004_C100712200v1 by isbangee

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									JBC Papers in Press. Published on December 21, 2001 as Manuscript C100712200

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Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy —Distinct behavior of granule motion in biphasic insulin release —

Mica Ohara-Imaizumi,* Yoko Nakamichi,* Toshiaki Tanaka,† Hitoshi Ishida,† and Shinya Nagamatsu*,‡

*Department of Biochemistry, Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, Japan
†

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Department of Internal Medicine (III), Kyorin University School of Medicine, Shinkawa

6-20-2, Mitaka, Tokyo 181-8611, Japan

‡

Corresponding author: Shinya Nagamatsu, M.D., Department of Biochemistry, Kyorin

University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, Japan Tel: +81-422-47-5511 (ext: 3437) Fax: +81-422-47-5538 E-mail: shinya@kyorin-u.ac.jp

Running title: Analysis of insulin granule motion in biphasic release

Abbreviations: ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; IAPP, islet amyloid polypeptide; KRB, Krebs Ringer Buffer.

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

2 SUMMARY To study insulin exocytosis by monitoring the single insulin secretory granule motion, evanescent wave microscopy was used to quantitatively analyze the final stage of insulin exocytosis with biphasic release. Green fluorescent protein (GFP)-tagged insulin transfected in MIN6 cells was packed in insulin secretory granules, which appeared to

preferentially dock to the plasma membrane. Upon fusion evoked by secretagogues,
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evanescent wave microscopy revealed that fluorescence of GFP-tagged insulin brightened, spread (within 300 ms), and then vanished. Under KCl stimulation, which represents 1st phase of release, the successive fusion events were seen mostly from previously docked granules for the first 1 min, followed by the recruitment of new granules to the plasmalemmal docking sites. Stimulation with glucose, in contrast, caused the fusion events from previously docked granules for the first 120 s, thereafter a continuous fusion (2nd phase of release) was observed over 10 min mostly from newly recruited granules that progressively accumulated on the plasma membrane. Thus, our data revealed the distinct behavior of the insulin granule motion during 1st and 2nd phase of release.

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INTRODUCTION Insulin is stored in large dense-core granules in pancreatic cells and is released by

exocytosis when blood glucose levels rise (1). We have previously demonstrated that SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptors) hypothesis (2) may be applicable to insulin exocytosis in pancreatic cells (3, 4), however, the dynamics of insulin granule exocytosis in live pancreatic cells is poorly understood.
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Imaging techniques are powerful tools for detecting vesicle exocytosis in live cells and have provided significant advances in understanding the mechanism of exocytosis. In previous studies, synaptic vesicles or granules were labeled with the weak base fluorescent dye acridine orange, which accumulates in acidic compartments in cells (5-7), though acridine orange labels not only secretory granules but also other acidic compartments in cells. To overcome the problem, we produced an expression vector encoding green fluorescent protein (GFP) fused to human preproinsulin. We found that the GFP-tagged insulin we produced here expressed in MIN6 cells can specifically label the insulin secretory granules. Therefore, we used this

probe combined with evanescent wave microscopy (also called total internal reflection microscopy) to acquire images of the docking and fusion of insulin granules. Evanescent wave microscopy selectively excites subcellular features close to (within ~ 100 nm) the plasma

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membrane at the cell–glass cover slip contact region (8). The advantage of this method over confocal laser microscopy is that only a thin layer (~ 100 nm) is illuminated, an excellent fluorescent signal-to-background is obtained, and photo damage is minimized. Because only granules close to the plasma membrane are visible, this method allows us to see with high resolution the single granules approaching, docking, and fusing with the plasma membrane. In the present study, we report the optical observation of exocytosis of insulin
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molecule itself stimulated by the physiological secretagogue using evanescent wave microscopy supplemented with the real-time images, thereby we reveal the distinct behavior of single insulin granule motion during 1st and 2nd phase of insulin release.

