the edge of the stretched muscle, and then the dyes were spread by capillary action along the muscle length.
Assessment of Fusion Pore Dynamics
Freshly prepared cells were placed on poly-L-lysine–coated coverslips and incubated with 3-kDa Texas Red-dextran (2 mg/ml) at 37°C. After stimulation, cells were fixed in 4% paraformaldehyde for 30 min. Then, 3-kDa fluorescein-dextran (4 mg/ml) was added either along with the Texas Red-dextran or at various times after the start of the acetylcholine (ACh) stimulation. To remove dye from the extracellular medium, cells were washed with fresh 4% paraformaldehyde every 5 min during cell fixation. To determine the green/red ratio, fluorescence intensity was measured in a region of interest (diameter, 0.5 µm) in granule and luminal areas. Immunostaining with a monoclonal anti-chymotrypsin antibody (1:50) (2100-0657; Serotec, Kidlington, United Kingdom) was performed after Triton X-100 permeabilization, as was phalloidin staining. The Shapiro–Wilks normality test was performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA), and the normal probability plots were produced by a coded algorithm in Excel (Microsoft, Redmond, WA).
Live-Cell Two-Photon Imaging
We used a custom-made, video-rate, two-photon microscope (Thorn et al., 2004) with a 60x oil immersion objective (NA 1.42; Olympus, Tokyo, Japan), providing an axial resolution (full width, half maximum) of 
1 µm. We imaged exocytotic events using 100 µM Cascade Blue (607 Da), 20 µg/ml sulforhodamine B (SRB) (558 Da), and fluorecein-dextrans as a membrane-impermeant fluorescent extracellular marker excited by femtosecond laser pulses at 800 nm, with fluorescence emission detected at 400–490 nm for Cascade Blue and 570–650 nm for sulforhodamine B or fluorescein-dextrans.
In all experimental protocols, control experiments with single dyes were used to assess cross-talk between fluorescence channels; in all cases, this was <7%. In the experiments with the fluorescein-dextrans (Figure 2), se corrected for this cross-talk. Background fluorescence signal was estimated from regions within the basal pole of the cell, and all fluorescence signals were background-subtracted.
Images (resolution of 10 pixels/µm; average of 15 video frames) were captured with VideoSavant (IO Industries, San Antonio, TX) and analyzed with the MetaMorph program (Molecular Devices, Sunnyvale, CA). An epifluorescent mercury light source provided high-intensity light to photobleach the extracellular dye in an 30-µm-diameter field at the image plane. Exocytotic event kinetics was measured from regions of interest (0.78 µm2; 100 pixels) over granules. Traces were rejected if extensive movement was observed. Photobleach recovery showed complex kinetics, presumably reflecting a multiexponential time course as dye diffused back through the tortuous acinar lumen. However, we found that linear regression (GraphPad Prism) approximated the recoveries well and robustly (Figure 7). All data are shown as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Thorn et al., 2004). Here, we have used extracellular lysine-conjugated fluorescent dyes fixed with paraformaldehyde to label granules (Turvey and Thorn, 2004; Pickett et al., 2005). When cells were stimulated with ACh for 1 min in an extracellular solution containing 3-kDa Texas Red-dextran and fixed, we observed fluorescent labeling of the branching lumen between acinar cells and spherical labeled structures apposed to this lumen, which we have previously characterized as zymogen granules (Thorn et al., 2004; Turvey and Thorn, 2004; Figure 1A).
