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The Actin-Myosin Cytoskeleton Mediates Reversible Agonist-Induced Membrane Blebbing

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The Actin-Myosin Cytoskeleton Mediates Reversible Agonist-Induced Membrane Blebbing Journal of Cell Science 111, 2911-2922 (1998) 2911 Printed in Great Britain © The Company of Biologists Limited 1998 JCS7297 The actin-myosin cytoskeleton mediates reversible agonist-induced membrane blebbing Rochelle R. Torgerson and Mark A. McNiven* Department of Biochemistry and Molecular Biology and The Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, MN 55905, USA *Author for correspondence (e-mail: [email protected]) Accepted 19 July; published on WWW 9 September 1998 SUMMARY Suprastimulation of pancreatic acinar cells with specific 30 minutes of suprastimulation, both basolateral actin and agonists inhibits zymogen secretion and induces the myosin II gradually increase to form a ring centered at the formation of large basolateral blebs. Currently the necks of the blebs. Immunocytochemical and biochemical molecular mechanisms that mediate this dramatic studies with a phospho-specific antibody to the myosin morphologic response are undefined. Further, it is unclear regulatory light chain reveal an activation of myosin II in if blebbing represents a terminal or reversible event. Using suprastimulated acini that is completely absent in resting computer-enhanced video microscopy of living acini we cells. Studies using cytoskeletal antagonistic drugs indicate have found that these large blebs form rapidly (within 2-3 that bleb formation and motility require actin remodeling minutes) and exhibit ameboid undulations. They are concomitant with an activation of myosin II. This aberrant induced by small increases in agonist concentration and activation and reorganization of the actin-myosin require an energy-dependent phosphorylation event. cytoskeleton is likely to have detrimental effects on acinar Remarkably, the blebs are rapidly absorbed when agonist cell function. Additionally, this mechanism of bleb levels are reduced, indicating that blebbing is a reversible formation may be conserved among other forms of response to a physiological stimulus, not a terminal event. physiological blebbing events. Morphological methods show that these dramatic changes in cell shape are accompanied by a marked reorganization of actin and myosin II at the basolateral domain. During Key words: Actin, Cytoskeleton, Myosin, Video microscopy INTRODUCTION However, despite the structural inhibition of morphological change, it is likely that the agonist-induced intracellular The secretagogue cholecystokinin and its octapeptide act on responses remain the same. pancreatic acinar cells through a G-protein-coupled receptor to In addition to this pathophysiological importance, increase intracellular calcium and elicit amylase secretion, cytoplasmic blebbing is a specialized form of cell protrusion characterized by a biphasic dose-response curve. Whereas that occurs physiologically in both healthy and dying cells. treating acinar cells with 10−10 M cholecystokinin octapeptide During early embryogenesis, blebs function in cell locomotion (CCK) (stimulation) induces maximal release of amylase, (Trinkaus, 1973). Blebs also form in cultured cells during cell increasing CCK concentrations further to 10−8 M CCK flattening, as seen during mitosis at the onset of cytokinesis (suprastimulation) causes a marked decrease in the percentage (Boss, 1955). Cytoplasmic blebbing is a characteristic of both of total amylase released. These suprastimulated acini undergo necrotic and apoptotic cell death (Kerr et al., 1972; Trump and aberrant basolateral blebbing (Adler et al., 1984; Burnham and Berezesky, 1995), possibly playing a role in cell-cell Williams, 1982; O’Konski and Pandol, 1990). This blebbing recognition in the latter case (Fadok et al., 1992). The phenomenon has been proposed to have clinical significance molecular mechanisms responsible for each of these different because animals infused with supramaximal doses of agonist types of blebbing have not been established, but lamellipodia develop an acute interstitial pancreatitis (Lampel and Kern, and filopodia, other forms of cell protrusions more extensively 1977). In this well-established in vivo model of pancreatitis, studied in the literature, have been found to be mediated by basolateral blebs do not form in response to suprastimulation actin-cytoskeletal dynamics (Mitchison and Cramer, 1996). (Savion and Selinger, 1978), most likely due to the integrity of Although bleb formation may be dependent on the actin the basal lamina, the confinement of cell packing, or the cytoskeleton, the mechanism would need to be unique as blebs restriction of surface membrane flow from the increased lack the actin meshwork that fills the other protrusions. number of cell-cell contacts (Tickle and