Myosin II is present in gastric parietal cells and required for lamellipodial dynamics associated with cell activation

Rihong Zhou,1 Charles Watson,1 Chuanhai Fu,2 Xuebiao Yao,1,2 and John G. Forte1

1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200; and 2Laboratory of Cell Dynamics, School of Life Science, University of Science and Technology of China, Hefei, China 230027

Submitted 3 March 2003 ; accepted in final form 19 April 2003


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nonmuscle myosin II has been shown to participate in organizing the actin cytoskeleton in polarized epithelial cells. Vectorial acid secretion in cultured parietal cells involves translocation of proton pumps from cytoplasmic vesicular membranes to the apical plasma membrane vacuole with coordinated lamellipodial dynamics at the basolateral membrane. Here we identify nonmuscle myosin II in rabbit gastric parietal cells. Western blots with isoform-specific antibodies indicate that myosin IIA is present in both cytosolic and particulate membrane fractions whereas the IIB isoform is associated only with particulate fractions. Immunofluorescent staining demonstrates that myosin IIA is diffusely located throughout the cytoplasm of resting parietal cells. However, after stimulation, myosin IIA is rapidly redistributed to lamellipodial extensions at the cell periphery; virtually all the cytoplasmic myosin IIA joins the newly formed basolateral membrane extensions. 2,3-Butanedione monoximine (BDM), a myosin-ATPase inhibitor, greatly diminishes the lamellipodial dynamics elicited by stimulation and retains the pattern of myosin IIA cytoplasmic staining. However, BDM had no apparent effect on the stimulation associated redistribution of H,K-ATPase from a cytoplasmic membrane compartment to apical membrane vacuoles. The myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) also did not alter the stimulation-associated recruitment of H,K-ATPase to apical membrane vacuoles, but unlike BDM it had relatively minor inhibitory effects on lamellipodial dynamics. We conclude that specific disruption of the basolateral actomyosin cytoskeleton has no demonstrable effect on recruitment of H,K-ATPase-rich vesicles into the apical secretory membrane. However, myosin II plays an important role in regulating lamellipodial dynamics and cortical actomyosin associated with parietal cell activation.

acid secretion; cytoskeleton; ion channels and pumps


THE DIVERSE FAMILY of myosin proteins is encoded by different genes, yet all members of the family share the phenotype of actin-based motor activity. So far, more than 13 family members have been identified, but only a few have been studied in detail. Among the most studied, skeletal muscle myosin II was the first myosin purified because it is the most abundant and easily purified myosin protein.

In nonmuscle cells, myosin II consists of two heavy chains (205 kDa) and four light chains (18.5 kDa). Activity of myosin II is regulated by phosphorylation of two regulatory light chains, which in turn are under control of myosin light chain kinase (MLCK). In higher eukaryotes nonmuscle myosin II occurs in two isoforms, myosin IIA and myosin IIB, that have been variously shown to participate in lamellipodial and filopodial extensions in neuronal cells (12, 31), formation of an actomyosin ring in the sub-tight junctional region of MDCK cells (9), and activation of the Na-K-2Cl cotransporter (NKCC1) in T84 intestinal cells (17). Using isoform-specific antisense oligonucleotide, Wylie and Chantler (32) showed that myosin IIA and myosin IIB have separate but linked functions in modulating adhesion and neurite outgrowth.

Acid secretion by the gastric parietal cell involves mass transit in the translocation of the H,K-ATPase-containing vesicular membranes from the cytoplasm to the apical plasma membrane (1416). Early studies using chemical inhibitors established a role for the actin cytoskeleton in this translocation process and remodeling of the plasma membrane associated with the stimulation (7). More recent studies using the actin modulator latrunculin B (Lat B) on cultured parietal cells revealed that there are at least two functionally distinct pools of filamentous actin (3): a Lat B-sensitive actin pool, primarily involved in motile function of the cultured cells, and a Lat B-resistant pool of apical microvillar filaments. However, the precise function and molecular organization of the actin cytoskeleton in these two pools and the possible interaction with myosin motors remain to be established.

In the present study, we show the presence of two nonmuscle myosin II isoforms in gastric parietal cells. In addition, we show that stimulation of cultured parietal cells leads to a redistribution of myosin II that is distinct from the well-known membrane translocation events involving H,K-ATPase. In resting parietal cells, staining for myosin II is broadly distributed throughout the cytoplasm. Stimulation of the cells via the histamine/cAMP pathway triggers a dynamic rearrangement of myosin II to the lamellipodial extensions at the cell periphery. Filamentous actin is also recruited to the lamellipodial extensions of stimulated cells. The myosin-ATPase inhibitor 2,3-butanedione monoximine (BDM) blocks the lamellipodial dynamics associated with parietal cell stimulation as well as the accumulation of myosin II and F-actin in the motile cell extensions. However, BDM does not interfere with stimulus-associated recruitment of H,K-ATPase to the apical membrane vacuoles and has only minor effect on the vacuolar swelling produced by stimulated HCl secretion. Myosin II dynamics and lamellipodial formation are specific events downstream from the PKA activation. We propose that myosin II plays an important role in the basolateral cytoskeletal dynamics accompanying secretory activation of the parietal cell.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials and chemicals. Isoform-specific antibodies reacting with myosin II heavy chain A (MIIHCA) and myosin II heavy chain B (MIIHCB) were purchased from Berkeley Antibody. Antibody against the {beta}-subunit of H,K-ATPase (2G11) was from Affinity Bioreagents. Minimal essential medium (MEM) was from Invitrogen. The myosin-ATPase inhibitor BDM and the MLCK inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) were from Sigma. All other reagents were of the highest grade available.

Purification of recombinant RalA protein. RalA protein was expressed in Escherichia coli BL21, and the purification of glutathione S-transferase (GST) fusion proteins with glutathione-Sepharose beads (Sigma) was carried out as described previously (4). Briefly, one liter of Luria Bertani medium (LB: 10 g NaCl, 10 g trypto peptone, 5 g yeast extract) was inoculated with bacteria transformed with wild-type GST-RalA. When the optical density (600 nm) of the bacterial culture reached 0.7–1.0, expression of protein was induced by addition of 0.5 mM isopropyl-{beta}-D-thiogalactopyranoside (IPTG). Bacteria were harvested 3 h after induction by centrifugation, resuspended in phosphate-buffered saline (PBS) containing 5 mg/ml protease inhibitors (leupeptin, pepstatin, chymostatin), and sonicated with four bursts of 10 s each with a probe tip sonicator. The lysis solution was cleared of insoluble material by centrifugation for 20 min at 10,000 g. The soluble fraction was applied to a column packed with glutathione-Sepharose beads, followed by extensive washes with PBS (20 times the bead volume).

Pull-down assay. The fusion proteins bound to glutathione-Sepharose beads were used as an affinity matrix. To identify proteins interacting with RalA, whole gland lysates, prepared as the Triton X-100-solubilized extract of gastric glands, were passed through glutathione-Sepharose beads coupled with GST-RalA bound to either GTP or GDP. After washing, the beads were boiled in SDS-PAGE sample buffer and bound proteins were separated on 6–16% gradient SDS-PAGE gels and visualized with Coomassie blue staining. Interesting bands were removed and subjected to in-gel digestion with trypsin (Promega).