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55 kDa, and, as would be expected, we found that when coapplied, 70-kDa fluorescein-dextran labeled the same exocytotic granules as 3-kDa Texas Red-dextran (Figure 1B). To probe the size of the fusion pore, we tested a range of fluorescein-dextrans of increasing molecular mass, up to 500 kDa (29 nm in diameter). In all experiments, these larger fluorescent dyes colabeled the same exocytotic granules as 3-kDa Texas Red-dextran (Figure 1C). However, there are two problems with the use of fluorescein-dextrans. First, they are polydisperse with a mixture of sizes present; and second, our diameter estimates are dependent on the dextran occupying a compact globular shape, which in reality they may not. To address the first issue, we ran the fluorescein-dextrans on a Sepharose CL-6B column, we compared the elution profiles obtained with those for standards of known molecular mass, and we selected the appropriate fractions as "purified" dyes for use in our experiments (Figure 2A, bars above graph indicate the fractions used for the purified dyes). Clearly, both the 2000- and 500-kDa fluorescein-dextrans are polydisperse, with a relatively wide spread across the fractions, suggesting the 2000-kDa dye has a substantial component of lower molecular mass and the 500-kDa dye has a component of higher molecular mass. Having purified the dyes, we then conducted a bio-exclusion assay by using freshly isolated mouse skeletal muscle (Launikonis and Stephenson, 2002) where the T-tubule system is open to the outside, and it is known to have a minimum dimension of 30 nm (Luff and Atwood 1971). Cascade Blue (607 Da) and the purified fluorescein-dextrans were applied individually to the extracellular media. Images showed the T-tubule system was apparent for Cascade Blue and 500-kDa fluorescein-dextran but not for the 2000-kDa fluorescein-dextran, indicating diffusive entry of the lower-molecular-mass dyes into the T-tubules (Figure 2B). We conclude that the 500-kDa fluorescein-dextran must have a size of <30 nm, consistent with the calculated diameter of 29 nm.
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These results indicate that the zymogen granule fusion pore has a diameter between 29 and 55 nm. This size is at odds with that from a previous report of 100–180 nm measured with atomic force microscopy (Schneider et al., 1997).
Large fusion pores have been described previously; for example, mast cells have pore diameters of >100 nm (Melikyan et al., 1995). However, these large pores are thought to occur at a terminal phase of exocytosis when the pore dilates and the granule collapses into the plasma membrane (Heuser and Reese, 1973; Curran et al., 1993). Semistable fusion pores, capable of closure, are thought to have much smaller diameters (Albillos et al., 1997; Alés et al., 1999). So, the next question we addressed was, is the large pore of the zymogen granule capable of closure?
Dynamics of the Fusion Pore
To directly test for the possibility of fusion pore closure, we developed a dual-labeling technique with extracellular 3-kDa Texas Red-dextran and 3-kDa fluorescein-dextran. In control experiments, cells were stimulated with ACh in the presence of both extracellular dyes. Both dyes labeled all granules (Figure 3A). In subsequent experiments Texas Red-dextran was present throughout the experiment, but fluorescein-dextran was applied at various times after ACh stimulation. The ACh stimulus was time limited to 1-min duration by the addition of a 10-fold excess of the antagonist atropine. We expected that a granule would be labeled with both dyes if the fusion pore remained open. However, if the fusion pore closed at time points after stimulation, the Texas Red-dextran would be trapped within the granule, whereas fluorescein-dextran (the second dye) would be excluded. In the example shown (Figure 3B), fluorescein-dextran was added 5 min after ACh. The overlay figures show that most granules are yellow, indicating that both dyes have entered the granule interior; however, a significant number of granules (15%) were labeled only with Texas Red-dextran.
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1.00 (Figure 4B). A Shapiro–Wilk normality test showed that this distribution was not significantly different from a Gaussian (p = 0.2).
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), and it indicates that the fusion pore does not open during the period of granule recovery. We tested for the possibility that the sulforhodamine B fluorescence signal might be lost due to granule acidification. In vitro experiments determined the sulforhodamine B fluorescence was nearly insensitive over a pH range from 7 to 2, with a decrease in fluorescence over that range of only 7.2% (data not shown). Therefore, our data indicate that fusion pore closure likely terminates the exocytotic process and that it is a prelude to endocytosis.
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). We stimulated exocytosis with 10 pM cholecystokinin in the presence of extracellular Cascade Blue, and we locally photobleached the dye in the granule and adjacent acinar lumen with high-intensity spot illumination for 5 s in 55-s cycles. After photobleaching, unbleached dye in the larger extracellular volume will diffuse back into the granule lumens, provided that the fusion pore is open (Thorn et al., 2004). The fluorescence signals, in regions of interest in the granule and in the acinar lumen, were followed over time for each cycle of photobleaching (Figure 7).
