Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini

Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca2+-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca2+ compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis. Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca2+-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca2+ compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis. Reorganization of the actin cytoskeleton plays an important role in a variety of cellular activities that range from cell migration and platelet aggregation to dendritic spine motility (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3875) Google Scholar, 2Halpain S. Trends Neurosci. 2000; 23: 141-146Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Reinhard M. Jarchau T. Walter U. Trend. Biochem. Sci. 2001; 26: 243-249Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Under resting conditions, the apical membrane of acinar cells in exocrine glands is coated with F-actin. Although this F-actin coat is thought to constitute a barrier to exocytosis (4Muallem S. Kwiatkowska K. Xu X. Yin H.L. J. Cell Biol. 1995; 128: 589-598Crossref PubMed Scopus (391) Google Scholar, 5Orci L. Gabbay K.H. Malaisse W.J. Science. 1972; 175: 1128-1130Crossref PubMed Scopus (283) Google Scholar, 6Trifaro J.M. Rodriguez del Castillo A. Vitale M.L. Mol. Neurobiol. 1992; 6: 339-358Crossref PubMed Scopus (71) Google Scholar, 7Valentijn K. Valentijn J.A. Jamieson J.D. Biochem. Biophys. Res. Commun. 1999; 266: 652-661Crossref PubMed Scopus (77) Google Scholar), secretory granules undergo exocytosis selectively at the apical membrane (8Ichikawa A. J. Cell Biol. 1965; 24: 369-385Crossref PubMed Scopus (149) Google Scholar, 9Palade G. Science. 1975; 189: 347-358Crossref PubMed Scopus (2354) Google Scholar, 10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar). Redistribution of F-actin is induced during intense secretory activity (4Muallem S. Kwiatkowska K. Xu X. Yin H.L. J. Cell Biol. 1995; 128: 589-598Crossref PubMed Scopus (391) Google Scholar, 11Schafer C. Ross S.E. Bragado M.J. Groblewski G.E. Ernst S.A. Williams J.A. J. Biol. 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Physiol. 2002; 282: G450-G460Crossref PubMed Scopus (41) Google Scholar), which is characterized at the cellular level by the appearance of vacuoles in, and disruption of the polarized secretion of digestive enzymes from, pancreatic acinar cells (17Kloppel G. Bradley III, E.L. Acute Pancreatitis: Diagnosis and Therapy. Raven, New York1994: 35-45Google Scholar). In animal models of this condition, protracted agonist stimulation results in both actin reorganization and vacuole formation (18Lampel M. Kern H.F. Virchows Arch. A Pathol. Anat. Histol. 1977; 373: 97-117Crossref PubMed Scopus (587) Google Scholar, 19Raraty M. Ward J. Erdemli G. Vaillant C. Neoptolemos J.P. Sutton R. Petersen O.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13126-13131Crossref PubMed Scopus (283) Google Scholar, 20Steer M.L. Baillieres Best Pract. Res. Clin. Gastroenterol. 1999; 13: 213-225Crossref PubMed Scopus (68) Google Scholar). However, the relation between vacuole formation and F-actin reorganization has not been clarified. Elucidation of the dynamic control of F-actin distribution during exocytosis in exocrine cells has not been possible by classical confocal microscopy because this technique lacks the tissue depth penetration necessary to visualize the fine organization of the apical plasma membrane within intact acini (10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar). In contrast, two-photon excitation microscopy has the ability to penetrate deep into tissues (10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar, 21Denk W. Strickler J.H. Webb W.W. Science. 1990; 248: 73-76Crossref PubMed Scopus (8397) Google Scholar) and allows simultaneous multicolor imaging with various combinations of fluorescent tracers (10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar, 49Takahashi N. Kishimoto T. Nemoto T. Kadowaki T. Kasai H. Science. 2002; 297: 1349-1352Crossref PubMed Scopus (228) Google Scholar). Taking advantage of these attributes of two-photon excitation microscopy, we have now investigated actin dynamics associated with physiological and pathological exocytosis in pancreatic acinar cells. Preparation of Mouse Pancreatic Acini—Clusters of acini were isolated from the pancreas of 5–7-week-old mice by a brief (4-min) digestion with collagenase (1 mg ml–1; Wako, Osaka, Japan) followed by gentle trituration. The acini were dispersed in a small chamber and superfused (1 ml min–1) with a solution (solution A) containing 150 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES-NaOH (pH 7.3), and 10 mm glucose. For the experiment shown in Fig. 3, G and H, dispersed acini were cultured under an atmosphere of 5% CO2 at 37 °C in Waymouth solution (Sigma) supplemented with penicillin (100 units ml–1; Invitrogen), streptomycin (0.1 mg ml–1; Invitrogen), 0.5 mm 3-isobutyl-1-methylxanthine (Sigma), soybean trypsin inhibitor (0.2 mg ml–1; Sigma), and 2.5% fetal bovine serum (Invitrogen). The culture dishes were coated thickly with a collagen gel (MatriGel; BD Bio-sciences) to preserve cell polarity. All experiments done were approved by the Institutional Animal Care and Use Committee at Okazaki National Research Institutes. Two-photon Excitation Imaging—For the visualization of exocytosis, pancreatic acini were immersed in solution A containing a fluid-phase polar tracer, either 0.5–1 mm sulforhodamine B (SRB 1The abbreviations used are: SRB, sulforhodamine B; CCK, cholecystokinin; NP-EGTA, o-nitrophenyl EGTA; LatA, latrunculin-A. ; or 0.5 mm Osaka, Japan) was in solution A containing and to cells a All experiments were at For the visualization of F-actin or acini were with in for with in for 10 with