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EXPERIMENTAL PROCEDURES Plasmid construction — To generate a construct in which GFP is located at the C terminus of preproinsulin, the coding region of human preproinsulin cDNA (a gift from Dr. G. I. Bell, University of Chicago) was amplified by polymerase chain reaction using originally designed primers, cleaved by restriction enzymes, and subcloned into vector pEGFP-N1 (Clontech) encoding enhanced GFP (EGFP) under cytomegalovirus immediate-early gene promoter. The
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resulting product was confirmed by automated DNA sequencer (Amersham Pharmacia Biotech), Cell culture and transfection — MIN6 cells (a gift from Dr. J.-i. Miyazaki, Osaka University, Osaka, Japan) at passage 15–30 were cultured as previously described (4) on fibronectine (KOKEN)-coated glass chamber slides (8 wells: Lab-Tek slides, Nunc) for imaging with confocal microscopy and on high refractive index glass (Olympus) for imaging with evanescent wave microscopy. MIN6 cells were transfected with the expression vector using Effectene transfection reagent (Qiagen), according to the manufacturer’s protocol. All experiments were performed between 2 and 3 days after transfection. Immunocytochemistry — Transfected cells were fixed, made permeable with 2% paraformaldehyde/0.1% Triton X-100, and processed for immunocytochemistry as described

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previously (4). The intrinsic fluorescence of GFP was maintained in this condition. Islet amyloid polypeptide (IAPP) was probed with anti-IAPP antibody (a gift from Dr. Donald F. Steiner, University of Chicago; diluted 1:50) and revealed with rhodamine-conjugated anti-rabbit IgG (DAKO; diluted 1:100). Immunofluorescence staining was detected with a Zeiss confocal laser-scanning microscope (LSM510). The emission signals were filtered with a 505–530-nm filter (GFP emission excited by argon laser at 488 nm) or with a long-pass
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590-nm filter (rhodamine emission excited by He/Ne laser at 543 nm). Evanescent wave microscopy — The Olympus total internal reflection system (9) was used with minor modifications. Light from an argon laser (488 nm, 3 mW) was introduced to an inverted microscope (IX70, Olympus) through a single mode fiber and two illumination lenses; the light was focused at the back focal plane of a high-aperture objective lens (Apo 100 OHR; NA 1.65, Olympus). The focal point was moved off-axis to the most peripheral position in the objective lens by simply shifting the position of the fiber. The transfected cells on the glass cover slip (refractive index n = 1.8 at 488 nm, Olympus) were mounted in an open chamber and incubated for 30 min at 37°C in Krebs Ringer Buffer (KRB) containing 110 mM NaCl, 4.4 mM KCl, 1.45 mM KH2PO4, 1.2 mM MgSO4, 2.3 mM calcium gluconate, 4.8 mM NaHCO3, 2.2 mM glucose, 10 mM HEPES (pH 7.4), and 0.3% bovine serum albumin. Cells were then

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transferred to the thermostat-controlled stage (37°C). Stimulation with KCl and glucose was achieved by addition of 100 mM KCl-KRB (NaCl was reduced to maintain the isotonicity of the solution) or 52 mM glucose-KRB into the chamber (final=50 mM KCl or 22 mM glucose). Diiodomethane sulfur immersion oil (n = 1.81 at 488 nm, Cargille Laboratories) was used to make contact between the objective lens and the cover slip. At a measured incidence angle of 61.3° with nglass = 1.8 and ncytosol = 1.38, a 488-nm beam had a calculated penetration depth of
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about 80 nm (8). Acquiring the images and analysis — Images were collected by a cooled charge-coupled-device camera (Micromax, MMX-512-BFT, Princeton Instruments; operated with Metamorph 4.5, Universal Imaging). Images were acquired every 300 ms. Most analyses, including tracking (single projection of difference images) and area calculations, were performed using Metamorph software. To analyze the data, fusion events were manually selected and the average fluorescence intensity of individual granules in a 1-µm 1-µm square

placed over the granule center was calculated. The number of fusion events was manually counted while looping 1000–2400 frame time-lapses. Sequences were exported as single TIFF files and further processed using Adobe Photoshop 6.0 or they were converted into Quick Time movies.

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Endogenous insulin release assay — After MIN6 cells were plated on 24-multiwell plates, cells were preincubated for 30 min in 2.2 mM glucose and then challenged with 2.2 mM glucose, 22 mM glucose, or 50 mM KCl. The media were collected at 1- or 2-min intervals. At the end of the stimulation period, the cells were disrupted by sonication and aliquots of media and cell extracts were analyzed by enzyme-linked immunosorbent assay (ELISA) as described previously (4). Released insulin is expressed as a percentage of total cellular content per
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minute.