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5 min. The bottom trace in Figure 7A shows the granule region of interest; the top trace shows the acinar lumen region of interest, and the image gallery above shows selected images of the appearance of the granule and then the bleaching and recovery of fluorescence at the time points indicated. The average rate of acinar lumen recovery was 0.412 ± 0.031 fluorescence units (arbitrary units [a.u.])/s (mean ± SEM; n = 33). The mean recovery in the granules, 0.457 ± 0.093 fluorescence units (a.u.)/s (n = 12), was not significantly different (Student's t test, p = 0.86). However, in 64% of exocytotic events (21/33), fluorescence recovery was not seen for at least one of the cycles of photobleaching, even though the fluorescence in the adjacent lumen did recover (Figure 7B). Lack of recovery was arbitrarily defined by a slope recovery of <0.2 fluorescence units (a.u.)/s (Figure 7, B and C, bottom traces; lack of recovery indicated by asterisk). The average slope in the granules ascribed to the population that did not recover fluorescence was 0.084 ± 0.019 fluorescence units (a.u.)/s (n = 21), significantly different from the lumen recovery rates (Student's t test, p < 0.001). Most of these events did not recover after a single (18/21) or subsequent bleach cycles, as shown in the example in Figure 7B. However, in three events, lack of recovery in one cycle of photobleaching was followed by recovery in the next cycle (Figure 7C, bottom trace). The fluorescent recoveries extracted from either the last photobleach cycle (in granules that always recovered fluorescence) or from the first photobleach cycle where recovery was not seen, were averaged together, and the data are shown in Figure 7, D and E.
Because in all cases the fluorescence of the adjacent acinar lumen did recover, at a rate greater than 0.2 fluorescence units (a.u.)/s, the only potential barrier to fluorescence recovery in the granule is a closed fusion pore. Furthermore, in those events that showed subsequent fluorescence recovery, either the fusion pore must have reopened or a new fusion pore must have formed. On the basis of these results, we conclude that majority of exocytotic events involve irreversible closure of the fusion pore within 5 min after the initial pore opening, supporting the idea that closure is usually the final event of the exocytotic process, although sometimes reopening can occur.
Relationship between Fusion Pore Dynamics and Loss of Granule Contents
Under physiological conditions, it would be expected that granule content would be lost before endocytosis. To test whether this is the case, we used immunohistochemistry of native chymotrypsinogen in combination with our fixed cell dual-labeling assay for fusion pore closure, fixing the cells 5 min after the onset of cholecystokinin stimulation (Figure 8A). Granule chymotrypsinogen content was estimated by comparison of chymotrypsinogen fluorescence in granules that had not undergone exocytosis (e.g., Figure 8Ai, enlarged in Figure 8Biii). We identified granules with open fusion pores as having normalized dye ratios of greater than 0.4, and those with closed fusion pores as having ratios <0.4 (Figure 8, A and B). We found that granules with open fusion pores still contained about half their original content, with remaining chymotrypsinogen levels at 52.8 ± 8.3% (n = 15). By contrast, granules with closed fusion pores had contents of only 15.1 ± 6.7% (n = 67), significantly less (Student's t test, p < 0.05) than the content of the granules with the open fusion pore. These differences in content in the two populations of granules do not reflect changes in granule size (Figure 8, C and D).
