Journal of Cell Science 111, 2911-2922 (1998) 2911 Printed in Great Britain © The Company of Biologists Limited 1998 JCS7297

The -myosin 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 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 (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 Image-1 software (Universal Imaging aberrant changes in actin-myosin distribution and cell shape. Corp., West Chester, PA) and recorded with a time-lapse sVHS The implications of these processes with regard to acinar cell videotape recorder (Panasonic, Secaucus, NJ). function and other forms of cytoplasmic blebbing are Immunofluorescence discussed. For studies designed to test the distribution of filamentous actin, isolated acini plated on glass coverslips were fixed for 10 minutes in 2% formaldehyde and 0.01% gluteraldehyde in a Pipes buffer. After MATERIALS AND METHODS rinsing with Dulbecco’s PBS (PBS), acini were permeabilized in 0.2% Triton X-100 in PBS for 10 minutes and rinsed in PBS again. Acini All chemicals and reagents were purchased from Sigma Chemical Co. were then incubated in blocking buffer (5% normal goat serum and (St Louis, MO), except the following: bovine albumin fraction V and 5% glycerol in PBS) for 60 minutes, followed by a 90-minute Hepes (Gibco BRL, Grand Island, NY); CCK (Kinevac incubation in rhodamine-labeled phalloidin. For studies designed to cholecystokinin octapeptide, Bracco Diagnostics, Princeton, NJ); test microtubule (Mt) and myosin II distribution, coverslips collagenase type I (Worthington Biochemical, Corp., Freehold, NJ); maintained at 37°C were dipped in Mt stabilizing buffer (MTSB) (100 2-deoxy-D-glucose (Aldrich Chemical Corp., Milwaukee, WI); mM Pipes, pH 6.95, 4 mM MgSO4, 1 mM EGTA, 10% (w/v) glycerol) formaldehyde and gluteraldehyde (Tousimis Research, Corp., and placed in permeabilization buffer (0.05% digitonin, 1 mM GTP Rockville, MD); Triton X-100 (Surfactant-Amps X-100, Pierce, in MTSB) for 2-4 minutes. Acini were fixed for 20 minutes in 2.5% Rockford, IL). Monoclonal anti-α tubulin antibodies were purchased formaldehyde, 0.075% gluteraldehyde and 1 mM GTP in a Pipes from Amersham Life Science Inc. (Cleveland, OH). Affinity-purified buffer, rinsed with PBS, and incubated in blocking buffer for 60 rabbit anti-human platelet myosin II antibodies were a kind gift from minutes. Acini were then incubated in monoclonal anti-α tubulin, Ira Herman (Tufts University, Boston, MA). Affinity-purified rabbit polyclonal anti-myosin II, or polyclonal anti-phospho-myosin anti-S19-phosphorylated MRLC antibodies were a generous gift from antibody for 60 minutes, rinsed repeatedly with PBS, and incubated Actin-myosin mediated reversible blebbing 2913 in FITC-labeled secondary antibody for 90 minutes. All coverslips RESULTS were rinsed extensively with PBS, dipped in water, and mounted on glass slides in SlowFade Light™ (Molecular Probes Inc., Eugene, Rapid and reversible changes in acinar cell shape OR). Pictures were taken with a Zeiss LSM-410 microscope. To define the changes in acinar cell shape during agonist Fluorescence quantitation treatment, acini were suprastimulated with CCK and examined Actin filament distribution was determined using a saturating using differential interference contrast (DIC) (Fig. 1A,B) and concentration of rhodamine-phalloidin, which preferentially binds scanning electron microscopy (Fig. 1A′,B′). DIC images of filamentous actin (see above). Images of resting and suprastimulated acini show that in the resting state (Fig. 1A) acinar cells had a acini used for quantitation were taken with a Zeiss LSM-410 pyramidal shape with a well-defined basolateral domain and a microscope using identical optical conditions. All attempts were made centrally located apical lumen surrounded by zymogen to use acini composed of 8-15 cells with the majority of their ductal granules. In the suprastimulated state (Fig. 1B) acini had large structures parallel to the coverslip so they could be seen in a single cytoplasmic blebs protruding from the basolateral domain. To optical section. Image-1 software was used to analyze images using resolve better these changes in cell shape, scanning electron two methods. First, boxes of a constant size (700 square pixels) were microscopy was performed. A resting acinus comprising 12- placed over the brightest region of the apical and basolateral domains, 15 well-rounded and easily defined cells is shown in Fig. 1A′. and the average intensity per pixel in each boxed region was ′ determined. Second, the entire apical and basolateral domains were Upon suprastimulation (Fig. 1B ), cytoplasmic blebs of various traced, and the average intensity per pixel in each traced region was sizes completely consumed the basolateral domain. Because determined. Three experiments were done, and 90 acini per condition the shape of suprastimulated acini was so complex, were used for each calculation. transmission electron microscopy was performed to define the relationship between the basolateral blebs and the main body SDS-PAGE and western blotting of the acinus (not shown). Images revealed large blebs with Acini were isolated as above, allowed