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RESULTS and DISCUSSION Localization of GFP-tagged insulin in MIN6 cells — Studies were carried out on the MIN6 cell line which responds to the physiological range of glucose (10, 11) with biphasic insulin release (see Fig. 3E). Firstly, in order to examine whether GFP-tagged insulin transiently expressed in MIN6 cells was localized to insulin secretory granules, the localization of the intrinsic fluorescence of GFP was compared with that of IAPP, a marker for
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insulin-containing dense-core secretory granules (12), in fixed MIN6 cells. The intense punctuate fluorescence of GFP was localized to > 94% of IAPP-containing granules stained with anti-IAPP antibodies, as detected by immunohistochemistry (Fig. 1). These results indicate that most of the GFP-tagged insulin expressed in MIN6 cells is correctly sorted to insulin secretory granules.

Fusion events induced by KCl-stimulation originate from previously docked granules —
We have then used evanescent wave microscopy to monitor the real-time docking-and-fusion process of single granules labeled with GFP-tagged insulin near the plasma membrane. Figure 2A shows a region in which GFP-tagged insulin transfected in MIN6 cells was adhered to a cover slip. Granules just beneath the plasma membrane (calculated distance from the cover slip is 80 nm) were visualized as spots of variable intensities. The average diameter is 0.39 ± 0.05

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µm (n = 4 cells), which is similar to that observed using electron microscopy (13), indicating that each spot shows the single vesicle. Under the unstimulated condition (2.2 mM glucose), many granules appeared to dock to the plasma membrane without fusion (see movie 1 in Supplementary data). Stimulation with KCl (50 mM), which represents 1st phase of insulin release (14), demonstrated the dramatic change in the dynamics of granule motion, in particular the fusion from previously docked
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granules. The fluorescence intensity of granules (boxed regions in Fig. 2A) transiently increased (observed as a bright flash in movie 1), spread, and then immediately disappeared (see movie 1). Figure 2C shows sequential images (1 µm 1 µm, 300-ms intervals) of the

single granules observed during KCl stimulation, where red and green boxes correspond to the same colored regions shown in Figure 2A. Note that fluorescent spots suddenly brightened and finally spread (within 300 ms) as GFP-tagged insulin diffused laterally on the plasma membrane. The spread of fluorescence in the single granules indicates exocytosis through fusion, as it represents the release of material from granules (15). The change of the fluorescence intensity of the fusing granule was relatively rapid: fluorescence transiently increased within about 900~1200 ms and vanished within about 300 ms (Fig. 2D), which is comparable to that observed in adrenal chromaffin cells (16).

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It is noteworthy that most fusion (90% of total fused granules) evoked by KCl stimulation occurred in the previously docked granules (red column in Fig. 2B, and red boxes 1 and 2 in Fig. 2C), with only some of the fusion occurring in newly recruited docked granules (green column in Fig. 2B, and green box 3 in Fig. 2C), whereas there was no difference in the kinetics of fusion between both types of docked granules (Fig. 2D). Fusion events evoked by KCl stimulation were almost always seen during the first 60 s (Fig. 2B), similar to the typical
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pattern of 1st phase of insulin release, which was consistent with the data of endogenous insulin release measured by ELISA (Fig. 2E). Daniel et al. have previously demonstrated by biochemical approach that KCl stimulation immediately evoked the fusion of docked granules (14). In the present study, we not only confirmed their results by visualizing technique, but also directly identified that previously docked granules are responsible for 1st phase of insulin release. Two types of granules are associated with glucose-stimulated biphasic insulin release — We then examined the dynamics of granule motion stimulated by the fuel secretagogue glucose. Glucose (22 mM) stimulation caused fluorescence intensity to increase, spread, and vanish (Fig. 3A; see also movie 2 in Supplementary data), as we observed under KCl stimulation. It is interesting that oscillation of fluorescent intensity in docked granules was observed during