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, Nemoto et al., 2004), a process that to date has no known function. To test possible F-actin involvement in fusion pore dynamics, we conducted the dual labeling method with the cells bathed in the first dye, fluorescein, and stimulated for 5 min before adding the second dye, tetramethylrhodamine. The test cells were pretreated with 10 µM latrunculin B for 10 min. Figure 9A shows typical control data in the top images with evidence for F-actin (stained with phalloidin) coating of granules. Pretreatment with latrunculin B reduced phalloidin staining (Figure 9A, bottom). Ratio analysis shows the control frequency histogram (Figure 9B, gray squares, n = 288 granules, >37 cells) with a similar distribution to the 5-min time point in Figure 5; that is, more granule events with low ratios, indicative of fusion pore closure. In the latrunculin B-treated cells (Figure 9B, black circles, n = 240 granules, >31 cells), the distribution shows a further shift to the left indicative of a significant increase (Student's t test, p < 0.001, comparing the control and test ratios) in the number of granules with evidence for fusion pore closure. We conclude that F-actin can influence fusion pore dynamics by stabilizing fusion pore opening.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In other systems, there is evidence that the fusion pore is dynamic, changing over time and existing in one of three distinct states (Fernandez et al., 1984, Spruce et al., 1990; Curran et al., 1993; Wang et al., 2001). It is first observed as a small pore with a conductance similar to that of an ion channel (Zimmerberg et al., 1987; Spruce et al., 1990). The pore can then rapidly expand to a severalfold larger second-stage pore (Spruce et al., 1990). In many cases, this second-stage pore is semistable, and it can fluctuate between open and closed states (Spruce et al., 1990; Curran et al., 1993; Melikyan et al., 1995). In some cell types, this second-stage fluctuating pore can last for a few seconds before resolving into either a closure (e.g., viral fusion, Melikyan et al., 1995 and chromaffin cells, Albillos et al., 1997) or a complete irreversible opening (e.g., mast cells; Spruce et al., 1990; Alvarez de Toledo et al., 1993). This third-stage likely involves dilation of the pore, giving it a large conductance, presumably as complete fusion of the two membranes occurs (Melikyan et al., 1995). These stages of fusion pore development have been seen in divergent examples of membrane fusion ranging from syncytia formation (Mohler et al., 1998) to influenza hemagglutinin-mediated viral fusion with a host cell (Melikyan et al., 1995) to chromaffin cell granule exocytosis (Albillos et al., 1997).
We have developed two new methods for the study of fusion pore dynamics. The dual-dye–labeling technique is used in fixed cells (Figures 3
–5 and 9) and combined with immunohistochemistry (Figure 8) or used in living cells (Figure 7). The photobleaching method is more complicated to perform, but it gives temporal information on pore dynamics, and it is essential to reveal pore reopening. The estimates of the proportion of closed fusion pores differ using the two techniques; 20% at 5 min in the fixed cell dual-dye method and 57% in the photobleaching method. These differences may have a number of explanations. First, in both cases, they are dependent on arbitrary thresholds. Increasing the green/red ratio threshold from 0.4–0.5 would obviously increase the proportion of granules identified with closed fusion pores. Second, in the fixed cell studies, the time course is not that well defined, because fixation takes place over many minutes. Pore opening during this time would lead to entry of the second dye and thereby underestimate the proportion of granules with a closed fusion pore. Whatever the basis for the differences in granule numbers, both techniques provide compelling evidence for pore closure.
Fusion pore dynamics are apparently under the influence of a variety of factors (Alés et al., 1999; Haller et al., 2001; Archer et al., 2002; Graham et al., 2002; Wang et al., 2003; Tsuboi et al., 2004). The large-diameter zymogen granule fusion pore, 29–55 nm, is larger than previously observed second-stage fluctuating pores, which raises the question of how such a large structure might close. Because of its size, the pore is unlikely to be exclusively proteinaceous (Wang et al., 2001); therefore, an ion channel-like mechanism for closure is not plausible. Here, we present the first evidence that the fusion pore dynamics is associated with the F-actin cytoskeleton, with latrunculin B treatment leading to fusion pore closure. Previous work has shown that F-actin coating of zymogen granules (Valentijn et al., 2000) occurs in all postfusion granules (Turvey and Thorn, 2004; Nemoto et al., 2004). A similar phenomenon has been observed in oocytes, and here there is evidence that coating involves actin nucleation (Sokac and Bement, 2006). Schneider et al. (1997) showed that actin depolymerization leads to a decrease in the size of apical depressions in pancreatic acinar cells, which they interpreted as an effect on the fusion pore diameter. However, our experiments now directly show effects of latrunculin B on fusion pore dynamics, and they suggest that F-actin stabilizes an open fusion pore presumably through a membrane-cytoskeleton linker protein. The coupling of the fusion pore to F-actin provides a possible site of regulatory control of pore dynamics perhaps through a motor protein. In support of this idea, in lacrimal acinar cells, myosin 2 has been shown to be associated with granules (Jerdeva et al., 2005); and in chromaffin cells, disruption of myosin 2 activity affects the kinetics of vesicular release of catecholamines (Neco et al., 2004).
