Non-Muscle Myosin IIA Differentially Regulates Intestinal Epithelial Cell Restitution and Matrix Invasion
2009; Elsevier BV; Volume: 174; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.080171
ISSN1525-2191
AutoresBrian A. Babbin, Stefan Koch, Moshe Bachar, Mary-Anne Conti, Charles A. Parkos, Robert Adelstein, Asma Nusrat, Andrei I. Ivanov,
Tópico(s)Wound Healing and Treatments
ResumoEpithelial cell motility is critical for self-rejuvenation of normal intestinal mucosa, wound repair, and cancer metastasis. This process is regulated by the reorganization of the F-actin cytoskeleton, which is driven by a myosin II motor. However, the role of myosin II in regulating epithelial cell migration remains poorly understood. This study addressed the role of non-muscle myosin (NM) IIA in two different modes of epithelial cell migration: two-dimensional (2-D) migration that occurs during wound closure and three-dimensional (3-D) migration through a Matrigel matrix that occurs during cancer metastasis. Pharmacological inhibition or siRNA-mediated knockdown of NM IIA in SK-CO15 human colonic epithelial cells resulted in decreased 2-D migration and increased 3-D invasion. The attenuated 2-D migration was associated with increased cell adhesiveness to collagen and laminin and enhanced expression of β1-integrin and paxillin. On the 2-D surface, NM IIA-deficient SK-CO15 cells failed to assemble focal adhesions and F-actin stress fibers. In contrast, the enhanced invasion of NM IIA-depleted cells was dependent on Raf-ERK1/2 signaling pathway activation, enhanced calpain activity, and increased calpain-2 expression. Our findings suggest that NM IIA promotes 2-D epithelial cell migration but antagonizes 3-D invasion. These observations indicate multiple functions for NM IIA, which, along with the regulation of the F-actin cytoskeleton and cell-matrix adhesions, involve previously unrecognized control of intracellular signaling and protein expression. Epithelial cell motility is critical for self-rejuvenation of normal intestinal mucosa, wound repair, and cancer metastasis. This process is regulated by the reorganization of the F-actin cytoskeleton, which is driven by a myosin II motor. However, the role of myosin II in regulating epithelial cell migration remains poorly understood. This study addressed the role of non-muscle myosin (NM) IIA in two different modes of epithelial cell migration: two-dimensional (2-D) migration that occurs during wound closure and three-dimensional (3-D) migration through a Matrigel matrix that occurs during cancer metastasis. Pharmacological inhibition or siRNA-mediated knockdown of NM IIA in SK-CO15 human colonic epithelial cells resulted in decreased 2-D migration and increased 3-D invasion. The attenuated 2-D migration was associated with increased cell adhesiveness to collagen and laminin and enhanced expression of β1-integrin and paxillin. On the 2-D surface, NM IIA-deficient SK-CO15 cells failed to assemble focal adhesions and F-actin stress fibers. In contrast, the enhanced invasion of NM IIA-depleted cells was dependent on Raf-ERK1/2 signaling pathway activation, enhanced calpain activity, and increased calpain-2 expression. Our findings suggest that NM IIA promotes 2-D epithelial cell migration but antagonizes 3-D invasion. These observations indicate multiple functions for NM IIA, which, along with the regulation of the F-actin cytoskeleton and cell-matrix adhesions, involve previously unrecognized control of intracellular signaling and protein expression. In the human intestine, a single layer of polarized epithelial cells creates a protective barrier that separates the body interior from the intestinal lumen. This barrier is capable of withstanding a variety of mechanical, chemical, and biological stressors and represents a dynamic self-renewing entity. The dynamic nature of the normal intestinal epithelium is illustrated by the fact that this epithelial sheet is constantly moving at a speed of 5 to 10 μm/h.1Heath JP Epithelial cell migration in the intestine.Cell Biol Int. 1996; 20: 139-146Crossref PubMed Scopus (151) Google Scholar This movement plays a vital role in the normal life cycle of intestinal epithelial cells. Indeed, epitheliocytes that originate from stem cells at the bottom of intestinal crypts differentiate and migrate along the crypt-surface axis and eventually shed from the intestinal surface.2Radtke F Clevers H Self-renewal and cancer of the gut: two sides of a coin.Science. 