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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
acid secretion; cytoskeleton; ion channels and pumps
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 0.71.0,
expression of protein was induced by addition of 0.5 mM
isopropyl--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 616% 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
-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 100200
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
1015 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
616% gradient gels and transferred onto nitrocellulose membrane.
Proteins were probed by appropriate primary antibodies and detected with
enhanced chemiluminescence (Pierce).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
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.
|
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.
|
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.
|
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.
|
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.
|
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.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-actin being
most abundant at the BL surface and
-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 |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Akagi K, Nagao
T, and Urushidani T. Responsiveness of -escin-permeabilized rabbit
gastric gland model: effects of functional peptide fragments. Am J
Physiol Gastrointest Liver Physiol 277:
G736G744, 1999.
3. Ammar DA,
Nguyen PN, and Forte JG. Functionally distinct pools of actin in secretory
cells. Am J Physiol Cell Physiol
281: C407C417,
2001.
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:
G23G33, 2002.
5. Berglindh T and Obrink KJ. A method for preparing isolated glands from the rabbit gastric mucosa. Acta Physiol Scand 96: 150159, 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:
9991008, 1997.
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: 595604, 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: 119122, 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: 557574, 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:
20352045, 2000.
11. Cramer LP and Mitchison TJ. Myosin is involved in postmitotic cell spreading. J Cell Biol 131: 179189, 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:
12071217, 2002.
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:
12871298, 2000.
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:
G349G358, 1981.
15. Forte JG and Yao X. The membrane recruitment and recycling hypothesis of gastric HCl secretion. Trends Cell Biol 6: 4548, 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: 941955, 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:
C441C447, 1999.
18. Higuchi H and Takemori S. Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle. J Biochem (Tokyo) 105: 638643, 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: 549551, 1989.[ISI][Medline]
20. Reenstra WW and Forte JG. Isolation of H+,K+-ATPase-containing membranes from the gastric oxyntic cell. Methods Enzymol 192: 151165, 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: 173179, 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:
837848, 2001.
23. Ruchhoeft ML and Harris WA. Myosin functions in Xenopus retinal ganglion cell growth cone motility in vivo. J Neurobiol 32: 567578, 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:
26052615, 1997.
25. Sellers JR. Myosins: a diverse superfamily. Biochim Biophys Acta 1496: 322, 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:
397415, 1997.
27. Thomas HA and Machen TE. Regulation of Cl/HCO3 exchange in gastric parietal cells. Cell Regul 2: 727737, 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:
C537C544, 1989.
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: 105113, 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: 863870, 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: 660663, 1996.[Abstract]
32. Wylie SR and Chantler PD. Separate but linked functions of conventional myosins modulate adhesion and neurite outgrowth. Nat Cell Biol 3: 8892, 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: 1296712972,
1998.
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: 153160, 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: 541557, 1995.[Abstract]
36. Yao X,
Thibodeau A, and Forte JG. Ezrin-calpain I interactions in gastric
parietal cells. Am J Physiol Cell Physiol
265: C36C46,
1993.