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glucose stimulation, which was not marked by KCl stimulation (see movie 1). This granule oscillation may be associated to the repetitive firing of Ca2+ induced by glucose metabolism, which usually correlates with oscillating insulin secretion (17-19). Although the relationship between oscillating Ca2+ and granule oscillation is unknown at present, the phenomenon of granule oscillation is the novel finding which is induced by glucose stimulation. It is quite intriguing that the glucose-induced fusion for the first 120 s occurred only
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from previously docked granules (red column in Fig. 3B, and red boxes 1 and 2 in Fig. 3C), but after 360 s fusion events from newly recruited docked granules markedly increased (green column in Fig. 3B, and green box 3 in Fig. 3C). As judged by the data of insulin release as measured by ELISA over the same time-course (Fig. 3E), fusion events after at least 360 s represent the 2nd phase of insulin release. It has been suggested that 2nd phase of release is due to granules that are translocated from a reserve pool to plasma membrane (14, 20), however, there has not been any reports to prove it. Here, our data with visualizing technique could clearly depict that fusion events during the 2nd phase of release occur mainly from newly recruited granules. On the other hand, fusion event for the first 120 s, which represents 1st phase of release, occurred mostly from previously docked granules as observed in KCl-stimulation. Thus, on the basis of analysis of single granule motion, we like to propose a

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theoretical hypothesis, two-compartmental model for the cell, which was originally proposed by Grodsky et al. (21, 22) and recently by Proks et al. (23). In the present study, fusing granules seem to be fully merged with plasma membrane, and no hemifusion was detected. Because insulin is stored in crystalline form (13), release of GFP-tagged insulin granules may require the expanded fusion pore (i.e., full fusion). Indeed, the rate of increase of fluorescence intensity in the study of INS1 cells using acridine orange
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was much faster (7) (33–100 ms; as well as that observed in synaptic vesicles; see Zenisek et al. (15)) than the rate observed in this study (900~1200 ms). Thus, it appears that the full fusion of insulin secretory granules predominantly occurs in pancreatic stimulation. Behavior of insulin granules near plasma membrane — Finally, we analyzed the insulin cells during physiological

granule motion in detail. Analysis of the behavior of single insulin granules on each sequential images allowed us to divide the docked granules into three types: the granules
fused, retreated (paused and then moved back inside the cell without fusing), or stayed in the place. By counting the individual granules of these three types on each sequential image (328 and 384 granules, stimulated by KCl or glucose stimulation, respectively), we could track the behavior of docked granules. Under KCl stimulation, the number of previously docked

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granules continuously decreased due to both fusion and retreat up to 150 s after KCl stimulation (red line in Fig. 4A), conversely, the number of newly recruited docked granules started to increase immediately to 150 s after KCl stimulation (green line in Fig. 4A). As a result, the total number of docked granules (previously docked granules + newly recruited docked granules) (black line in Fig. 4A) was kept at the same level before and after KCl stimulation (see also superimposed images in Fig. 4A).
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On the other hand, during glucose (22 mM) stimulation, the number of previously docked granules slowly decreased due to fusion and retreat (red line in Fig. 4B), whereas the number of newly recruited docked granules rapidly and progressively increased from 20 s after stimulation (green line in Fig. 4B). Subsequently, the total number of docked granules increased up to approximately 140% of the initial number of docked granules during the glucose stimulation (black line; see also superimposed images in Fig. 4B). The activation of recruitment of new granules to docking sites may contribute to refilling of the docked granule pool (ready releasable pool) during the 2nd phase of glucose-stimulated insulin release, and the rate of recruitment of granules from the reserve pool to the docked pool exceeds the rate of exocytosis. Thus, these findings also coincide with a two-compartmental model for the cell.

In conclusion, by combining the highly sensitive evanescent wave microscopy and the

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strongly fluorescent GFP-labeled granule system, we have clearly shown the motion of single insulin secretory granules in the process of docking and fusion during physiological stimulation. Our data confirm a two-compartmental model for the cell, further, this method

will be useful for clarifying the molecular steps, preceding glucose-induced insulin exocytosis in pancreatic cells.

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ACKNOWLEDGMENTS We thank Olympus and Nippon Roper for help in setting up the evanescent wave microscopy. This study was supported by a Grant-in-Aid for Scientific Research (C) (11670148) from the Japanese Ministry of Education, Science and Culture; a grant from the Research for the Future Program (JSPS-RFTF97I00201) from the Japan Society for the Promotion of Science; a grant from the Research Fund of the Mitsukoshi Health and Welfare
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Foundation 2000.