In other cell types, fusion pore closure is thought to be a prerequisite for whole-granule recapture in a kiss-and-run type of exocytosis (Ceccarelli et al., 1972). However, although this model has been suggested for acinar cells (Cho et al., 2002), we have never seen recapture of previously fused whole granules into cells. Instead, the data in Figures 6 and 7 indicate that fusion pore closure is the terminal point of exocytosis and the prelude to a different form of endocytosis. In Figure 6, after following single-color granules (indicating fusion pore closure) for protracted periods, we eventually observed a decrease in their fluorescence, which we have previously interpreted as piecemeal pinching back of small subresolution vesicles. These vesicles may then be recovered into the cell and recycled through the Golgi complex, a mechanism that would preserve the lipid and protein identity of the granule membrane (Thorn et al., 2004). What is new here is that during this recapture phase, the granule color does not change; that is, the fusion pore must remain closed throughout endocytic recovery. In support of this suggestion, the photobleaching experiments of Figure 7 show that after closure, fusion pore reopening is a rare event. When correlated with granule content, it is clear that fusion pore closure is associated with low levels of chymotrypsinogen, again supporting the view that pore closure is the terminal event in exocytosis. Although these data do not exclude the possibility that some granules collapse, it does indicate a new form of granule behavior. This proposed sequence of events—opening of the fusion pore, loss of content, and then fusion pore closure followed by a piecemeal recovery of granule membrane—is remarkably similar to the process of trichocyst exocytosis in Paramecium, suggesting either conservation or parallel evolution of a mechanism that operates in a simple single-cell animal through to eukaryotes (Plattner et al., 1985).
Our previous work indicates that much of the granule content is lost quickly (Thorn and Parker, 2005). We now show that, at 5 min after stimulation, granules with an open fusion pore still have 50% of their content remaining (Figure 8). After fusion pore closure significantly less granule content is present, but still 10% of chymotrypsinogen remains. This value is consistent with observations showing that heterologously expressed granule content proteins can still be seen after fusion pore closure (Perrais et al., 2004; Obermuller et al., 2005). Perhaps of more physiological relevance is the finding that native proteins can also be found in the granules of lactotrophs (Bauer et al., 2004) after exocytosis and recapture. What the benefit of partial content release might be is unclear. In lactotrophs, it has been speculated that retrieved exocytosed granules may use residual prolactin to seed the development or maturation of new granules at the Golgi complex (Bauer et al., 2004). Indeed, there is a study in acinar cells, suggesting that zymogens are recovered by the cell and recycled in further rounds of secretion (Romagnoli and Herzog, 1987), a process that is likely to involve the Golgi complex.
In conclusion, the data presented point to a new model for exocytosis in acinar cells that is not consistent with the classical model of simple fusion pore dilation and granule collapse. Instead, we observe fusion pore closure, and we provide evidence that this is a prelude to endocytosis. Further work will be essential to understand secretion in these cells and the regulatory mechanisms controlling fusion pore behavior.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work. 
Address correspondence to: Peter Thorn (p.thorn{at}uq.edu.au).
Abbreviations used: ACh, acetylcholine.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alés, E., Tabares, L., Poyato, J. M., Valero, V., Lindau, M., and Alvarez de Toledo, G. (1999). High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism. Nat. Cell. Biol 1, 40–44.[CrossRef][Medline]
Alvarez de Toledo, G., Fernández-Chacón, R., and Fernández, J. M. (1993). Release of secretory products during transient vesicle fusion. Nature 363, 554–558.[CrossRef][Medline]
Archer, D. A., Graham, M. E., and Burgoyne, R. D. (2002). Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis. J. Biol. Chem 277, 18249–18252.