and with or in For the simultaneous imaging of both F-actin and acini were immersed in a solution containing and for and (Sigma) were in and into solution A. Two-photon excitation imaging of pancreatic acinar cells was (10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar). In cells were with an Japan) and a with a A with an of was to the of the the of the was for by a of The at the was and the excitation was for SRB, and and for and The of or was at that of or was at For the experiments with and the was at and was by Japan) in the and were The of at the apical membrane was by in the membrane region by imaging Fig. The were and with or of the and either of the or A and and to the from a of the by by the in the of were with coating of zymogen a two-photon under resting of a pancreatic with the of the the of in the region in A during stimulation with two-photon imaging of of the shown in which was to a solution containing 0.5 mm and with were at the to the in B. 1 for a to these and two-photon imaging of of F-actin in cells and in cells with for of at 10 with an of 1 and between and from the surface of the and confocal of F-actin in the acini shown in and The were with the and used in and but was with a at of with a at For the simultaneous imaging of and exocytosis, acini were for with in solution A. The were from the of (10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar). A was used an for the caged-Ca2+ compound o-nitrophenyl The was to a The from the was by a and an A in was triggered by photolysis of NP-EGTA, which was by of acini with 10 in solution A for F-actin of excitation imaging allows visualization of zymogen granule exocytosis in pancreatic acini A and that have been immersed in a solution containing the polar SRB, that the rapidly into granules that have with the plasma membrane (10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar). Two-photon imaging also allows simultaneous of the (100 induced in which in triggered exocytosis of zymogen granules at the apical membrane of acinar cells in a Granules within of the cell thus exocytosis by with the of granules that with the apical in the formation of of structures (10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar). The of granules were for an of Fig. the of this of the we investigated F-actin organization by of with which to F-actin. In resting cells, a coat of F-actin was at the apical membrane but not secretory granules J. Cell Biol. Google Scholar). stimulation with CCK, F-actin also coated secretory granules to the apical membrane In to the two-photon confocal microscopy was not to F-actin coating of granules and a of the intense by zymogen granules (10Nemoto T. Kimura R. Ito K. Tachikawa A. Miyashita Y. Iino M. Kasai H. Nat. 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Stimulation of exoenzyme acini with also induced the formation of which were not coated with F-actin The Rho-dependent F-actin coating of granules undergoing exocytosis thus to necessary to vacuole formation. that actin occurred at the apical plasma The of F-actin at the apical membrane was thus reduced in acini with 10 for agonist stimulation cells, with that in control acini In contrast, of acini for 2 with exoenzyme C3 did not in a in F-actin coating at the apical membrane The of at the apical membrane in acini did not from that in control acini of cultured acini to exoenzyme C3 for the F-actin coat at the apical membrane with that in control cultured cells the of in the by with 3, G and Rho-dependent actin thus at the apical membrane but at a and it in the granule membrane exocytosis of investigated the formation of vacuoles by imaging of acini immersed in a solution containing formation was in acini with for to 10 In contrast, vacuoles with a of were by the of granules within stimulation with in of acini that been with for The vacuoles of and at and vacuole formation and we structures with a of or The of vacuoles was and the of and The were and in control and at the of stimulation with with of actin polymerization, J.A. 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The of is with the of vacuoles in cells because these did not the of exocytosis, which was and in control and and because the granules that induced exocytosis did not into the plasma membrane 1 and In of the granules that were in exocytosis were also and to vacuole formation. stimulation of acini with a high of triggered the of vacuoles the was at stimulation and the of induced by stimulation with 10 was reduced with that of by stimulation with agonist A in the of vacuole formation was under conditions, with the of vacuoles at stimulation with that in cells. that stimulation of acini with a high of results in a of by of the F-actin coating of granule membranes vacuole formation was in the acini by photolysis of a caged-Ca2+ compound in which in for The of vacuole was at the that vacuole formation not from in but of by F-actin of the F-actin coat of the apical plasma membrane did not by exocytosis, that did not exocytosis not the number of exocytic in acini was not by with A and The number of exocytic at the apical membrane or Fig. during stimulation with (100 for 10 was thus not by of acini with for the of F-actin at the apical membrane was reduced by the number of or exocytic or exocytosis, did not between acini that been with and that not the dynamic F-actin coating of granule membranes 3, A and results that the of the of granules was not either by of the F-actin coat of the apical membrane or by prevention of the F-actin coating of granule the of exocytosis, we the latency to exocytosis the between the of the in and the formation of the and we have the latency to or exocytic the between the The of exocytosis induced by (100 was by with reduced by was that with the in W. 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