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Ishihara, H., Asano, T., Tsukuda, K., Katagiri, H., Inukai, K., Anai, M., Kikuchi, M., Yazaki, Y., Miyazaki, J.-I., and Oka, Y. (1993) Diabetologia, 36, 1139-1145

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554-578 22. Grodsky, G.M. (2000) in Diabetes Mellitus, (LeRoith, D., Taylor, S.I. and Olefsky, J.M., eds) 2nd Ed., pp. 2-11, Lippincott Williams and Wilkins, PA 23. Proks, P., Eliasson, L., Ammala, C., Rorsman, P., Ashcroft, F.M. (1996) J. Physiol. 496, 255-264

19 FIGURE LEGENDS Fig. 1. Colocalization of GFP-tagged insulin and IAPP in MIN6 cells. After MIN6 cells were transfected with expression vector encoding GFP-tagged insulin, they were fixed with paraformaldehyde, and immunostained with anti-IAPP antibody as described in Materials and Methods. There was significant overlap (yellow) of the GFP-insulin (green) and IAPP (red) fluorescence intensity. Scale bar: 10 µm. Fig. 2. Evanescent wave images and analysis of GFP-labeled insulin granule motion during KCl (50 mM) stimulation. The fluorescence of GFP-labeled insulin granules was imaged close to the plasma membrane, and images were acquired every 300 ms (see movie 1 in Supplementary data; time 0 indicates the addition of KCl [50 mM]). (A) Image observed before stimulation. Red and green boxes indicate that the granules, which will be fused with the plasma membrane, originated from previously docked granules (red) and newly recruited docked granules (green), respectively. (B) Histogram of the number of fusion events (n = 3 cells) at 10-s intervals after KCl stimulation. Red column is from previously docked granules and green column from newly recruited docked granules. (C) Sequential images (1 µm 1 µm,

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300-ms intervals) of single granules (the color and number correspond to boxes 1, 2, 3 in Fig. 2A) observed after stimulation. (D) Changes of the fluorescence intensity in the respective

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boxed regions were plotted with time. The average of fluorescence intensity prior to fusion was taken as 100%. (E) Time-course of endogenous insulin release exposed to KCl solution measured by insulin ELISA. Data are the mean ± SEM of eight determinations from individual wells. Fig. 3. Image and analysis of GFP-labeled insulin granules during glucose (22 mM) stimulation with evanescent wave microscopy. Images were acquired every 300 ms over 10
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min (see movie 2 in Supplementary data; glucose was added at time 0). (A) Image observed before stimulation. Red and green boxes indicate the granules to be fused from previously docked granules and newly recruited docked granules, respectively. (B) Histogram of the number of fusion events (n = 4 cells) at 1-min intervals after glucose stimulation. (C) Sequential images (1 µm 1 µm, 300-ms intervals) of single granules (boxes 1, 2, 3 in A)

observed after stimulation. (D) Time-course of the fluorescence change of the respective boxed regions. The average of fluorescence intensity prior to fusion was taken as 100%. (E) Time-course of endogenous insulin release exposed to glucose solution measured by insulin ELISA. Data are the mean ± SEM of eight determinations from individual wells. Fig. 4. Time-dependent change of the number of docked granules during (A) KCl (50 mM ) and (B) glucose stimulation (22 mM). The number of previously docked granules (red

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line) and that of newly recruited granules (green line) during stimulation were determined by counting of granules on each sequential image (n=3 cells each). Black line shows the total number of docked granules, which corresponds to the sum of red and green lines in the time-course. Time 0 indicates the addition of secretagogues. The number of previously docked granules at time 0 was taken as 100 % (61, 81 and 52 granules, respectively in each 3 cells in A; 68, 45 and 63 granules, respectively in each 3 cells in B). The typical images before and after
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stimulation were superimposed. Data are the mean ± SEM.

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Supplemental Information Supplemental movie 1 Image of GFP-labeled insulin granule motion during KCl (50 mM)-stimulation with evanescent microscopy. Images acquired every 300 ms. Timestamp (minute: second. millisecond) and calibration bar (5 µm) were overlaid on the movie. Stimulation started at 0 s. Same granules as in Fig. 2A.
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Supplemental movie 2 Image of GFP-labeled insulin granule motion during glucose (22 mM)-stimulation with evanescent microscopy. Images acquired every 300 ms. Timestamp (minute: second. millisecond) and calibration bar (5 µm) were overlaid on the movie. Stimulation started at 0 s. Same granules as in Fig. 3A. This movie was shown from 1 min to 4 min after the stimulation.

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