to equilibrate to 37°C, wide necks of attachment extending from the basolateral incubated in KRH ± CCK for 5 minutes, and pelleted. Acini were then domain of suprastimulated acini. These blebs contained resuspended in buffer (20 mM Tris, pH 7.2, 300 mM sucrose, 2 mM mitochondria, significant amounts of endoplasmic reticulum, MgCl2, 5 mM EGTA) containing Complete™ protease inhibitor cocktail (Boehringer-Mannheim, Indianapolis, IN), sonicated, and occasionally nuclei. These transmission electron incubated in 1% SDS for 10 minutes at 4°C, and centrifuged for 10 micrographs demonstrated the continuity between the blebs minutes at 16,000 g. The protein concentrations of the post-nuclear and the main body of the acini and were consistent with many supernatents were determined using bicinchoninic acid, as per of the observations of Adler et al. (1984), Burnham and instructions from the manufacturer (Pierce, Rockford, IL). SDS- Williams (1982) and O’Konski and Pandol (1990). PAGE was conducted using a buffer system based on the method of To view the suprastimulated blebbing as it occurred and to Laemmli (1970). Western blotting was performed as described determine whether or not blebbing was a terminal or reversible previously (Henley and McNiven, 1996) except that 10% nonfat dry event, we used DIC, computer-enhanced video microscopy to milk was used as a blocking reagent. Densitometric quantitation was view individual acini as they were treated with CCK. The video done utilizing a Bio-Rad GS-700 imaging densitometer and Molecular photomicrograph series in Fig. 2A shows a 3-5 cell acinus Analyst™ software (Bio-Rad Laboratories, Hercules, CA). Each under resting conditions, which was suprastimulated with myosin light chain value was normalized to the corresponding heavy −8 chain value, and then suprastimulated values were normalized to 10 M CCK and then allowed to recover in stimulatory −10 resting values for each experiment. concentrations of CCK (10 M). In the resting state, the

Fig. 1. Dramatic changes in acinar cell shape are induced by a supramaximal concentration of a secretory agonist. Differential interference contrast (A and B) and scanning electron micrographs (A′ and B′) of acini resting or suprastimulated (10−8 M CCK) for 30 minutes. Resting acini (A and A′) had a clearly defined pyramidal shape with an apical lumen (A, arrow) surrounded by zymogen granules (A, arrowhead). In the suprastimulated state (B and B′), the basolateral domains were completely consumed by blebs which contained nuclei (N) and other organelles. 2914 R. R. Torgerson and M. A. McNiven

Fig. 2. Cytoplasmic blebs induced by high concentrations of CCK are fully reversible and display cytoplasmic streaming. (A) DIC, computer- enhanced video micrographs of a single acinus resting (0 M), suprastimulated (10−8 M), and then stimulated (10−10 M) sequentially with CCK. Blebbing occurred within 3 minutes of suprastimulation (arrowheads) and was fully reversed within 10 minutes upon reducing the concentration of CCK to stimulatory levels. (B) High magnification video reveals that blebs exhibited by suprastimulated acini displayed active shape changes with membrane undulation, organelle movement and cytoplasmic streaming. The asterisk indicates a bleb that is absorbed within 1 minute of returning to stimulatory conditions (see text). acinus had a centrally located lumen, apically clustered event due to a nonspecific toxic insult, we examined the zymogen granules, and a well-defined basolateral domain. requirements for agonist concentration, intracellular signals, Within only 2 minutes of suprastimulation, protrusions cellular energy levels and cell viability. To determine the appeared along the basolateral domain, which rapidly enlarged concentration of CCK necessary to cause these dramatic shape into well-formed blebs. Throughout the 12 minutes of changes, a dose-response curve was performed to quantitate the suprastimulation, additional blebs formed. Cytoplasmic amount of cell blebbing induced by increasing concentrations organelles can be resolved within some of the blebs, although of CCK (Fig. 3A). Whereas stimulatory concentrations of CCK many organelles appeared constrained by the main body of the (10−10 M) induced less than 10% of the acini to bleb, treatment cell as if tethered at the necks of the blebs. Strikingly, these with CCK at five times this concentration (5×10−10 M) caused cytoplasmic blebs were extremely dynamic, displaying active, blebbing in approximately 50% of the acini. Nearly 100% of non-Brownian motion. Fig. 2B is a higher magnification series the acini blebbed when treated with 5×10−9 M CCK. Thus, revealing movement of fully formed blebs from another acinus. modest increases in CCK caused large increases in the Over the course of 5 minutes, these large blebs displayed percentage of blebbed acini. Although it has been shown that ameboid movements and cytoplasmic streaming. Organelles suprastimulated blebbing can be blocked by the CCK-receptor were translocated along rapidly with the streaming cytosol. antagonist Loxiglumide (Wang et al., 1996), little is known Suprastimulated acini allowed to recover in