Gastric mucosal cell fractions. Gastric mucosal cell fractions were obtained from rabbit stomach as previously described and in accordance with procedures approved by the local Animal Care and Use Committee (20). Gastric mucosa was homogenized in 25 volumes of buffer containing (in mM) 5 piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES)-Tris, pH 6.7, 125 mannitol, 40 sucrose, and 1 EDTA. The homogenate was centrifuged at 80 g for 10 min to remove whole cells and connective tissue, and the resulting supernatant was used to generate three crude particulate fractions and a final supernatant by sequential centrifugation steps: P1 (4,300 g for 10 min) containing large cellular structures, including nuclei and plasma membranes; P2 (13,000 g for 10 min) containing mainly mitochondria and large granules; and P3 (100,000 g for 1 h) containing microsomes, such as endoplasmic reticulum and H,K-ATPase-rich tubulovesicles (TV). The final high-speed supernatant (S3) was saved as the cytosol. An enriched plasma membrane fraction (PM) was prepared from P1 by centrifugation at 100,000 g for 1 h through a density step gradient made of 12% and 18% Ficoll (type 400) with membranes collected from atop each layer (20). A fraction of H,K-ATPase-rich TV was prepared from P3 by density gradient centrifugation on a sucrose step gradient as described previously (29).

In-gel digestion and matrix-assisted laser desorption delayed-extraction reflectron time of flight mass spectrometry of RalA-binding protein peptides. In-gel digestion was a modification of the method of Rosenfeld et al. (21). Protein bands (~0.5 µg) were excised and minced with a clean scalpel, and the pieces were destained with three washes of 50% acetonitrile-12.5 mM NH4HCO3 (~10 min each). The destained gel pieces were dried in a Speedvac vacuum without heating, followed by rehydration in 50 µlof25mMNH4HCO3 (pH 8.0) that included 0.025 µg/ml trypsin. The pieces were overlaid with an additional 40 µl of 25 mM NH4HCO3 and incubated for 4 h at 37°C. Peptides were recovered by successive extractions of the digestion mixture with 50% acetonitrile-5% trifluoroacetic acid (TFA), 75% acetonitrile-5% TFA, and 95% acetonitrile-5% TFA, respectively. All supernatants were pooled and concentrated to ~5 µl in a Speedvac and brought back up to 50 µl in 5% acetonitrile-0.1% TFA. The sample was loaded onto a zip-tip (Millipore) equilibrated with 5% acetonitrile-0.1% TFA, washed three times with 5% acetonitrile-0.1% TFA, and eluted with 6 µl of 50% acetonitril-0.09% TFA. The eluate containing the peptide mix was stored at –20°C until further analysis.

Aliquots of tryptic digests were cocrystallized with {alpha}-cyano-4-hydroxycinnamic acid and analyzed with a matrix-assisted laser desorption (MALDI) delayed-extraction reflectron time of flight (TOF) instrument (Voyager Elite mass spectrometer; Perseptive Biosystems, Framingham, MA) equipped with a nitrogen laser. Measurements were performed in a positive ionization mode. All MALDI spectra were externally calibrated with a standard peptide mixture. The data were analyzed by software developed in the University of California-San Francisco Mass Spectrometry Facility and are available at http://prospector.ucsf.edu. Both the National Center for Biotechnology Information protein database and Swiss Prot database were searched. Search parameters included the putative protein molecular weight and a peptide mass tolerance of 100–200 parts/million.

In addition, mass and sequence data were obtained by liquid chromatography/mass spectrometry on a Q-star Pulsar (MDS Sciex, Toronto, ON, Canada) using the same peptide digests except that application and elution from the zip-tips were done with formic acid substituted for TFA. These data were analyzed with Mascot search software (http://www.matrixscience.com/).

Cosedimentation of MIIHCA with actin filaments. Stored G-actin samples were thawed and centrifuged at 333,000 g (TLA 100.3 rotor; Beckman Instruments) for 40 min to remove any polymerized or aggregated actin. Aliquots of monomeric actin were incubated for 2 h at 4°C with or without MIIHCA-enriched rabbit gastric subcellular fraction S3 in polymerizing buffer (in mM: 5 Tris, pH 7.5, 0.5 ATP, 2 MgCl2, 100 KCl, and 0.2 DTT) to promote polymerization. The filaments were then pelleted by centrifugation at 333,000 g for 40 min. The pellets were washed three times with PBS. For visualization and quantitation of actin and its binding proteins, pellets and supernatants were solubilized in SDS gel sample buffer and subjected to electrophoresis. Gels were stained with Coomassie blue and dried between sheets of cellulose for visualization and quantitative scanning.

Removal of MIICHA from actin-related subcellular fractions. Rabbit gastric subcellular fraction P3 was centrifuged at 230,000 g for 30 min to bring down the microsomal membranes. The membrane pellets were resuspended and incubated with 150 mM NaHCO3 pH 8.5, PBS, or PBS with 0.5 mM ATP at 4°C for 30 min. The suspensions were centrifuged at 230,000 g for 20 min. Pellets and supernatants were solubilized in SDS gel sample buffer and subjected to electrophoresis. Gels were transferred to nitrocellulose membrane and probed with actin and MIIHCA antibodies.

Isolation of gastric glands and parietal cells. Isolated gastric glands and parietal cells were prepared as previously described (36) from New Zealand White rabbits by a combination of high-pressure perfusion via the abdominal aorta and collagenase digestion. Briefly, the perfused gastric mucosa was scraped, minced, and washed twice with MEM containing 20 mM HEPES, pH 7.4 (MEM-HEPES). The minced mucosa was digested in MEM-HEPES containing 0.25 mg/ml collagenase (Sigma) and 0.25 mg/ml BSA at 37°C for ~30 min. Large elements of debris were removed by straining the suspension through a 40-µm mesh. Because of their large size, relatively intact gastric glands settle out in 10–15 min, leaving individual cells suspended in the medium. The cell suspension was removed to harvest cells for culture (see Primary culture of gastric parietal cells), and the glands were washed by settling three times in HEPES-MEM.

To test the effects of BDM on acid secretory function the isolated glands were permeabilized with streptolysin O (SLO) as previously described (4). Intact glands in HEPES-MEM were washed two times by setting at 4°C in ice-cold K buffer (in mM: 10 Tris base, 20 HEPES acid, 100 KCl, 20 NaCl, 1.2 MgSO4, 1 NaH2PO4, 40 mannitol, pH 7.4). SLO was added to a final concentration of 1 µg/ml, and the glands were mixed by inversion and incubated on ice until the glands had completely settled. The supernatant containing excess SLO was removed, and after being washed in ice-cold K buffer the glands were finally resuspended in K buffer including 1 mM pyruvate and 10 mM succinate and then incubated at 37°C for 3 min to allow the SLO pores to form.

Acid secretion by SLO-permeabilized gastric glands was quantified with the aminopyrine (AP) uptake assay of Berglindh and Obrink (5) as modified (4). Briefly, aliquots (0.35 ml) of gland suspension were added to preweighed 1.5-ml Eppendorf tubes already containing 4 x 107 M (46 nCi/ml) of 14C-labeled AP. The glands were either held in a resting state in the presence of 100 µM cimetidine, a histamine antagonist, or stimulated with 0.1 mM cAMP and 1 mM ATP. After oxygenation, the glands were shaken at 37°C for 20 min and then pelleted by centrifugation. The pellets were dried and weighed, and 14C was counted in a Beckman liquid scintillation counter. The AP uptake ratio (ratio of the concentration of [14C]AP in the pellet vs. the solution) was used as an indicator of acid secretion.