Bauer, R. A., Overlease, R. L., Lieber, J. L., and Angleson, J. K. (2004). Retention and stimulus-dependent recycling of dense core vesicle content in neuroendocrine cells. J. Cell. Sci 117, 2193–2202.
Ceccarelli, B., Hurlbut, W. P., and Mauro, A. (1972). Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation. J. Cell. Biol 54, 30–38.
Cho, S.-J., Cho, J., and Jena, B. (2002). The number of secretory vesicles remains unchanged following exocytosis. Cell Biol. Int 26, 29–33.[CrossRef][Medline]
Curran, M. J., Cohen, F. S., Chandler, D. E., Munson, P. J., and Zimmerberg, J. (1993). Exocytotic fusion pores exhibit semi-stable states. J. Membr. Biol 133, 61–75.[Medline]
Fernandez, J. M., Neher, E., and Gomperts, B. D. (1984). Capactiance measurements reveal stepwise fusion events in degranulating mast cells. Nature 312, 453–455.[CrossRef][Medline]
Graham, M. E., O'Callaghan, D. W., McMahon, H. T., and Burgoyne, R. D. (2002). Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size. Proc. Natl. Acad. Sci. USA 99, 7124–7129.
Haller, T., Dietl, P., Pfaller, K., Frick, M., Mair, N., Paulmichl, M., Hess, M. W., Furst, J., and Maly, K. (2001). Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells. J. Cell. Biol 155, 279–289.
Heuser, J. E., and Reese, T. S. (1973). Evidence for recycling of synaptic vesicle membrane during transmitter release at frog neuromuscular junction. J. Cell. Biol 57, 315–344.
Holroyd, P., Lang, T., Wenzel, D., De Camilli, P., and Jahn, R. (2002). Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells. Proc. Natl. Acad. Sci. USA 99, 16806–16811.
Jerdeva, G. V., Wu, K., Yarber, F. A., Rhodes, C. J., Kalman, D., Schechter, J. E., and Hamm-Alvarez, S. F. (2005). Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells. J. Cell. Sci 118, 4797–4812.
Launikonis, B. S., and Stephenson, D. G. (2002). Tubular system volume changes in twitch fibres from toad and rat skeletal muscle assessed by confocal microscopy. J. Physiol 538, 607–618.
Luff, A. R., and Atwood, H. L. (1971). Changes in the sarcoplasmic reticulum and transverse tubular system of fast and slow skeletal muscles of the mouse during postnatal development. J. Cell Biol 51, 369–383.
Melikyan, G. B., Niles, W. D., Ratinov, V. A., Karhanek, M., Zimmerberg, J., and Cohen, F. S. (1995). Comparison of transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar membranes. J. Gen. Physiol 106, 803–819.
Mohler, W. A., Simske, J. S., Williams-Masson, E. M., Hardin, J. D., and White, J. G. (1998). Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis. Curr. Biol 8, 1087–1090.[CrossRef][Medline]
Neco, P., Giner, D., Viniegra, S., Borgesi, R., Villarroel, A., and Gutierrez, L. M. (2004). New roles of myosin II during vesicle transport and fusion in chromaffin cells. J. Biol. Chem 279, 27450–27457.
Nemoto, T., Kimura, R., Ito, K., Tachikawa, A., Miyashita, Y., Iino, M., and Kasai, H. (2001). Sequential-replenishment mechanism of exocytosis in pancreatic acini. Nat Cell. Biol 3, 253–258.[CrossRef][Medline]
Nemoto, T., Kojima, T., Oshima, A., Bito, H., and Kasai, H. (2004). Stabilization of exocytosis by dynamic F-actin coating of zymogen granules in pancreatic acini. J. Biol. Chem 279, 37544–37550.
Obermuller, S., Lindqvist, A., Karanauskaite, J., Galvanovkis, J., Rorsman, P., and Barg, S. (2005). Selective nucleotide-release from dense-core granules in insulin-secreting cells. J. Cell Sci 118, 4271–4282.