reduced, about the more distal signaling requirements. To this end, acini stimulatory concentrations of CCK (or in the absence of CCK), were treated with the protein kinase inhibitor staurosporine rapidly regained their original shape. Within 1 minute of (100 nM) and suprastimulated (10−8 M) with CCK. After 30 returning to stimulatory concentrations of CCK, the acinus in minutes, the acini were fixed, and blebbing was quantitated. Fig. 2A absorbed one of the blebs (note asterisk). The Staurosporine was able to significantly reduce blebbing in remaining blebs reduced in size until the acinus reformed a suprastimulated acini to resting levels (Fig. 3B). Staurosporine well defined basolateral domain. Only 12 minutes were needed is able to block apoptotic blebbing (Mills et al., 1998), and its for a complete recovery. The finding that suprastimulated effect on acini may indicate that suprastimulated blebbing blebbing is reversible supports studies by Savion and Selinger requires a phosphorylation event. Furthermore, (1978), which showed that secretory potential can be suprastimulated blebbing, unlike necrotic blebbing, is an reestablished when suprastimulated pancreatic slices are energy-requiring process. Acini treated with sodium azide and returned to stimulatory concentrations of agonist. 2-deoxy-D-glucose to deplete cellular ATP did not bleb when To establish that supramaximal blebbing is a reversible suprastimulated (Fig. 3B). Acini maintained in an incubator response to a physiologic agonist as opposed to a terminal throughout the day until cell death became prominent were Actin-myosin mediated reversible blebbing 2915 slow to bleb or did not bleb (not shown). Additionally, cells minutes (Fig. 4C) a strong, continuous basolateral actin ring within an acinus that were exposed to mechanical manipulation had formed. As the cytoplasmic blebs increased in size, the with a micropipette did not bleb when suprastimulated, apical and basolateral actin appeared to constrict until the whereas unmanipulated cells within the same acinus did (not transformation was complete at 30 minutes (Fig. 4D). Little shown). The fact that blebbing requires freshly isolated and change in cell shape or actin distribution was seen beyond 30 unperturbed cells, is induced by small increases in agonist minutes, and no rhodamine-phalloidin staining was seen within concentration, and can be inhibited by depleting cellular ATP the blebs. Quantitative measurements (see Materials and or blocking CCK signaling, indicates that suprastimulated methods) of the apical and basolateral actin distribution after blebbing results from the over-stimulation of a physiological 30 minutes of suprastimulation showed that there was a 60% signaling cascade, as opposed to a unrelated toxic effect. increase in basolateral actin, which correlated with the appearance of the basolateral actin ring. Though the area of the Agonist-induced alterations in actin distribution region occupied by apical actin often decreased (Fig. 4A-D), Because the actin cytoskeleton is a well-known determinant of there was no change in the intensity per unit area of apical actin cell shape, we next investigated the effect of suprastimulation staining. on actin distribution using immunofluorescence confocal microscopy. To correlate changes in cell shape and actin, we Effects of actin cytoskeletal altering drugs on cell conducted a time-course study which entailed suprastimulating shape changes acini, then fixing and staining with rhodamine-phalloidin at 5- Changes in the distribution of filamentous actin in acini minute intervals (Fig. 4). In the resting state (Fig. 4A), correlated with agonist-induced shape changes. To determine filamentous actin was located apically outlining the lumen; if these cytoskeletal alterations were necessary to produce very little actin was found along the basolateral domain. As suprastimulated blebbing, we pretreated acini with cyt D, a blebs began to form after 5 minutes (Fig. 4B), the basolateral drug which caps the barbed ends of actin filaments. Following actin increased giving the acinus a striking, honeycomb a 15-minute pretreatment with cyt D, acini were appearance. Basolateral actin continued to increase, and by 15 suprastimulated in the presence of the drug for 30 minutes, and the percentage of blebbed acini was quantitated (Fig. 5F). Using 5 µg/ml of cyt D, which maximally reduced filamentous actin by immunofluorescence (not shown), we were able to block suprastimulated blebbing. Cyt D treatment reduced