2005; 307: 1904-1909Crossref PubMed Scopus (583) Google Scholar In addition to its role in normal intestinal homeostasis, epithelial cell migration significantly contributes to the pathophysiology of intestinal disorders such as inflammatory bowel disease and colorectal cancer. In the former disease, a collective movement of the epithelial sheet plays a major role in closure of mucosal wounds, which is known as epithelial restitution.3Blikslager AT Moeser AJ Gookin JL Jones SL Odle J Restoration of barrier function in injured intestinal mucosa.Physiol Rev. 2007; 87: 545-564Crossref PubMed Scopus (421) Google Scholar, 4Wilson AJ Gibson PR Epithelial migration in the colon: filling in the gaps.Clin Sci (Lond). 1997; 93: 97-108PubMed Google Scholar In the latter pathology, invasion of epithelial cancer cells into underlying tissues results in tumor dissemination.5Condeelis J Singer RH Segall JE The great escape: when cancer cells hijack the genes for chemotaxis and motility.Annu Rev Cell Dev Biol. 2005; 21: 695-718Crossref PubMed Scopus (283) Google Scholar, 6Sahai E Illuminating the metastatic process.Nat Rev Cancer. 2007; 7: 737-749Crossref PubMed Scopus (441) Google Scholar Thus, epithelial cell motility is a fundamental feature of normal intestinal mucosal physiology, mucosal repair, and cancer metastasis. Cell migration is generally considered as a cyclic process initiated by extension of protrusions in the direction of migration and completed by the retraction of the trailing end of the cell.7Lauffenburger DA Horwitz AF Cell migration: a physically integrated molecular process.Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3286) Google Scholar, 8Ridley AJ Schwartz MA Burridge K Firtel RA Ginsberg MH Borisy G Parsons JT Horwitz AR Cell migration: integrating signals from front to back.Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3860) Google Scholar Reorganization of actin filaments drives the entire migration cycle by generating forces to extend membrane protrusions and to move the cell body forward. This reorganization of filamentous (F)-actin is mediated by two major mechanisms: the so-called F-actin “treadmilling” that involves actin polymerization and depolymerization at opposite filament ends, and contraction of filaments driven by the myosin II motor.7Lauffenburger DA Horwitz AF Cell migration: a physically integrated molecular process.Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3286) Google Scholar, 8Ridley AJ Schwartz MA Burridge K Firtel RA Ginsberg MH Borisy G Parsons JT Horwitz AR Cell migration: integrating signals from front to back.Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3860) Google Scholar, 9Small JV Resch GP The comings and goings of actin: coupling protrusion and retraction in cell motility.Curr Opin Cell Biol. 2005; 17: 517-523Crossref PubMed Scopus (144) Google Scholar Whereas F-actin treadmilling is known to mediate protrusions at the migrating cell front, the roles of myosin II in cell motility appear to be more diverse and involve regulation of protrusion dynamics, cell-matrix adhesions, and forward translocation of the cell body.7Lauffenburger DA Horwitz AF Cell migration: a physically integrated molecular process.Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3286) Google Scholar, 8Ridley AJ Schwartz MA Burridge K Firtel RA Ginsberg MH Borisy G Parsons JT Horwitz AR Cell migration: integrating signals from front to back.Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3860) Google Scholar, 9Small JV Resch GP The comings and goings of actin: coupling protrusion and retraction in cell motility.Curr Opin Cell Biol. 2005; 17: 517-523Crossref PubMed Scopus (144) Google Scholar, 10Kaverina I Krylyshkina O Small JV Regulation of substrate adhesion dynamics during cell motility.Int J Biochem Cell Biol. 2002; 34: 746-761Crossref PubMed Scopus (222) Google Scholar Therefore, myosin II-driven contractility can be considered as a key mechanism integrating different steps of cell migration. Myosin II is a motor protein that utilizes ATP to move actin filaments. This motor functions as a heterohexamer composed of two heavy chains and two pairs of light chains.11De La Cruz EM Ostap EM Relating biochemistry and function in the myosin superfamily.Curr Opin Cell Biol. 2004; 16: 61-67Crossref PubMed Scopus (223) Google Scholar, 12Maciver SK Myosin II function in non-muscle cells.Bioessays. 1996; 18: 179-182Crossref PubMed Scopus (61) Google Scholar The heavy chain consists of a globular head that binds to actin and hydrolyzes ATP and an extended tail that coils together with another heavy chain tail to form a rigid rod-like structure. The tails of multiple myosin II molecules readily self-associate, creating bipolar myosin aggregates that are crucial for actin filament movement and bundling.11De La Cruz EM Ostap EM Relating biochemistry and function in the myosin superfamily.Curr Opin Cell Biol. 2004; 16: 61-67Crossref PubMed Scopus (223) Google Scholar, 12Maciver SK Myosin II function in non-muscle cells.Bioessays. 1996; 18: 179-182Crossref PubMed Scopus (61) Google Scholar Epithelial cells express non-muscle myosin (NM) II, which is characterized by three different heavy chain isoforms: IIA, IIB, and IIC.13Golomb E Ma X Jana SS Preston YA Kawamoto S Shoham NG Goldin E Conti MA Sellers JR Adelstein RS Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family.J Biol Chem. 2004; 279: 2800-2808Crossref PubMed Scopus (259) Google Scholar, 14Phillips CL Yamakawa K Adelstein RS Cloning of the cDNA encoding human nonmuscle myosin heavy chain-B and analysis of human tissues with isoform-specific antibodies.J Muscle Res Cell Motil. 1995; 16: 379-389Crossref PubMed Scopus (107) Google Scholar These isoforms possess a high degree (64% to 80%) of sequence similarity, but have different enzymatic/biochemical properties.15Ivanov AI Bachar M Babbin BA Adelstein RS Nusrat A Parkos CA A unique role for nonmuscle myosin heavy chain IIA in regulation of epithelial apical junctions.PLoS ONE. 2007; 2: e658Crossref PubMed Scopus (129) Google Scholar, 16Kovacs M Wang F Hu A Zhang Y Sellers JR Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform.J Biol Chem. 2003; 278: 38132-38140Crossref PubMed Scopus (188) Google Scholar As a result, different NM II heavy chains may have either unique15Ivanov AI Bachar M Babbin BA Adelstein RS Nusrat A Parkos CA A unique role for nonmuscle myosin heavy chain IIA in regulation of epithelial apical junctions.PLoS ONE. 2007; 2: e658Crossref PubMed Scopus (129) Google Scholar, 17Cai Y Biais N Giannone G Tanase M Jiang G Hofman JM Wiggins CH Silberzan P Buguin A Ladoux B Sheetz MP Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow.Biophys J. 2006; 91: 3907-3920Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 18Jana SS Kawamoto S Adelstein RS A specific isoform of nonmuscle myosin II-C is required for cytokinesis in a tumor cell line.J Biol Chem. 2006; 281: 24662-24670Crossref PubMed Scopus (46) Google Scholar, 19Sandquist JC Swenson KI Demali KA Burridge K Means AR Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration.J Biol Chem. 2006; 281: 35873-35883Crossref PubMed Scopus (146) Google Scholar, 20Togo T Steinhardt RA Nonmuscle myosin IIA and IIB have distinct functions in the exocytosis-dependent process of cell membrane repair.Mol Biol Cell. 2004; 15: 688-695Crossref PubMed Scopus (88) Google Scholar or interchangeable roles21Bao J Jana SS Adelstein RS Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis.J Biol Chem. 2005; 280: 