Trinkaus, 1977). Several past studies have investigated, both in vitro and in 2912 R. R. Torgerson and M. A. McNiven vivo, the cytoskeletal changes which occur during Fumio Matsumura (Rutgers University, Piscataway, NJ). Secondary suprastimulation of pancreatic acini. Those studies which FITC-conjugated goat anti-rabbit antibodies were purchased from focused on the role of the actin cytoskeleton provided Cappel, Organnon Teknika Corp. (Durham, NC). contradictory results. Burnham and Williams (1982) studied Acini isolation the effects of high concentrations of secretagogue on the morphology of isolated pancreatic acini. They hypothesized Pancreatic acini were isolated by a modification of the methods of Jena et al. (1991) and Roettger et al. (1995). For each experiment, one that suprastimulated blebbing was mediated by a contractile 90-110 g male Sprague-Dawley rat was killed by decapitation. The process involving the microfilament system because blebbing pancreas was removed and placed in ice-cold Krebs Ringer Hepes could be blocked by cytochalasin B, an agent which disrupts Buffer (KRH) (25 mM Hepes, 100 mM NaCl, 5 mM KCl, 1 mM microfilaments. Conversely, Jungermann et al. (1995) utilized KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 1.2 mM MgSO4, 2.5 mM an in vivo model of suprastimulation of the rat exocrine glucose, essential and nonessential amino acids, 0.2% bovine albumin pancreas and found that within 30 minutes of suprastimulation, fraction V, and 0.01% soybean trypsin inhibitor, pH 7.4). Following actin filaments were disassembled, aggregated and degraded. injection of 5 ml of collagenase (40 units/ml in KRH), the pancreas Finally, others (Burnham and Williams, 1982; Savion and was minced, transferred to a 25 ml polypropylene Erlynmeyer flask, Selinger, 1978) have reported a disassembly of the apical actin oxygenated with 100% O2, and placed in a 22°C shaking water bath (120 cycles/minute). After 5 minutes, pancreatic tissue was matrix of suprastimulated acini. transferred to a 15 ml conical tube, oxygenated, shaken by hand for Thus, while these relevant studies have provided important 10 minutes, then filtered through 200 µm nylon mesh. Acini were information implicating the actin cytoskeleton in pelleted at 30 g for 3 minutes and washed 3 times with KRH. Acini suprastimulation-induced blebbing, the precise molecular to be used for microscopy were plated on glass coverslips in protein- mechanisms responsible for these substantial, agonist-induced free KRH (Cornell-Bell et al., 1993). After 1-2 minutes, protein-free morphological changes remain undefined. Further, it is unclear medium was replaced by complete KRH. Acini were used if the blebbing process represents a terminal or reversible immediately after isolation. alteration in cell shape. To address these issues, we have Scanning electron microscopy combined computer-enhanced video microscopy, immunofluorescence confocal microscopy, biochemical Acini were isolated, plated on coverslips as above, incubated in KRH ± CCK for 30 minutes at 37°C, and fixed in 3% gluteraldehyde in a methods and cytoskeletal inhibitory drugs to perform 37°C Pipes buffer (0.1 M Pipes, 3 mM EGTA, 3 mM MgSO4, pH mechanistic studies on dispersed rat pancreatic acini. As 6.95) for at least 1 hour. Acini were then rinsed for 20 minutes in two observed by others, suprastimulation induces dramatic changes of 0.1 M phosphate buffer, pH 7.2, dehydrated in progressive basolateral blebbing. Most surprisingly, we have found that the concentrations of ethanol, mounted on specimen stubs, and coated membrane blebs undergo rapid ameboid undulations and are with Au/Pd. Micrographs were taken on a JOEL 6400 operating at 15 readily absorbed upon reduction of the agonist concentration. kV. Blebbing is accompanied by a marked reorganization of cytoplasmic actin as well as a reorganization and Video microscopy phosphorylation of myosin II. Finally, the formation of these For microscopic observation of living specimens, dissociated blebs is inhibited by cytochalasin D (cyt D), the myosin pancreatic acini on coverslips were mounted in chambers in KRH and placed on a heated microscope stage (37°C). A perfusion apparatus ATPase inhibitor butanedione monoxime (BDM) (20 mM) and µ allowed for addition and changing of agonists and drugs. Time-lapse the myosin light chain kinase inhibitor ML-9 (100 M), computer-enhanced video microscopy was performed with a Zeiss suggesting that actin and myosin II dynamics are required. This Axiovert 35 (Carl Zeiss Inc., Thornwood, NY) equipped with a study indicates that over-stimulation of the CCK G-protein- Hamamatsu XC-77 CCD camera and a C2400-68 intensifier coupled receptor signaling cascade in pancreatic acini induces (Hamamatsu Phototonics K.K., Hamamatsu-city, Japan). Images were a supraphysiological stimulus, resulting in dramatic and enhanced and captured via
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