Fig. 4. Actin reorganization accompanies cytoplasmic blebbing. Fig. 3. Basolateral blebs form in response to moderate over- Immunofluorescence confocal microscopy of acini suprastimulated stimulation of a physiological signaling cascade and require ATP. (10−8 M) with CCK for 0, 5, 15 or 30 minutes then fixed and stained (A) Blebbed acini were counted (double blind) after 30 minutes of with rhodamine-phalloidin for filamentous actin. (A) Prominent treatment with increasing concentrations of CCK. The percentage of apical actin surrounded the lumen in resting acini, whereas blebbed acini substantially increased with only a fivefold increase in basolateral actin (arrows) was minimal. (B) Within 5 minutes lateral the concentration of CCK above that of stimulation (10−10 M). n≥100 and basolateral actin (arrows) increased, and blebs began to form for each treatment. (B) Treating acini with staurosporine (100 nM) or (arrowheads). (C) By 15 minutes, a basolateral ring of actin formed 5 mM sodium azide/5 mM 2-deoxy-D-glucose (−ATP) reduced the (arrows). There were breaks in the ring centered at the necks of the percentage of blebbed acini to control levels (asterisks; P<0.0001). blebs (arrowheads). (D) From 15-30 minutes, the blebs (arrowheads) Results are the mean ± s.d. for three experiments; n=300 for each increased in size while the basolateral ring of actin (arrows) appeared treatment. to constrict. 2916 R. R. Torgerson and M. A. McNiven blebbing from nearly 100% in suprastimulated controls to continued presence of cyt D, because the removal of cyt D 15%, a level similar to that of resting cyt D-treated acini. These allowed the blebs to be absorbed within only 4 minutes (Fig. results support the findings of Burnham and Williams (1982) 5E). This recovery time is significantly shorter than the 10-15 and indicate that the accumulation of basolateral actin is minutes required for an acinus that has been returned to KRH necessary for bleb formation. medium without a pretreatment with cyt D (Fig. 3A). To determine whether filamentous actin is also involved in bleb motility and reversibility, we used DIC, computer- Microtubules protrude into cytoplasmic blebs enhanced video microscopy to view individual acini as they The microtubule (Mt) cytoskeleton, in addition to the actin were treated with or without CCK and cyt D. The video cytoskeleton, is known to play a role in the determination of photomicrograph series in Fig. 5A-E shows a 3-4 cell acinus cell shape. The presence of Mts within suprastimulated blebs resting in KRH medium that was first suprastimulated with was indicated by the linear, directed movements of cytoplasmic 10−8 M CCK, then treated with cyt D, first in the continued organelles imaged with DIC computer-enhanced video presence and then in the absence of CCK, and finally allowed microscopy. This suggested Mts might play a role in bleb to recover in KRH medium. In the resting state (Fig. 5A) the formation, motility and reversibility. We utilized confocal acinus had a well-defined basolateral domain. Upon immunofluorescence microscopy to determine the effect of suprastimulation, highly motile blebs exhibiting ameboid suprastimulation on the distribution of Mts (Fig. 6A,B). Acini undulation formed (Fig. 5B). Within only 2 minutes of the were suprastimulated for 30 minutes, fixed and stained for α- addition of cyt D (Fig. 5C), the blebs became static and tubulin and filamentous actin. In the resting state (Fig. 6A), Mts spherical, and the acinus maintained this shape even after the originated near the apical plasma membrane and extended out CCK had been removed. 20 minutes after the removal of CCK toward the basolateral domain (Marlowe et al., 1998). Upon (Fig. 5D), only one small bleb had been absorbed and the large suprastimulation (Fig. 6B), this distribution remained blebs had only slightly reduced in size. The failure of the unchanged; Mts extended from the constricted apical region acinus to regain its original shape was most likely due to the out through the basolateral ring of actin into the blebs. The

Fig. 5. Cyt D blocks CCK-induced bleb formation, motility and reversibility. (A-E) DIC, computer-enhanced video micrographs of a single acinus treated sequentially in the presence or absence of CCK and with or without Cyt D. Resting acini (A) maintained a well-defined basolateral domain. Within 3 minutes of suprastimulation with CCK (B) motile, basolateral blebs formed (arrowheads). Upon addition of Cyt D in the continued presence of CCK (C) blebs became static and spherical. 20 minutes after the removal of CCK, acini that remained in the presence of Cyt D (D) only absorbed small blebs and slightly reduced the size of large blebs (arrowheads). Remarkably, acini rapidly absorbed all blebs within 4 minutes (E) once Cyt D was removed. (F) Quantitation of cyt D effects on blebbing. Acini resting or suprastimulated (10−8 M) with CCK for 30 minutes in the presence or absence of cyt D were fixed, and blebbing was quantitated. Cyt D significantly reduced suprastimulated blebbing compared to control (asterisk; P<0.0001) to levels Cyt D seen in resting acini. Results are the mean ± s.d. for three 5 µg ml experiments; n=300 for each treatment. Actin-myosin mediated reversible blebbing 2917