19594-19599Crossref PubMed Scopus (90) Google Scholar, 22Bao J Ma X Liu C Adelstein RS Replacement of nonmuscle myosin II-B with II-A rescues brain but not cardiac defects in mice.J Biol Chem. 2007; 282: 22102-22111Crossref PubMed Scopus (68) Google Scholar in regulating cell shape, cell adhesion, cytokinesis, and vesicular traffic. Several recent studies have yielded conflicting data on the involvement of NM II in epithelial cell migration. Thus, pharmacological inhibition of NM II with blebbistatin was shown to attenuate migration of pancreatic and renal epithelial cells,23Duxbury MS Ashley SW Whang EE Inhibition of pancreatic adenocarcinoma cellular invasiveness by blebbistatin: a novel myosin II inhibitor.Biochem Biophys Res Commun. 2004; 313: 992-997Crossref PubMed Scopus (65) Google Scholar, 24Gupton SL Waterman-Storer CM Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration.Cell. 2006; 125: 1361-1374Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar but reportedly did not affect motility of mammary and prostate epithelial cells.25Bastian P Lang K Niggemann B Zaenker KS Entschladen F Myosin regulation in the migration of tumor cells and leukocytes within a three-dimensional collagen matrix.Cell Mol Life Sci. 2005; 62: 65-76Crossref PubMed Scopus (35) Google Scholar In other studies, small interfering (si)RNA-mediated knockdown of the NM II heavy chain A isoform (hereafter referred to as NM IIA) was found to suppress migration of mammary epithelial cells26Betapudi V Licate LS Egelhoff TT Distinct roles of nonmuscle myosin II isoforms in the regulation of MDA-MB-231 breast cancer cell spreading and migration.Cancer Res. 2006; 66: 4725-4733Crossref PubMed Scopus (189) Google Scholar but enhanced the motility of lung epithelial cells.19Sandquist JC Swenson KI Demali KA Burridge K Means AR Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration.J Biol Chem. 2006; 281: 35873-35883Crossref PubMed Scopus (146) Google Scholar These contradictory data may reflect peculiar behavior of different cell lines, as well as different experimental conditions used to study cell migration.24Gupton SL Waterman-Storer CM Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration.Cell. 2006; 125: 1361-1374Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 27Nakayama M Amano M Katsumi A Kaneko T Kawabata S Takefuji M Kaibuchi K Rho-kinase and myosin II activities are required for cell type and environment specific migration.Genes Cells. 2005; 10: 107-117Crossref PubMed Scopus (65) Google Scholar Despite its biological importance, the role of NM II in migration of intestinal epithelial cells has not yet been studied. The present study was designed to investigate the role of NM IIA in intestinal epithelial cell migration. This myosin II isoform was shown to be a major generator of traction forces in motile cells17Cai Y Biais N Giannone G Tanase M Jiang G Hofman JM Wiggins CH Silberzan P Buguin A Ladoux B Sheetz MP Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow.Biophys J. 2006; 91: 3907-3920Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar and it is abundantly expressed in both well-differentiated epithelial cells15Ivanov AI Bachar M Babbin BA Adelstein RS Nusrat A Parkos CA A unique role for nonmuscle myosin heavy chain IIA in regulation of epithelial apical junctions.PLoS ONE. 2007; 2: e658Crossref PubMed Scopus (129) Google Scholar and their embryonic precursors.28Conti MA Even-Ram S Liu C Yamada KM Adelstein RS Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-A in mice.J Biol Chem. 2004; 279: 41263-41266Crossref PubMed Scopus (271) Google Scholar The role of NM IIA was examined by using two different models of cell migration: a two-dimensional (2-D or planar) wound closure assay and a three-dimensional (3-D) Matrigel invasion assay, resembling restitution of injured epithelial sheets, and metastatic dissemination of colorectal tumors, respectively. We report that inhibition of NM IIA oppositely affects epithelial cell restitution