Palade, G. (1975). Intracellular aspects of process of protein-synthesis. Science 189, 347–368.
Perrais, D., Kleppe, I. C., Taraska, J. W., and Almers, W. (2004). Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells. J. Physiol 560, 413–428.
Pickett, J. A., Thorn, P., and Edwardson, J. M. (2005). The plasma membrane Q-SNARE syntaxin 2 enters the zymogen granule membrane during exocytosis in the pancreatic acinar cell. J. Biol. Chem 280, 1506–1511.
Plattner, H., Pape, R., Haacke, B., Olbricht, K., Westphal, C., and Kersken, H. (1985). Synchronous exocytosis in paramecium-cells. 6. Ultrastructural analysis of membrane resealing and retrieval. J. Cell. Sci 77, 1–17.[Abstract]
Romagnoli, P., and Herzog, V. (1987). Reinternalization of secretory proteins during membrane recycling in rat pancreatic acinar-cells. Eur. J. Cell. Biol 44, 167–175.[Medline]
Schneider, S. W., Sritharan, K. C., Geibel, J. P., Oberleithner, H., and Jena, B. P. (1997). Plasma membrane structures involved in exocytosis, observation by the atomic force microscope. Proc. Natl. Acad. Sci. USA 94, 316–321.
Sokac, A. M., and Bement, W. M. (2006). Kiss-and-coat and compartment mixing, Coupling exocytosis to signal generation and local actin assembly. Mol. Biol. Cell 17, 1495–1502.
Spruce, A. E., Breckenridge, L. J., Lee, A. K., and Almers, W. (1990). Properties of the fusion pore that forms during exocytosis of a mast-cell secretory vesicle. Neuron 4, 642–654.
Taraska, J. W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S., and Almers, W. (2003). Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proc. Natl. Acad. Sci. USA 100, 2070–2075.
Thorn, P., and Parker, I. (2005). Two phases of zymogen granule lifetime in mouse pancreas, ghost granules linger after exocytosis of contents. J. Physiol 563, 433–442.
Thorn, P., Fogarty, K. E., and Parker, I. (2004). Zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity. Proc. Natl. Acad. Sci. USA 101, 6774–6779.
Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V., and Petersen, O. H. (1993). Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate. Cell 74, 661–668.[CrossRef][Medline]
Tsuboi, T., McMahon, H. T., and Rutter, G. A. (2004). Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells. J. Biol. Chem 279, 47115–47124.
Turvey, M., and Thorn, P. (2004). Lysine-fixable dye tracing of exocytosis shows F-actin coating is a step that follows granule fusion in pancreatic acinar cells. Pflügers Archiv 448, 552–555.[Medline]
Valentijn, J. A., Valentijn, K., Pastore, L. M., and Jamieson, J. D. (2000). Actin coating of secretory granules during regulated exocytosis correlates with the release of rab3D. Proc. Natl. Acad. Sci. USA 97, 1091–1095.
Venturoli, D., and Rippe, B. (2005). Ficoll and dextran vs. globular proteins as probes for testing glomerular permselectivity: effects of molecular size, shape, charge, and deformability. Am. J. Physiol 288, F605–F613.
Wang, C-T., Grishanin, R., Earles, C. A., Chang, P. Y., Martin, T.F.J., Chapman, E. R., and Jackson, M. B. (2001). Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles. Science 294, 1111–1115.
Wang, C-T., Lu, J. C., Bai, J., Martin, T.F.J., Chapman, E. R., and Jackson, M. B. (2003). Different domains of synaptotagmin control the choice between kiss-and-run and full fusion. Nature 424, 943–947.[CrossRef][Medline]
Zimmerberg, J., Curran, M., Cohen, F. S., and Brodwick, M. (1987). Simultaneous electrical and optical measurements show that membrane-fusion precedes secretory granule swelling during exocytosis of beige mouse mast-cells. Proc. Natl. Acad. Sci. USA 84, 1585–1589.
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