intensity of Mt staining at the breaks in the basal actin ring indicated that Mts were densely packed in the necks of the blebs. To determine if the presence of Mts was necessary to produce suprastimulated blebbing, we utilized nocodazole to disassemble Mts and prevent the nucleation of new Mts. When used at 10 µg/ml, only short stubs of Mts remained at the lumenal border (Marlowe et al., 1998). Despite most Mts being disassembled, nearly 100% of suprastimulated acini actively blebbed (Fig. 6C). These blebs displayed ameboid movement and cytoplasmic streaming, but directed, linear movement of organelles was not exhibited (not shown). Interestingly, blebs which formed in the presence of nocodazole were reversible but required as much as twice as long as controls to be absorbed (not shown). Agonist-induced alterations in myosin II distribution and phosphorylation accompany changes in actin The dramatic ameboid undulations exhibited by suprastimulated acini are reminiscent of actin-myosin- mediated, cytoplasmic streaming events (Jansen and Taylor, 1993). To determine the effect of suprastimulation on the distribution of myosin II, we utilized confocal immunofluorescence microscopy of acini stained with an antibody to myosin II heavy chain (Fig. 7A,B). In the resting state (Fig. 7A), myosin II was located apically, surrounding the lumen. After 30 minutes of suprastimulation, most of the apical myosin II was redistributed to a basolateral ring. The progressive increase in basolateral myosin, which culminated in the formation of a basolateral ring centered at the necks of the blebs, paralleled the reorganization of actin (Fig. 4). Immunocytochemistry with a phospho-specific antibody was utilized to determine if myosin II was phosphorylated during suprastimulation. This antibody has been shown to be highly specific for myosin light chain kinase (MLCK)-phosphorylated Suprastimulated myosin regulatory light chain (MRLC) and has been used to examine with high spatial resolution the subcellular distribution of activated myosin during cell locomotion and mitosis (Matsumura et al., 1998). MLCK phosphorylates the regulatory light chain of myosin on serine 19, a reaction which stimulates the myosin ATPase and force generation in both smooth muscle and nonmuscle cells (Adelstein and Eisenberg, 1980; Itoh et al., 1989). When acini in the resting state were fixed and stained with this phospho-specific antibody (Fig. 8A), very low levels of a punctate cytoplasmic staining were observed. None of the apical myosin II surrounding the lumen (Fig. 7A) appeared to 1 µg/ml 10 µg/ml be phosphorylated. Upon suprastimulation (Fig. 8B), the level of phosphorylated myosin increased significantly, highlighting Fig. 6. Microtubules are not required for cytoplasmic blebbing. the prominent basolateral ring. Immunofluorescence confocal microscopy of acini resting or These morphological results were supported by quantitative suprastimulated (10−8 M) with CCK for 30 minutes then fixed and western blotting using the same phospho-specific antibody (Fig. stained with an antibody to α-tubulin (green) and rhodamine- 8C). Western blot analysis with an antibody to myosin heavy phalloidin for filamentous actin (red). In resting acini (A), Mts chain revealed a single band at 200 kDa, as expected. However, originated at the apical lumen (arrowhead) and extended through the the phospho-specific antibody recognized two proteins in the toward the basolateral domain (arrow). This distribution 20-25 kDa range. The lower 20 kDa band is probably remained unchanged in the suprastimulated state (B). Mts extended phosphorylated MRLC, but the identity of the upper band is from the lumen (arrowhead) beyond the basolateral ring of actin unknown. The two proteins do respond in a similar manner to (arrows) into the blebs (B). (C) Acini resting or suprastimulated (10−8 M) with CCK for 30 minutes in the presence or absence of suprastimulation by CCK, and thus may be isoforms of MRLC. nocodazole (Noc) were fixed, and blebbing was quantitated. However, the antibody to phosphorylated MRLC has been Nocodazole had no effect on suprastimulated blebbing compared to shown to be highly specific for the mono-phosphorylated control acini. Results are the mean ± s.d. for three experiments; n = form (Matsumura et al., 1998) so it is unlikely that the 300 for each treatment. bands represent different phospho-isoforms. Densitometric 2918 R. R. Torgerson and M. A. McNiven

Fig. 7. Myosin II reorganization parallels that of actin. Immunofluorescence confocal microscopy of acini either resting or suprastimulated (10−8 M) with CCK for 30 minutes then fixed and stained with an antibody to myosin II heavy chain. Myosin II in resting acini (A) mainly surrounded the apical lumen (arrowheads). In the suprastimulated state (B) myosin II formed a prominent basolateral ring at the necks of the blebs (arrows), whereas minimal myosin II remained in the apical region. quantitation of MRLC phosphorylation revealed that acini treated with 20 mM BDM in the continued presence of phosphorylation increased 5- to 8-fold within 5 minutes of 10−8 M CCK. Upon addition of BDM, the blebs became less suprastimulation. These results support the findings of Burnham motile and were absorbed back into the acinus (not shown). et al. (1988), who reported an 80% increase in myosin light These results indicated that the ATPase activity of myosin II chain phosphorylation in suprastimulated acini and also was necessary for the maintenance and motility of obtained an unidentified phosphorylated 21 kDa protein. suprastimulated blebs. Effects of myosin inhibitory drugs on cell shape changes DISCUSSION To test directly if myosin II activation is required