Proton uptake and leakage by H,K-ATPase-rich TV. Proton transport by H,K-ATPase-rich TV was monitored by acridine orange quenching as described previously (20). An aliquot of TV (~20 µg) was added to the uptake medium (140 mM KCl, 2.5 mM PIPES-Tris, pH 7.0, 1 mM MgATP, 25 µM EDTA, 1 µM acridine orange). Fluorescence changes (excitation 493 nm, emission 540 nm) were continuously monitored with a spectrofluorimeter (SPEX) at 37°C. Because of limited permeability of TV to K+, little proton uptake (quenching of fluorescence) occurs until the addition of 2 µM valinomycin (20). After a suitable pH gradient had developed the H,K-ATPase pump was blocked by chelating Mg2+ with an additional 4 mM EDTA and the proton leak rate could be measured. At this time the effects of BDM and ML-7 on the proton leak rate were measured by following the relaxation of the quenching signal. At the end of all experiments, the equilibrium fluorescence was measured by adding the potent K+/H+ exchange ionophore nigericin.

Primary culture of gastric parietal cells. Preparations relatively enriched in parietal cells were recovered from the cell suspension described in Isolation of gastric glands and parietal cells by three repetitions of gentle centrifugation at 200 g for 5 min, followed by resuspension in fresh HEPES-MEM. The suspended cells were strained, washed three times with MEM-HEPES, harvested, and transferred to culture as described previously (1). Cells were treated with 25 µg/ml amphotericin B in medium B [DMEM-F-12 (Invitrogen) supplemented with 20 mM HEPES, 0.2% BSA, 10 mM glucose, 8 nM epidermal growth factor, 1x selenite, insulin, and transferrin (SITE) medium (Sigma), 1 mM glutamine, 100 U/ml penicillin-streptomycin, 400 µg/ml gentamicin sulfate, and 15 µg/l geneticin or 20 µg/ml novobiocin, pH 7.4] to prevent yeast infection. Plating onto Matrigel (Collaborative Biomedical)-coated coverslips followed, and cells were thereafter incubated at 37°C in medium A (without amphotericin B). To destroy contaminating yeast and bacteria, the cells were incubated for 30 min in medium B [DMEM-F-12 (GIBCO) supplemented with 20 mM HEPES, 0.2% BSA, 10 mM glucose, 8 nM epidermal growth factor, 1x SITE medium (Sigma), 1 mM glutamine, 100 U/ml penicillin-streptomycin, 400 µg/ml gentamicin sulfate, 25 µg/ml amphotericin B, and 15 µg/l geneticin or 20 µg/ml novobiocin, pH 7.4]. Cells were plated onto Matrigel-coated coverslips in 12-well plates and incubated at 37°C in culture medium A (medium B with amphotericin B).

Primary cultures of gastric parietal cells from rabbit were harvested and maintained as described previously (1). The suspended cells were strained and washed three times with MEM-HEPES. Cells were treated with 25 µg/ml amphotericin B in medium B [DMEM-F-12 (Invitrogen) supplemented with 20 mM HEPES, 0.2% BSA, 10 mM glucose, 8 nM epidermal growth factor, 1x SITE medium, 1 mM glutamine, 100 U/ml penicillin-streptomycin, 400 µg/ml gentamicin sulfate, and 15 µg/l geneticin or 20 µg/ml novobiocin, pH 7.4] to prevent yeast infection. Plating onto Matrigel-coated coverslips followed, and cells were thereafter incubated at 37°C in medium A (without amphotericin B). Cultures enriched in parietal cells were obtained from the above cell suspension.

Immunofluorescence microscopy of MIIHCA in isolated gastric glands and parietal cells. Isolated rabbit gastric glands were first treated either with cimetidine to maintain a resting state or with histamine plus IBMX for maximal stimulation. Glands were then fixed with 4% paraformaldehyde for 10 min and permeabilized in 0.2% Triton X-100 for 10 min. The fixed, permeabilized glands were blocked with 2% BSA in PBS for 30 min before incubation with primary antibodies. MIIHCA was detected by 2-h incubation with affinity-purified polyclonal antibodies. The primary antibody was visualized by either FITC- or rhodamine-conjugated goat anti-rabbit IgG (Jackson Labs). Actin was detected by coincident incubation with 80 nM rhodamine- or FITC-labeled phalloidin (Sigma) Cells were visualized by Zeiss confocal microscopy to collect images.

To localize myosin II proteins in gastric parietal cells and to test for the possible relocation of proteins associated with the acid secretion, primary cultures of parietal cells were treated with secretagogues and/or inhibitors to effect different physiological states: 1) 100 µM cimetidine to maintain a resting state, 2) 100 µM histamine plus 50 µM IBMX to effect maximal stimulation of HCl secretion, or 3) 100 µM histamine plus 50 µM IBMX with 5 µM SCH28080, a proton pump inhibitor (1). To test the function of myosin II in parietal cells, we additionally added 20 mM BDM, a myosin-ATPase inhibitor, and 10 µM ML-7, a MLCK inhibitor, to cells that had been treated regularly with cimetidine or histamine plus IBMX with or without SCH28080, as indicated above. The cells were fixed with 4% paraformaldehyde for 10 min followed by permeabilization in 0.2% Triton X-100 for 10 min. The fixed, permeabilized cells were blocked with 2% BSA in PBS for 30 min before the incubation of primary antibodies. Further steps for blocking with BSA, incubation with MIIHCA affinity-purified polyclonal antibodies, and secondary antibodies were all as described for gastric glands. The labeled cells were visualized by epifluorescence microscopy with a Nikon Microphot FX-2 camera and ISee Imaging software.

Measurement of apical membrane vacuoles and basolateral membrane extensions. Morphometric data were obtained from three separate parietal cell culture preparations and included cells that were treated with various stimulants. The apical membrane vacuole diameter was measured in micrometers for all vacuoles that were visible in cross section. Vacuole diameter was calculated as the mean of the major and minor axes of the F-actin-stained vacuoles. Lamellipodia and filopodia were measured by focusing down on the plane of the coverslip where cells were attached. The area of each F-actin-stained cellular extension was measured in square micrometers by tracing its periphery; areas were summed for each cell.

Western blots. Samples were subjected to SDS-PAGE on ~6–16% gradient gels and transferred onto nitrocellulose membrane. Proteins were probed by appropriate primary antibodies and detected with enhanced chemiluminescence (Pierce).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification of myosin II in gastric parietal cells. Our discovery of myosin II in parietal cells came as an offshoot of studies to determine the role of SNARE proteins in regulated HCl secretion. A small GTPase known as RalA had been shown to be a useful affinity matrix for proteins involved in membrane trafficking processes (8). We generated bacterial recombinant RalA protein and used it as an affinity ligand to pull down RalA-binding proteins from gastric parietal cells. The binding data are shown in Fig. 1. A control indicated that the GST-RalA protein migrated as a single band at an apparent molecular size of 47 kDa, with perhaps some smaller breakdown products. After incubation with whole gastric gland lysates, a number of proteins appeared to associate with the GST-RalA beads. One band of apparent 200 kDa consistently associated with the GST-RalA beads and simultaneously was depleted from the starting material (Fig. 1A). Binding of the 200-kDa band is specific for the RalA protein because glutathione-Sepharose beads themselves did not absorb the protein (data not shown). Moreover, the same protein band was adsorbed independent of whether the RalA was in the GTP- or GDP-bound form.