and invasion via multiple mechanisms that involve alterations in cell-matrix adhesion and F-actin organization, as well as profound changes in intracellular signaling and protein expression. The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect matrix adhesion, cytoskeletal, and signaling proteins by immunoblotting and immunofluorescence labeling: anti-NM IIA pAb (Covance, Berkley, CA); anti-paxillin, anti-β1-integrin, anti-focal adhesion kinase (FAK), and anti-vinculin mAbs (BD Biosciences, San Jose, CA); anti-phosphorylated (Tyr118) paxillin, anti-phospho-(Tyr397) FAK, anti-total extracellular signal-regulated kinase (ERK) 1/2, anti-phospho-ERK1/2, anti-total Raf-1, anti-phospho-(Ser338) Raf-1, and anti-matrix metalloprotease (MMP)-2 pAbs (Cell Signaling Technology Inc., Beverly, MA); anti-calpain-1 and anti-calpain-2 pAbs (Santa Cruz Biotechnology, Santa Cruz, CA); anti-MMP-9 pAb (Upstate Biotechnology, Lake Placid, NY); anti-MMP-2 and anti-MMP-7 mAbs (EMD Chemicals Inc., Gibbstown, NJ); and, anti-β-actin pAb and anti-α-tubulin mAb (Sigma-Aldrich, St. Louis, MO). Alexa-488 or Alexa-568 dye-conjugated, donkey anti-rabbit and goat anti-mouse secondary antibodies, and Alexa-labeled phalloidin, were obtained from Invitrogen (Carlsbad, CA); horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). S(-)-blebbistatin was obtained from Sigma; U0126, PD 098059, N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal (ALLN), and calpeptin were purchased from EMD Biosciences; and GM-6001 was obtained from Biomol International (Plymouth Meeting, PA). All other reagents were of the highest analytical grade and were obtained from Sigma. SK-CO15, a transformed human colonic epithelial cell line,29Le Bivic A Real FX Rodriguez-Boulan E Vectorial targeting of apical and basolateral plasma membrane proteins in a human adenocarcinoma epithelial cell line.Proc Natl Acad Sci USA. 1989; 86: 9313-9317Crossref PubMed Scopus (150) Google Scholar was a gift from Dr. Enrique Rodriguez-Boulan (Weill Medical College of Cornell University, NY). Caco-2 and T84-transformed human colonic epithelial cell lines, IEC-6, a non-tumorigenic rat intestinal epithelial cell line and COS-7, green monkey kidney epithelial cells were purchased from the American Type Culture Collection (Manassas, VA). SK-CO15, Caco-2, and Cos-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 15 mmol/L HEPES, 1% nonessential amino acids, 40 μg/ml penicillin, and 100 μg/ml streptomycin, pH 7.4. For IEC-6 culture, 0.1% insulin was added to the Dulbecco's modified Eagle's medium. T84 cells were cultured in the 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 5% newborn calf serum, 10 mmol/L HEPES, 14 mmol/L Na HCO3, 40 μg/ml penicillin, and 100 μg/ml streptomycin, pH 7.4. For immunolabeling experiments, epithelial cells were grown on collagen-coated coverslips. For other assays, the cells were cultured on plastic plates. SK-CO15 cells were grown to confluency on collagen-I-coated 24-well culture plates after which a single linear wound was created through the monolayers using a sterile pipette tip. Monolayers were washed to remove cellular debris and placed in complete media. Sites at which wounds were to be measured were marked on the undersurface of the wells to ensure that measurements were taken at the same place. Wounds were imaged at 0 and 6 hours on a Zeiss Axiovert microscope with an attached CCD-camera. Stock solutions of blebbistatin and other inhibitors were diluted in cell culture medium and added to cell monolayers immediately after wounding. SK-CO15 cells transfected with NM IIA-specific or control siRNA were wounded at 84 hours post-transfection. Wound widths were measured from the images using Scion Image software (Scion Image Corp., Frederick, MD). Ten measurements along the wound length were averaged to determine wound widths and the distance (μm) the wound edges migrated into the wound space. For each experimental group, the migrating distances of two different cell monolayers