for suprastimulated blebbing, we utilized two myosin inhibitory Suprastimulated blebbing of acinar cells is a drugs. The first, BDM, is a well-characterized inhibitor of reversible event induced by a physiological agonist myosin II ATPase activity that is able to inhibit postmitotic cell The observation that pancreatic acini bleb in response to spreading (Cramer and Mitchison, 1995) and growth cone suprastimulatory concentrations of agonist has been well motility (Ruchhoeft and Harris, 1997). The second drug, ML-9, documented (Adler et al., 1984; Burnham and Williams, 1982; is a MLCK-specific protein kinase inhibitor (Saitoh et al., 1987). O’Konski and Pandol, 1990). However, it was unclear whether To determine if myosin II function was necessary to produce this dramatic agonist-induced morphological alteration suprastimulated blebbing, acini were pretreated with BDM or represented a terminal or reversible event. Furthermore, the ML-9 for 15 minutes, suprastimulated in the continued presence molecular mechanisms supporting this morphological response of the drug for 30 minutes, and fixed for quantitation of blebbed were poorly understood. In this study we have utilized multiple acini (Fig. 8D). Both drugs effectively blocked suprastimulated experimental methods to better characterize the blebbing process blebbing in 85-90% of suprastimulated acini. For these and define the cytoskeletal systems responsible. Importantly, we quantitative studies, 50 mM BDM was used because drug provide the first look at suprastimulated blebbing in living acini activity needed to be maintained throughout pretreatment and and show that it is a reversible phenomenon. Within 2 minutes suprastimulation. However, during video microscopy we used a of suprastimulation with CCK, basolateral protrusions rapidly perfusion chamber, which allowed for periodic replenishing of expand to form large, undulating blebs, which completely the drug. In this experimental system, we found 20 mM BDM consume the basolateral domain and are quickly absorbed upon to be more than sufficient to block suprastimulated blebbing. agonist removal (Fig. 2). Further, we have observed that Immunocytochemistry on suprastimulated, ML-9-treated acini filamentous actin redistributes to the basolateral domain to form with an antibody to myosin II heavy chain or a phospho-specific a ring centered at the necks of the blebs (Fig. 4). This antibody showed that not only did ML-9 block blebbing, but it reorganization is necessary for dynamic blebbing as cyt D also significantly blocked the basolateral reorganization of inhibits bleb formation, movement and reversibility (Fig. 5). myosin (Fig. 8E). Although apical myosin became disorganized Finally we identify the molecular motor enzyme responsible for and sub-plasmalemmal myosin patches formed, these changes this response by demonstrating that myosin II redistributes in a were modest compared to those seen in suprastimulated acini not manner similar to actin, is dramatically phosphorylated (Figs 7, treated with ML-9 (Fig. 7B). Additionally, despite a punctate 8), and is required for blebbing. It is important to stress that cytoplasmic staining, the newly accumulated basolateral myosin suprastimulated blebbing is not a terminal event in response to patches were not phosphorylated (Fig. 8F). Thus, the results of a nonspecific toxic insult, but rather a reversible response to the our studies with myosin inhibitory drugs show that both myosin over-stimulation of a G-protein coupled signaling cascade by a II ATPase function and MRLC phosphorylation are necessary physiological agonist (Fig. 3). for bleb formation. To determine whether myosin II was also involved in bleb A role for actin and myosin in acinar cell motility and reversibility, we utilized DIC, computer-enhanced suprastimulated blebbing video microscopy to observe actively blebbing, suprastimulated Interactions between the actin-myosin cytoskeleton are known Actin-myosin mediated reversible blebbing 2919

Fig. 8. Suprastimulation induces a marked phosphorylation of MRLC which is required for cytoplasmic blebbing. Immunofluorescence confocal microscopy of acini either resting or suprastimulated (10−8 M) with CCK for 30 minutes then fixed and stained with a phospho-specific antibody to myosin II (A,B). Myosin II in resting acini (A), which mainly surrounded the apical lumen, was not phosphorylated (arrowheads). In the suprastimulated state (B) the basal ring of myosin was extensively phosphorylated (arrows). (C) Post-nuclear supernatants from acini resting or suprastimulated (10−8 M) with CCK for 5 minutes were analyzed by western blotting. An antibody to myosin heavy chain recognized a 200 kDa protein (MHC), and the phospho- specific antibody revealed two bands. The lower 20 kDa protein is probably phosphorylated myosin light chain (P- MLC), but the upper 23 kDa protein remains unidentified (U). Data from two experiments showed that suprastimulation induced a 5- to 8-fold increase in phosphorylated myosin light chain, respectively. (D) Acini resting or suprastimulated (10−8 M) with CCK for 30 minutes in the presence or absence of the myosin inhibitor BDM