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Fig. 1. Glutathione-S-transferase (GST)-RalA proteins pull down myosin IIA from gastric cell lysates. Isolated gastric glands were lysed in phosphate-buffered saline (PBS) with 0.5% Triton X-100 and sonicated on ice 3 times for 15 s, with 1 min between 2 sonications. The gland lysate was centrifuged at 10,000 g for 5 min to remove particulate material, and the supernatant was used for the pull down. Bacterially recombinant RalA protein coupled to glutathione agarose beads was used as an affinity matrix. The beads were preincubated with gastric glandular lysates (SM) in the presence of either GTP (GTP-RalA) or GDP (GDP-RalA). Flow-through material was taken as unbound (NB), and the material bound to the beads (B) was removed by boiling in SDS-PAGE sample buffer. A: bound proteins were fractionated by SDS-PAGE and stained with Coomassie blue. Presumed positions of myosin II [myosin II heavy chain A (MIIHCA)] and GST-RalA protein are shown by arrowheads at right. The high-molecular-weight polypeptide of ~200 in the bound fraction was removed and subjected to in-gel digestion with trypsin for analysis by a matrix-assisted laser desorption (MALDI) delayed-extraction reflectron time of flight (TOF) mass spectrometry. Molecular weight (mw) standards are shown in the first lane from left. B: a duplicate SDS-PAGE gel of A was used for transblotting to nitrocellulose membrane and probed with a specific antibody against MIIHCA.

 

The 200-kDa RalA-binding band was excised from the Coomassie blue-stained gel and subjected to in-gel digestion with trypsin, and the resulting peptides were assayed by MALDI-TOF and by LC MS/MS mass spectrometry. Through both approaches it was apparent that a large number of the peptide signals were consistent with MIIHCA, yielding a statistically significant match. For MALDI 64% of the identified peptide masses were consistent with MIIHCA, and sequence data from LC MS/MS predicted that 28 of the individual peptides were from MIIHCA (data not shown).

To confirm myosin II as a RalA-binding partner, a gel of GST-RalA-binding and -nonbinding materials was blotted to nitrocellulose and probed with a specific antibody against myosin II. Similar to the Coomassie blue-stained gel, myosin II was present in the starting lysate and was bound to the GST-RalA beads in both the GTP- and GDP-bound forms (Fig. 1B). Thus our studies revealed the association of myosin II with RalA in a GTP-independent manner.

Both myosin IIA and myosin IIB are present in gastric epithelial cell fractions. Nonmuscle myosin IIA and IIB are major isoforms existing in nonmuscle cells. To determine whether both isoforms are expressed in gastric epithelial cells, we carried out Western blots with isoform-specific antibodies to detect the two isoforms in various subcellular fractions derived from homogenates of rabbit gastric glands. As shown in Fig. 2, myosin IIA is present in several crude particulate fractions, P1, P2, and P3, as well as in the cytosolic supernatant fraction, S3. Myosin II was de-enriched in density gradient-purified membrane fractions derived form the crude fractions. For example, the plasma membrane-rich fraction from P1, and the TV H,K-ATPase-rich fraction from P3 were markedly depleted of myosin II. Figure 2 also shows that myosin IIB is present and has a similar distribution profile among the gastric particulate fractions; however, the myosin IIB isoform is virtually absent from the cytosolic supernatant fraction. The difference in subcellular distribution suggests the possibility that the two isoforms of myosin II may function differently.



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Fig. 2. Both myosin IIA and myosin IIB are present in gastric epithelial cell fractions. Subcellular fractions from gastric mucosa were separated by SDS-PAGE. Crude cell fractions included large structures such as nuclei and plasma membranes (P1; 4,300 g x 10 min), mitochondria and large granules (P2; 13,000 g x 10 min), microsomes (P3; 100,000 g x 1 h), and the cytosolic final high-speed supernatant (S3). Purified membrane fractions included a fraction enriched in plasma membranes (PM) prepared from P1 by Ficoll density-gradient centrifugation and a tubulovesicle fraction (TV) enriched in H,K-ATPase prepared from P3 by sucrose density-gradient centrifugation. An aliquot of each fraction, amounting to 35 µgof protein, was added to each lane. Gels were transblotted to nitrocellulose and probed with antibodies specific for either myosin II isoform, MIIHCA or myosin II heavy chain B (MIIHCB). Western blots show that both myosin II isoforms show a similar distribution pattern among the cell fractions, except that myosin IIA (but not IIB) is enriched in cytosolic fraction S3. Although the myosin II isoforms were relatively abundant in the crude particulate fractions, P1, P2, and P3, they were clearly de-enriched in the membrane fractions purified from P1 (PM) and P3 (TV).

 

To further characterize the biochemical properties of myosin II from gastric fractions, we carried out actin binding experiments. Some general characteristics of the myosin II in gastric epithelial cells are exemplified in Fig. 3. When the gastric cytosolic supernatant fraction was incubated with purified actin under conditions predicted to polymerize G-actin into F-actin, most of the myosin IIA was harvested with the F-actin pellet (Fig. 3A). Inclusion of ATP in the incubation led to diminution of myosin IIA recovered in the pellet. Thus soluble myosin in the cytosol will bind to F-actin and be released by ATP. In an analogous manner, most of the myosin IIA that sedimented with the membrane pellets was released by incubation with ATP, whereas neither NaCl nor NaHCO3 (pH 8.5) was effective in dissociating myosin IIA from the pellet (Fig. 3B). These data are consistent with the known effects of ATP on the binding interaction between the myosin head portion and F-actin. Furthermore, they suggest that the myosin II associated with particulate fractions is bound by association with F-actin.



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Fig. 3. Gastric mucosal myosin II has the same general biochemical properties as conventional myosin II. A: aliquots of monomeric actin (75 µg G-actin) were incubated as indicated with or without the S3 cytosolic fraction of gastric mucosa, with or without polymerizing buffer to promote polymerization (K+ + Mg2+), and with or without 0.5 mM ATP. After 2-h incubation at room temperature the samples were centrifuged at 333,000 g for 40 min and washed 3 times with PBS to pellet the polymerized F-actin. The supernatant (S) and pellet (P) from each sample were solubilized in SDS gel sample buffer and subjected to SDS-PAGE. Gels were stained with Coomassie blue. Arrowheads at right indicate the presumed respective positions of myosin II and G-actin on the gel. B: the nature of myosin II association with the gastric microsomal fraction was probed by incubating the P3 fraction at 4°C with 150 mM NaHCO3 pH 8.5 (NaHCO3), PBS, or PBS including 0.5 mM ATP (PBS + ATP). After 30 min the suspensions were centrifuged at 230,000 g for 20 min. Pellets were washed 3 times with PBS. The pellet (P) and supernatant (S) from all samples were solubilized in SDS gel sample buffer and subjected to SDS-PAGE. Gels were transferred to nitrocellulose and probed with actin and MIIHCA antibodies. For comparison, a separate lane includes the starting microsomal pellet (P3 pellet).

 

BDM inhibits AP uptake by gastric glands and increases proton leakage across vesicles. BDM is the best-characterized inhibitor of myosin ATPase activity (11, 18, 34). BDM has been used to block myosin II-dependent processes, such as epithelial cell spreading (11), vesicle recruitment during Ca2+-regulated exocytosis (6), locomotion and lamellipodial formation in leukocytes (13, 30), and retrograde actin flow in neuronal growth cones (23), whereas BDM does not inhibit actin filament polymerization or kinesin-ATPase or dynein-ATPase activity (11). Accordingly, we tested the effects of BDM on stimulus-dependent acid accumulation by isolated gastric glands. Freshly isolated gastric glands were permeabilized by SLO and stimulated by including cAMP and ATP in the incubation medium, and the accumulation of AP was measured with and without BDM. Figure 4A shows the dose-response curve, with an EC50 of ~8 mM BDM, consistent with a possible role for myosin II in acid secretory function.



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Fig. 4. BDM inhibits aminopyrine (AP) uptake by isolated gastric glands and increases proton leakage across gastric tubulovesicles. A: acid secretory activity was assayed in streptolysin O (SLO)-permeabilized gastric glands by the AP uptake ratio. Nonsecreting glands (0.1 mM cimetidine, cim) were compared with those stimulated by cAMP + ATP (0.1 and 1 mM, respectively), in the absence or presence of increasing concentrations of the myosin ATPase inhibitor 2,3-butanedione monoximine (BDM). B: effect of BDM on proton leakage. Purified H,K-ATPase-rich gastric tubulovesicles were assayed for proton uptake and proton leakage rate by the acridine orange uptake method. Vesicles were initially incubated with 140 mM KCl, 1 mM Mg2+-ATP, and 5 µM acridine orange. At a designated time 5 µM valinomycin (val) was added, effecting a rapid proton uptake. Addition of 4 mM EDTA chelates Mg2+ and blocks the pump, resulting in a slow leak of protons toward equilibrium. BDM was added at the indicated concentrations, and the increase in proton leakage was noted. Finally, nigericin (nig) was added to dissipate all proton gradients. C: effect of 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) on proton leakage. H,K-ATPase-mediated proton uptake was measured as in B. Addition of 10 µM ML-7 induced a rapid dissipation of the proton gradient.

 

Relatively high doses have been the rule for BDM inhibitory effects on myosin ATPase activity (18) and on myosin-dependent cell processes (11, 13); however, we were concerned that high concentrations of the drug might influence the AP response in some other way, such as an increased proton leakage. Therefore, we tested the effects of BDM on proton leakage of isolated gastric TV. Figure 4B presents the results of proton uptake experiments on isolated gastric TV. When vesicles are incubated with ATP, Mg2+, and acridine orange in high-KCl buffer there is little fluorescence quenching until the addition of a K+ ionophore like valinomycin. The rapid quenching signifies rapid proton uptake and development of a large pH gradient resulting from H+/K+ exchange by H,K-ATPase. When the pump is blocked by chelating Mg2+ there is a small endogenous rate of proton leakage. The subsequent additions of BDM clearly indicate a concentration-dependent increase in the rate of proton leakage produced by BDM (Fig. 4B). Although the protonophoric effect of BDM is not nearly as dose effective as that produced by ML-7 (cf. Fig. 4C), the leakage effect undermines the reliability of the AP uptake data.

Myosin IIA is found throughout cytoplasm of parietal cells. We next sought to define the cellular location of myosin II in parietal cells in different physiological states. Gastric glands were treated with cimetidine or histamine plus IBMX to maintain glands in the resting or stimulated states, respectively, and then immunoprobed with MIIHCA-specific primary antibody and double-stained for F-actin with an FITC-labeled phalloidin probe. (Experiments to immunostain gastric cells with antibodies against MIIHCB were unsuccessful; thus the cytolocalization data reported here are confined to MIIHCA.)

Confocal images collected from resting gastric glands show that myosin IIA is relatively diffusely distributed throughout the cytoplasm of parietal cells, although there is some minor degree of localization to plasma membranes (Fig. 5). However, in histamine-stimulated gastric glands, the myosin IIA probe showed bright basolateral membrane staining in parietal cells. Stimulation cleared the cloudy appearance of myosin IIA from the cytoplasm and moved it to the basolateral membrane. We also observed that glandular nonparietal cells (chief cells and mucous neck cells) have a distinctive deposition of myosin IIA that colocalized with F-actin along their apical borders lining the gland lumen. This nonparietal cell staining did not appear to be altered by stimulation.



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Fig. 5. Myosin IIA is relocated to the basolateral pole of parietal cells after stimulation. Isolated gastric glands were either treated with 100 µM cimetidine to maintain a resting state (top) or treated with histamine + IBMX to produce a stimulated state (bottom). After fixation and permeabilization, glands were incubated with a myosin IIA-specific antibody followed by a rhodamine-conjugated goat anti-rabbit IgG. The cells were also counterstained with FITC-labeled phalloidin to visualize the filamentous actin cytoskeleton. Confocal images from the 2 fluorophores were collected with a Zeiss 510 confocal microscope. Parietal cells are clearly obvious as large bulging cells with a network of F-actin-positive apical canalicular membrane branching throughout the cell. In resting parietal cells myosin IIA staining is seen throughout the cell, although there is some localization to plasma membranes. Arrowheads indicate the basolateral membrane of parietal cells that show staining for F-actin in both resting and stimulated cells and for myosin IIA in stimulated cells. Arrows point to the apical membrane of nonparietal cells that show definitive, stimulation-independent F-actin and myosin IIA staining that lines the entire gastric lumen but virtually no positive staining through the rest of the cell. Bar, 20 µm.

 

We next shifted our attention to the cultured parietal cell system, in which stimulation-associated redistribution of membranes and proteins can be studied in greater detail. Figure 6 shows a montage of epifluorescent images collected from cultured parietal cells that were maintained in three functional states: 1) the resting, nonsecreting state, 2) the fully activated and secreting state (histamine + IBMX), and 3) the activated state in the presence of the H+ pump inhibitor SCH28080 to minimize water flow into the apical vacuoles. In all cases the cultured cells were immunoprobed with a MIIHCA-specific primary antibody and visualized with a rhodamine-labeled secondary antibody. Cells were then double-stained either for F-actin with FITC-phalloidin or for H,K-ATPase with FITC-coupled H,K-ATPase antibody.



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Fig. 6. Myosin IIA is relocated to parietal cell lamellipodia after stimulation of acid secretion. Cultured gastric parietal cells were treated with 100 µM cimetidine to maintain the resting state (A), 100 µM histamine + 50 µM IBMX to effect the maximal stimulated state (B), or histamine + IBMX in the presence of the proton pump inhibitor SCH28080 (5 µM; C), which allows for translocation of H,K-ATPase to apical membrane vacuoles but greatly diminishes vacuolar swelling. After 20-min treatment at 37°C, cells were fixed, permeabilized, and incubated with a myosin IIA-specific antibody followed by FITC-conjugated goat anti-rabbit IgG. The cells were also counterstained with either rhodamine-labeled phalloidin to visualize the filamentous actin cytoskeleton or rhodamine-labeled H,K-ATPase antibody. A: resting cells show distinct F-actin (F-act)-labeled apical membrane vacuoles and broad distribution of H,K-ATPase (HK) throughout the cells. Myosin II (MII) was also broadly distributed throughout resting cells, with virtually no colocalization with F-actin or H,K-ATPase. B: in maximally stimulated cells apical membrane vacuoles, rich in both F-actin and H,K-ATPase staining, are greatly swollen and lamellipodia, rich in F-actin and myosin II, extend from the surrounding plasma membrane. C: when cells were stimulated in the presence of the pump inhibitor SCH28080 F-actin and H,K-ATPase colocalized to the apical membrane vacuoles, which have only minimal swelling. A subset of F-actin staining, along with myosin II staining, is also seen in the large lamellipodial extensions that are characteristic of this treatment. Bar, 20 µm.

 

Consistent with previous studies (see, e.g., Refs. 1 and 3), the F-actin staining shown for resting cells in Fig. 6A appears as ringlike structures representing the apical vacuoles. There is also somewhat weaker F-actin staining at the basolateral membrane surrounding the cells. However, the staining of myosin IIA revealed a pattern distinctly different from that of F-actin. In resting cells, myosin IIA is located throughout the cytoplasm, with some suggestive staining in the regions immediately adjacent to the apical membrane vacuoles (Fig. 6A). Overall, there was relatively little coincidence in the distribution of F-actin with that of myosin IIA.