or the MLCK inhibitor ML-9 were fixed, and blebbing was quantitated. BDM and ML-9 significantly reduced suprastimulated blebbing compared to control (asterisk; P<0.0001). Results are the mean ± s.d. for three experiments; n=275 for each treatment. (E,F) Immunofluorescence confocal microscopy of acini pretreated with ML- 9 (100 µM), suprastimulated (10−8 M) with CCK for 30 minutes, then fixed and stained with an antibody to myosin II heavy chain (E) or a phospho-specific antibody to myosin II (F). Suprastimulated acini (E) displayed a decrease and disorganization of apical myosin (arrowheads) as well as an accumulation of sub-plasmalemmal patches (arrows). However, in the presence of ML-9 (F) neither the apical (arrowheads) nor basolateral myosin (arrows) were phosphorylated. to play an important role in the support of both regulated regulated secretion, cells are believed to modulate access of secretion and cell shape determination (Cramer et al., 1994; secretory granules to the plasma membrane through the Iida et al., 1997; Maciver, 1996; Mitchison and Cramer, 1996; controlled disassembly of the subcortical actin matrix Spudich, 1994; Stossel, 1993; Vitale et al., 1991). During (Muallem et al., 1995; Vitale et al., 1995). Additionally, 2920 R. R. Torgerson and M. A. McNiven phosphorylation of MRLC correlates with secretion in beta with fully formed motile blebs, the blebs become static. This cells of the pancreas and rat basophilic leukemia cells and may effect is most prominent with cyt D (Fig. 4), which causes indicate a role for myosin II in the translocation of secretory blebs to become spherical and non-motile within 2 minutes of granules or cell shape changes (Choi et al., 1994; Iida et al., addition. Bleb absorption seems to require the disassembly of 1997; Spudich, 1994). The cell shape changes that occur during the actin-myosin ring. As the contractile force weakens, the events such as cytokinesis (Fujiwara and Pollard, 1976; body of the acinus increases in size causing the blebs to shrink. Moores et al., 1996) and cell migration (Cramer et al., 1994; The myosin inhibitor BDM causes blebs to be absorbed even Mitchison and Cramer, 1996; Stossel, 1993) are mediated by in the continued presence of suprastimulatory concentrations actin-myosin interactions as well. of CCK. However, cyt D does not cause bleb absorption Past studies that have examined the role of actin dynamics (Fig. 4). This contradiction can be explained by in the pancreatic acinar cell during suprastimulation have immunofluorescence studies, which show that cyt D induces a produced seemingly conflicting results (see Introduction). disorganization of the actin within the basal ring without These apparent discrepancies may arise from differences in the causing a significant disassembly (not shown). These region of the cell being investigated. Burnham and Williams interpretations of the mechanisms of bleb motility and (1982) concentrated on the role of actin in morphological reversibility emphasize the role of actin and myosin II within changes and found that Cyt B inhibited blebbing, implicating the basolateral ring and are based on our immunofluorescence an active role for actin in the shape change. This would support studies, which show that filamentous actin and myosin II are our findings of actin-myosin redistribution and activation in the absent within the blebs. However, it is possible that short actin basolateral domain. Alternatively, other studies focused on the polymers and unconventional myosins are present and play a dynamics of the apical actin meshwork (Burnham and role in the ameboid undulations displayed by motile blebs and Williams, 1982; Jungermann et al., 1995; Savion and Selinger, bleb reversibility (Poucell-Hatton et al., 1997). 1978) and found that suprastimulation induced actin depolymerization or degredation. We found an apparent Suprastimulated blebbing compared to other forms shrinkage of the apical domain (Figs 4D, 6B), which may of blebbing support these findings. Therefore, the apparent discrepancy in Blebbing is a specialized form of cell protrusion that occurs the past findings can be resolved if the complexity of the actin during many cell events. Originally, blebbing was solely cytoskeleton is acknowledged. Though there is a prominent considered to be a pathological response to nonspecific toxic meshwork of actin filaments regulating access of zymogen insults which signaled the onset of necrotic cell death. The granules to the apical plasma membrane, there is also a concept of blebbing as an important physiological form of cortical, basolateral actin skeleton. It is possible that these two protrusion came when Trinkaus (1973) described bleb regions of actin respond in different ways to agonist treatment. formation and bleb-mediated locomotion in fundulus deep For instance, as the apical actin disassembles to allow zymogen cells during blastulation. Blebbing has also been implicated as granules to fuse with the plasma membrane during secretion a hallmark of the execution stage of (Kerr et al., (Muallem et al., 1995), the basolateral cytoskeleton may be 1972). Our results suggest a common