Resting parietal cells were also double-stained for H,K-ATPase and MIIHCA (Fig. 6A). Because H,K-ATPase is specifically localized to the tubulovesicular membranes of parietal cells, the staining of H,K-ATPase also serves as a marker for this compartment. Parietal cells marked by positive H,K-ATPase staining are also positive for MIIHCA staining (although nonparietal cells were also found to stain positive for myosin II). In resting parietal cells myosin IIA is distributed as relatively diffuse cytoplasmic staining, whereas H,K-ATPase staining appears punctuate with a limited degree of localization to apical membrane vacuoles. There is little overlap in the distribution profile of the two proteins, but it is unclear to which intracellular organelles myosin IIA associates. Thus we conclude that myosin IIA is present in nonsecreting gastric parietal cells with a distribution pattern distinctly different from both H,K-ATPase and F-actin.

Myosin IIA is relocated concomitant with lamellipodial dynamics associated with stimulation. Stimulation of cultured parietal cells with histamine results in relocation and insertion of H,K-ATPase-containing vesicles into the apical membrane vacuoles (1). Stimulation also results in significant lamellipodial dynamics at the basolateral membrane (3). To test whether the stimulation affects localization of myosin IIA we carried out double immunofluorescence staining on stimulated parietal cells. Figure 6B shows that the apical membrane vacuoles are greatly enlarged in the cells stimulated by histamine plus IBMX. H,K-ATPase is localized to the expanded vacuoles along with F-actin. Long F-actin filament bundles are also prominent in the lamellipodial extensions that typically emanate from stimulated cells. Stimulation results in relocation of the majority of MIIHCA to the same lamellipodia, except that MIIHCA staining tends to be more weblike than filamentous and is distributed in greater abundance to the lamellipodial regions proximal to the cell body. Quantitative data for average diameter of vacuoles and surface area of lamellipodia and filopodia are given in Table 1 for all conditions of stimulation and inhibition.


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Table 1. Apical membrane vacuoles and lamellipodial extensions are greatly enlarged in stimulated parietal cells, and these enlargement responses are variably diminished by myosin II inhibitors

 

Studies in which cells had been stimulated in the presence of the proton pump inhibitor SCH28080, shown in Fig. 6C, verify that MIIHCA relocates, along with F-actin, to the lamellipodial membrane space whereas H,K-ATPase traffics to the apical membrane vacuoles. In this treatment, with the pump inhibitor present, the vacuoles are not grossly swollen, the lamellipodia are in their most elaborate state, and it is easy to distinguish H,K-ATPase migration to the apical vacuoles from myosin IIA migration to the lamellipodia. We conclude that myosin IIA is involved in lamellipodial dynamics in response to histamine stimulation.

Myosin IIA is essential for lamellipodial dynamics associated with cell activation. Histamine stimulation elicited translocation of MIIHCA from the cytosol to the cortical region of the basal membrane. To address the function of myosin II in such dynamic processes, we used BDM, a known inhibitor of myosin II and myosin V ATPase activities (11), in the parietal cell culture model. As shown in Fig. 7A the most striking change was that BDM virtually abolished the redistribution of myosin IIA to lamellipodia that normally occurred with stimulation. In fact, formation of lamellipodia, along with incorporation of F-actin, was greatly diminished by BDM, as confirmed by the quantitative data in Table 1. However, addition of BDM to parietal cell cultures did not alter the translocation of H,K-ATPase to apical membrane vacuoles associated with stimulation. In addition, stimulation-induced swelling of the vacuoles also occurred in the presence of BDM, although average vacuole size was somewhat smaller than for control stimulated parietal cells (Table 1), but this may be due to some protonophoric action at the concentration used.



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Fig. 7. BDM inhibits lamellipodial dynamics associated with parietal cell stimulation. Cultured gastric parietal cells were treated as described in Fig. 5 to establish conditions of rest (A), stimulation (B), or stimulation in the presence of the pump inhibitor SCH28080 (C), all in the presence of 20 mM BDM, a myosin ATPase inhibitor. Fixed and permeabilized cells were probed for myosin IIA, F-actin, and H,K-ATPase as in Fig 5. The presence of BDM did not appear to alter the stimulation-associated redistribution of H,K-ATPase to the apical membrane vacuoles, although the "stimulated" vacuoles did appear to be somewhat smaller than the control cells of Fig. 5. However, formation of, and relocation of myosin II to, lamellipodia were greatly diminished in BDM-treated cells. Bar, 20 µm.

 

The phosphorylation of myosin II light chain is known to regulate myosin II contractility and contribute to the formation of lamellipodia [see review by Sellers (25)]. To test for the role of myosin phosphorylation in parietal cell function, we treated cultured cells with ML-7, a MLCK inhibitor. Like BDM, treatment with ML-7 did not alter the localization of F-actin or H,K-ATPase in the resting cells or the redistribution of H,K-ATPase to the apical vacuoles after stimulation (Fig. 8). However, unlike BDM treatment, the histamine-stimulated expansion of apical vacuole membranes was greatly attenuated by ML-7. This effect on vacuolar expansion may be due to a potent protonophoric effect reported for ML-7 (2), but it is clear that the stimulation-associated translocation of H,K-ATPase to the apical membrane is not altered by the MLCK inhibitor. ML-7 did appear to reduce the relative abundance of lamellipodia (Table 1), and the redistribution of myosin IIA to lamellipodia was not as distinct as for stimulated control preps. We conclude that myosin II is essential for lamellipodial dynamics, but not for apical membrane remodeling, associated with histamine stimulation.



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Fig. 8. ML-7 does not alter stimulation-associated H,K-ATPase translocation but does prevent vacuole dilation associated with stimulation. Cultured gastric parietal cells were treated as described in Fig. 5 to establish conditions of rest (A), stimulation (B), or stimulation in the presence of the pump inhibitor SCH28080 (C), all in the presence of 10 µM ML-7, a myosin light chain kinase (MLCK) inhibitor. Fixed and permeabilized cells were probed for myosin IIA, F-actin, and H,K-ATPase as in Fig. 5. ML-7 did not alter the stimulation-associated redistribution of H,K-ATPase to the apical membrane vacuoles, although vacuolar swelling was eliminated. ML-7 appeared to have only minimal effect on lamellipodial extensions and recruitment of myosin II to the lamellipodia. Bar, 20 µm.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myosin II is a founding member of the myosin superfamily whose function has been implicated in a variety of cellular dynamics. The nonmuscle myosin IIA and IIB proteins play important roles in lamellipodial formation during neurite growth (12, 31, 33). Here we provide the first evidence that myosin IIA is present in gastric parietal cells and occurs as a soluble protein and/or associated with the actin cytoskeleton in these cells. Furthermore, our studies show that in response to stimulation by histamine myosin IIA is rapidly relocated to the basolateral cortex of parietal cells of intact gastric glands and to the lamellipodial extensions of cultured parietal cells. Finally, we offer the first demonstration that both myosin dynamics and lamellipodial formation are abolished when a myosin ATPase inhibitor is included, suggesting that myosin II plays an important role in the basolateral membrane dynamics associated with parietal cell activation.

A major role for the actin cytoskeleton in the secretory processes of parietal cells has been implied from studies using actin disruptors and sequestering agents that disorganize the actin filaments within the apical microvilli and inhibit acid secretion (3, 7). Highly organized microfilaments are typical features of apical microvilli within the secretory canaliculi of the parietal cell. In going from a resting to secreting state, there are major changes in the apical canalicular membrane area of parietal cells, especially apparent in the elongation of microvilli (16). Consistent with the morphological changes, the recruitment of H,K-ATPase-rich cytoplasmic vesicles into the apical membrane is the basis for the major functional change at the onset of secretion (14). Thus the apical surface of the parietal cell has been a prominent focus of research in recent years. When we first identified myosin II in parietal cells, we expected that it might participate as a motor protein in the regulated cytoskeletal reorganization and secretory dynamics at the apical surface. We were surprised to find that stimulation of parietal cell secretion correlated with a mobilization of MIIHCA to the basolateral domain.

In fact, many important transport events and cytoskeletal specializations occur at the basolateral (BL) membrane of parietal cells. For each mole of acid delivered into the secretory lumen a mole of base must be moved across the BL membrane. For the most part, this is accommodated by the abundant and active exchangers at the BL membrane (22, 27, 28). Water of the gastric secretory volume must also be accommodated by an equivalent flow of water into the cell across the BL membrane. Thus there are interesting osmotic questions to be accounted for. Several cytoskeletal proteins have been localized to the region of the BL membrane. A layer of cortical F-actin is readily apparent in the BL membrane of parietal cells, whereas F-actin is very scant at the BL region of other cells within the gastric gland (Fig. 5; see Ref. 35). Moreover, there is a polarization of actin isoforms in parietal cells, with {gamma}-actin being most abundant at the BL surface and {beta}-actin most abundant at the apical surface (35). The 40-kDa cAMP-dependent phosphoprotein LASP-1 is also polarized to the basolateral membrane in resting parietal cells, although it is relocated away from the site on stimulation (10). From the present studies myosin IIA is another cytoskeletal protein that is distributed toward the BL surface of the parietal cell, with this preferred distribution being a function of stimulation.

A unique feature of the conventional myosins, i.e., the myosin II subfamily, is their ability to form bipolar filaments via self-association of their tail domains. Such double-ended structures have obvious functional significance in interacting with actin filaments to effect contractile events. In nonmuscle cells myosin II has been linked to a variety of motile functions, including normal furrow formation preceding cytokinesis (24) and contraction of the circumferential ring of actin filaments located at the zonula adherens of epithelial cells (9). In Dictyostelium cells lacking myosin II, the plasma membrane surface was much more easily deformed than in the wild type, suggesting that myosin II plays a role in "stiffening" the cell cortex (19). Myosin II has also been shown to have an important role in cell migration, and a number of studies have examined the interactions of actin and the myosins in cellular extensions such as lamellipodia and filopodia (12, 26, 32). Svitkina et al. (26) carefully examined the dynamics of actin and myosin II in the motility of fish epidermal keratocytes in culture. A gradient of discrete, variable-size clusters of bipolar myosin II minifilaments were identified in actin-rich lamellipodial extensions. Larger myosin II filaments were apparent in the region proximal to the cell body, whereas smaller bipolar filaments extended to the periphery, where myosin II was much less abundant. These authors developed a model of cell motility that proposed dynamic network contraction to draw the cell body toward the leading edge. The model is consistent with 1) the observed actin and myosin filament orientation and 2) the migration of keratocytes in culture. However, there remains the question of the functional role for the motile events in situ.

What might be the function of myosin II in parietal cell stimulation? In both freshly isolated gastric glands and cultured parietal cells, stimulation by the histamine/cAMP pathway leads to accumulation of myosin IIA toward the BL surface, tending to colocalize with F-actin at that surface. This stimulus-associated distribution did not occur with other gastric epithelial cells; in fact, myosin IIA in chief cells and mucous neck cells tended to be localized to the apical pole along with apical F-actin (these nonparietal cells had virtually no F-actin at the BL surface). Redistribution of myosin II in the cultured parietal cells was more dramatic than in the glands as stimulation was often associated with elaborate, actin-rich, lamellipodial extensions. Previous work showed that lamellipodial F-actin has distinctly different properties from the apical microvillar F-actin in parietal cells (3). The former is a high-turnover pool, now known to include myosin II motor protein, and the latter is a very stable F-actin pool of bundled microfilaments. The recruitment and association of myosin with basolateral F-actin may be a mechanism to structurally "rigidify" the basolateral membrane to provide a physical advantage to the cell. In the isolated gland, or in situ, the development of a stabilized BL surface may help counteract osmotic effects that occur as water is transported across the apical surface. When parietal cells are laid down in culture, the polarity is reorganized as a part of the BL membrane attaches to the matrix, analogous to the in situ attachments with basement membrane and connective tissue. Rather than the recruitment to the cortex that one sees in situ, stimulation of cultured parietal cells via the histamine/cAMP pathway recruits myosin to the motile lamellipodial extensions that seek increased surface contact.

Our initial finding that BDM inhibited AP uptake in SLO-permeabilized gastric glands suggested that myosin might have a direct role in acid secretory function (Fig. 4A); however, the effects of BDM on increased proton leakage cast doubt on these data (Fig. 4B). Application of BDM to the parietal cell culture system provided much more distinctive data regarding stimulus-dependent morphological changes as well as location and redistribution of myosin II within the cells. Disruption of myosin ATPase activity with BDM virtually abolished lamellipodial formation in cultured parietal cells but had no obvious effect on the recruitment of H,K-ATPase-rich membranes to the apical surfaces. This suggests that myosin motors, at least those known to be inhibited by BDM (myosin II and V), are not essential for the regulated apical trafficking associated with HCl secretion. The 25% reduction in size of stimulated vacuoles of BDM-treated cells (Table 1) may be due to the protonophoric action of the reagent and not to an effect on myosin per se. Moreover, the fact that BDM clearly disturbed stimulus-dependent myosin II redistribution and lamellipodial formation indicates that the drug was having its well-known effect on myosin ATPase at the concentrations typically required.

The MLCK inhibitor ML-7 had relatively minor effects on lamellipodial activity associated with stimulation. This suggests that myosin II does not require phosphorylation of its regulatory light chain for activation and incorporation into lamellipodia or that the light chain is primarily in an active state before stimulation. Moreover, ML-7 had no effect on the stimulation-associated translocation of H,K-ATPase; the marked inability of vacuolar swelling is almost certainly due to the potent protonophoric effects reported for ML-7 (2) and confirmed in the present study. Together, these data demonstrate stimulation-related structural effects for myosin II in the cortical cytoskeleton at the BL membrane of parietal cells. Specific disruption of the BL actomyosin cytoskeleton had no demonstrable effect of the recruitment of H,K-ATPase-rich vesicles into the apical secretory membrane.


    DISCLOSURES
 
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56292 (to X. Yao) and DK-10141 and DK-38972 (to J. G. Forte). The mass spectral data were provided by the University of California-San Francisco Mass Spectrometry Facility (A. L. Burlingame, Director) supported by the Biomedical Research Technology Program of the National Center for Research Resources (RR-01614 and RR-12961).