mechanism of blebbing increasing to regulate plasma membrane tension or receptor shared by healthy suprastimulated acinar cells and these dynamics. Thus, disassembly in one region of the cell does not physiological forms of blebbing. preclude assembly in another. Indeed, the contrasts between classic necrotic cell blebbing We believe that suprastimulated blebbing is an aberrant cell and suprastimulated blebbing in the acinar cell are numerous shape change mediated by the over-stimulation of a and striking. Necrotic blebbing, which can occur in response physiological actin-myosin force-generating mechanism to toxic insults such as lipid peroxidation (VanWinkle et al., normally utilized to promote secretion. The cytoskeletal 1994), anoxia (Friedman and Haddad, 1993) and energy reorganization that accompanies suprastimulated blebbing is depletion (Friedman and Haddad, 1993; Johnson et al., 1994; an augmentation of changes seen in the stimulated state. Marcussen, 1996), produces non-motile, empty plasma During normal stimulation (10−10 M CCK), when acini secrete membrane blebs that frequently detach from the cell surface maximally, apical actin partially disassembles (Muallem et al., (Trump and Berezesky, 1995). Although the mechanisms 1995) and basolateral actin and myosin II increase modestly to behind necrotic blebbing are not fully understood, they are form sub-plasmalemmal patches (not shown). This basolateral hypothesized to involve cortical solation, which is potentiated accumulation may provide a mild contractile force which by cyt D (Cunningham, 1995; Hale and Wuthier, 1987). This promotes apical secretion. However, during suprastimulation premise is supported by the observation that cells lacking the (10−8 M CCK), basolateral actin and myosin II increase actin filament cross-linking protein ABP-280 (nonmuscle substantially beyond this to form a circumferential ring (Figs filamin) display extensive, circumferential blebbing of the 4D, 7B). If this build up of basolateral actin and myosin is plasma membrane, which decreases as the amount of blocked by the actin-myosin inhibitors cyt D, BDM or ML-9, polymerized actin in the cell periphery increases (Cunningham, blebs are not formed. Thus, the contractile dynamics of the 1995; Cunningham et al., 1992). Unlike necrotic blebs, which actin-myosin ring appear to be responsible for bleb formation. are empty static protrusions, suprastimulated acinar cell blebs Once mature blebs are formed, regulation of the aberrant are extremely dynamic and filled with cytoplasmic organelles. basolateral actin and myosin II may also be responsible for Whereas necrotic blebbing is potentiated by cyt D and ATP bleb motility and bleb absorption. Modulating the contractile depletion, suprastimulated blebbing requires an ordered forces within the ring could affect fluid dynamics within the reorganization of the actin cytoskeleton (Figs 4, 5) and is cells to promote cytoplasmic streaming and bleb undulation. If inhibited by cyt D-induced cortical solation (Fig. 5) and energy the actin-myosin inhibitors cyt D or BDM are added to acini depletion (Fig. 3B). Finally, acinar cells need to be healthy in Actin-myosin mediated reversible blebbing 2921 order to bleb. Aged cells and cells manipulated by have found that block suprastimulated blebbing (cyt D and micropipettes do not bleb. ML-9) also reduce secretion in the stimulated state and thus In contrast to these disparities, the physiologic blebbing seen cannot be used to assess whether secretory potential is restored both during blastulation in fundulus deep cells (Trinkaus, if blebbing is inhibited (not shown; Burnham and Williams, 1973) and at the onset of the execution phase of apoptosis (Kerr 1982; Savion and Selinger, 1981). Defining the mechanism by et al., 1972; Mills et al., 1998) share many similarities with which agonists activate the actin-myosin matrix of the suprastimulated blebbing. Blebs that form on deep cells do so pancreatic acinar cell will provide important insights into rapidly in 2-3 minutes, are devoid of actin filaments, but have normal regulated secretion and pathophysiological responses. a band of actin stretching across their base. Importantly, the Additionally, it may help to guide further investigation into the formation of these blebs is inhibited by cyt B. As with regulatory mechanisms employed in other forms of physiologic pancreatic acini, these features indicate a role for an actin- blebbing. based mechanism of bleb formation. Unfortunately, to our knowledge, the role of myosin in deep cell blebbing has not We would like to thank Dr R. V. Rao (Mayo Clinic, Rochester, MN) been investigated. During the apoptotic course, a thick cortical and Ms B. J. Oswald (Mayo Clinic, Rochester, MN) for help and actin ring forms at the base of organelle-filled blebs (Kerr et technical assistance, as well as Ira Herman (Tufts University, Boston, al., 1972; Laster and Mackenzie, 1996). These blebs are MA) and Fumio Matsumura (Rutgers University, Piscataway, NJ) for providing reagents. formed, absorbed and reformed, eventually pinching off from the cell body. 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