    ACKNOWLEDGMENTS
 
We thank Dr. Yoshito Kaziro (Tokyo Institute of Technology, Yokohama, Japan) for the RalA cDNA construct. We thank Dr. Curtis Okamoto for discussion and suggestions during this study.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Forte, Dept. of Molecular and Cell Biology, 245 LSA, MC#3200, Univ. of California, Berkeley, CA 94720 (E-mail: jforte{at}uclink.berkeley.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Agnew BJ, Duman JG, Watson CL, Coling DE, and Forte JG. Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade. J Cell Sci 112: 2639–2646, 1999.[Abstract/Free Full Text]

2. Akagi K, Nagao T, and Urushidani T. Responsiveness of {beta}-escin-permeabilized rabbit gastric gland model: effects of functional peptide fragments. Am J Physiol Gastrointest Liver Physiol 277: G736–G744, 1999.[Abstract/Free Full Text]

3. Ammar DA, Nguyen PN, and Forte JG. Functionally distinct pools of actin in secretory cells. Am J Physiol Cell Physiol 281: C407–C417, 2001.[Abstract/Free Full Text]

4. Ammar DA, Zhou R, Forte JG, and Yao X. Syntaxin 3 is required for cAMP-induced acid secretion: streptolysin O-permeabilized gastric gland model. Am J Physiol Gastrointest Liver Physiol 282: G23–G33, 2002.[Abstract/Free Full Text]

5. Berglindh T and Obrink KJ. A method for preparing isolated glands from the rabbit gastric mucosa. Acta Physiol Scand 96: 150–159, 1976.[ISI][Medline]

6. Bi GQ, Morris RL, Liao G, Alderton JM, Scholey JM, and Steinhardt RA. Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis. J Cell Biol 138: 999–1008, 1997.[Abstract/Free Full Text]

7. Black JA, Forte TM, and Forte JG. The effects of microfilament disrupting agents on HCl secretion and ultrastructure of piglet gastric oxyntic cells. Gastroenterology 83: 595–604, 1982.[ISI][Medline]

8. Brymora A, Cousin MA, Roufogalis BD, and Robinson PJ. Enhanced protein recovery and reproducibility from pull-down assays and immunoprecipitations using spin columns. Anal Biochem 295: 119–122, 2001.[ISI][Medline]

9. Castillo AM, Lagunes R, Urban M, Frixione E, and Meza I. Myosin II-actin interaction in MDCK cells: role in cell shape changes in response to Ca2+ variations. J Muscle Res Cell Motil 19: 557–574, 1998.[ISI][Medline]

10. Chew CS, Parente JA Jr, Chen X, Chaponnier C, and Cameron RS. The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia. J Cell Sci 113: 2035–2045, 2000.[Abstract/Free Full Text]

11. Cramer LP and Mitchison TJ. Myosin is involved in postmitotic cell spreading. J Cell Biol 131: 179–189, 1995.[Abstract]

12. Diefenbach TJ, Latham VM, Yimlamai D, Liu CA, Herman IM, and Jay DG. Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone. J Cell Biol 158: 1207–1217, 2002.[Abstract/Free Full Text]

13. Eddy RJ, Pierini LM, Matsumura F, and Maxfield FR. Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration. J Cell Sci 113: 1287–1298, 2000.[Abstract/Free Full Text]

14. Forte JG, Black JA, Forte TM, Machen TE, and Wolosin JM. Ultrastructural changes related to functional activity in gastric oxyntic cells. Am J Physiol Gastrointest Liver Physiol 241: G349–G358, 1981.[Abstract/Free Full Text]

15. Forte JG and Yao X. The membrane recruitment and recycling hypothesis of gastric HCl secretion. Trends Cell Biol 6: 45–48, 1996.[ISI]

16. Forte TM, Machen TE, and Forte JG. Ultrastructural changes in oxyntic cells associated with secretory function: a membrane-recycling hypothesis. Gastroenterology 73: 941–955, 1977.[ISI][Medline]

17. Hecht G and Koutsouris A. Myosin regulation of NKCC1: effects on cAMP-mediated Cl secretion in intestinal epithelia. Am J Physiol Cell Physiol 277: C441–C447, 1999.[Abstract/Free Full Text]

18. Higuchi H and Takemori S. Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle. J Biochem (Tokyo) 105: 638–643, 1989.[Abstract]

19. Pasternak C, Spudich JA, and Elson EL. Capping of surface receptors and concomitant cortical tension are generated by conventional myosin. Nature 341: 549–551, 1989.[ISI][Medline]

20. Reenstra WW and Forte JG. Isolation of H+,K+-ATPase-containing membranes from the gastric oxyntic cell. Methods Enzymol 192: 151–165, 1990.[Medline]

21. Rosenfeld J, Capdevielle J, Guillemot JC, and Ferrara P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203: 173–179, 1992.[ISI][Medline]

22. Rossmann H, Bachmann O, Wang Z, Shull GE, Obermaier B, Stuart-Tilley A, Alper SL, and Seidler U. Differential expression and regulation of AE2 anion exchanger subtypes in rabbit parietal and mucous cells. J Physiol 534: 837–848, 2001.[Abstract/Free Full Text]

23. Ruchhoeft ML and Harris WA. Myosin functions in Xenopus retinal ganglion cell growth cone motility in vivo. J Neurobiol 32: 567–578, 1997.[ISI][Medline]

24. Sabry JH, Moores SL, Ryan S, Zang JH, and Spudich JA. Myosin heavy chain phosphorylation sites regulate myosin localization during cytokinesis in live cells. Mol Biol Cell 8: 2605–2615, 1997.[Abstract/Free Full Text]

25. Sellers JR. Myosins: a diverse superfamily. Biochim Biophys Acta 1496: 3–22, 2000.[ISI][Medline]

26. Svitkina TM, Verkhovsky AB, McQuade KM, and Borisy GG. Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J Cell Biol 139: 397–415, 1997.[Abstract/Free Full Text]

27. Thomas HA and Machen TE. Regulation of Cl/HCO3 exchange in gastric parietal cells. Cell Regul 2: 727–737, 1991.[ISI][Medline]

28. Thomas HA, Machen TE, Smolka A, Baron R, and Kopito RR. Identification of a 185-kDa band 3-related polypeptide in oxyntic cells. Am J Physiol Cell Physiol 257: C537–C544, 1989.[Abstract/Free Full Text]

29. Tyagarajan K, Chow DC, Smolka A, and Forte JG. Structural interactions between alpha- and beta-subunits of the gastric H,K-ATPase. Biochim Biophys Acta 1236: 105–113, 1995.[ISI][Medline]

30. Urwyler N, Eggli P, and Keller HU. Effects of the myosin inhibitor 2,3-butanedione monoxime (BDM) on cell shape, locomotion and fluid pinocytosis in human polymorphonuclear leucocytes. Cell Biol Int 24: 863–870, 2000.[ISI][Medline]

31. Wang FS, Wolenski JS, Cheney RE, Mooseker MS, and Jay DG. Function of myosin-V in filopodial extension of neuronal growth cones. Science 273: 660–663, 1996.[Abstract]

32. Wylie SR and Chantler PD. Separate but linked functions of conventional myosins modulate adhesion and neurite outgrowth. Nat Cell Biol 3: 88–92, 2001.[ISI][Medline]

33. Wylie SR, Wu PJ, Patel H, and Chantler PD. A conventional myosin motor drives neurite outgrowth. Proc Natl Acad Sci USA 95: 12967–12972, 1998.[Abstract/Free Full Text]

34. Yagi N, Takemori S, Watanabe M, Horiuti K, and Amemiya Y. Effects of 2,3-butanedione monoxime on contraction of frog skeletal muscles: an X-ray diffraction study. J Muscle Res Cell Motil 13: 153–160, 1992.[ISI][Medline]

35. Yao X, Chaponnier C, Gabbiani G, and Forte JG. Polarized distribution of actin isoforms in gastric parietal cells. Mol Biol Cell 6: 541–557, 1995.[Abstract]

36. Yao X, Thibodeau A, and Forte JG. Ezrin-calpain I interactions in gastric parietal cells. Am J Physiol Cell Physiol 265: C36–C46, 1993.[Abstract/Free Full Text]