was measured and averaged in a particular experiment, and the same experiment was independently performed three times. Cell invasion assay was performed using commercially available modified Boyden chambers layered with growth factor-reduced Matrigel (BD Biosciences) according to the established protocol.30Hotary KB Yana I Sabeh F Li XY Holmbeck K Birkedal-Hansen H Allen ED Hiraoka N Weiss SJ Matrix metalloproteinases (MMPs) regulate fibrin-invasive activity via MT1-MMP-dependent and -independent processes.J Exp Med. 2002; 195: 295-308Crossref PubMed Scopus (181) Google Scholar Chambers were hydrated and blocked in serum-free media containing 0.1% bovine serum albumin (BSA) for 2 hours at 37°C. SK-CO15 cells were trypsinized, washed in serum-free media, and counted. An equal number of cells (1 × 105 cells) were loaded into upper chambers in serum-free media containing 0.1% BSA. Compete media was placed in lower wells. After a 24-hour incubation at 37°C (5% CO2), the upper chamber was cleared of cells using a cotton swab tip. The inserts were then fixed in a solution of 3.7% paraformaldehyde containing 0.1% crystal violet. The number of cells on the undersurface of membranes (ie, invaded cells) was counted using bright field microscopy. Cell number in three different ×100 objective fields were counted and averaged for each group in a particular experiment, and each experiment was independently performed three times. In experiments involving pharmacological inhibitors, the inhibitors were added to both the upper and lower chambers. SK-CO15 cells transfected with NM IIA-specific or control siRNAs were loaded into invasion chamber at 72 hours post-transfection. The cell adhesion assay was performed as previously described.31Yokosaki Y Palmer EL Prieto AL Crossin KL Bourdon MA Pytela R Sheppard D The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.J Biol Chem. 1994; 269: 26691-26696Abstract Full Text PDF PubMed Google Scholar Briefly, SK-CO15 cells were transfected with siRNA and, 84 hours post transfection, were trypsinized and washed in HEPES-buffered Hanks balanced salt solution (HBSS). Cells were resuspended in HBSS containing 0.1% BSA and equal numbers of cells (2.5 × 104) were plated in collagen I or laminin-coated 96 well plates that had been blocked with the HBSS-BSA for 2 hours. Cells were then incubated at 37°C for 1 hour then gently washed three times with HBSS. Adherent cells were fixed and stained with 3.7% paraformaldehyde containing 0.1% crystal violet. Cell adhesion was then assessed using a microplate reader by analyzing absorbance at 570 nm. Cell monolayers were fixed/permeabilized in 100% methanol (−20°C for 20 minutes), blocked in HBSS containing 1% BSA (blocking buffer) for 60 minutes at room temperature, and incubated for another 60 minutes with primary antibodies diluted in blocking buffer. Monoclonal and polyclonal primary antibodies were used at final concentrations of 2.5 to 5 μg/ml and 0.8 to 2 μg/ml respectively. Cells were then washed, incubated for 60 minutes with Alexa dye-conjugated secondary antibodies, rinsed with HBSS and mounted on slides with ProLong Antifade medium (Molecular Probes). For fluorescent double-labeling of myosin II isoforms with F-actin, monolayers were fixed in 100% ethanol (−20°C for 20 minutes) and sequentially stained with primary anti-myosin II heavy chain and Alexa dye-conjugated secondary antibodies, whereas F-actin was labeled with Alexa-conjugated phalloidin. For tissue labeling, frozen sections (5 μm thickness) of normal human colonic mucosa were mounted on glass coverslips, air-dried, fixed in 100% ethanol (−20°C for 20 minutes), and immunolabeled as described above. In addition, H&E staining of colonic mucosa sections was performed to observe general tissue architecture and orientation. Stained cell monolayers and tissue sections were examined using a Zeiss LSM510 laser scanning confocal microscope (Zeiss Microimaging Inc., Thornwood, NY) coupled to a Zeiss 100M axiovert and ×63 or ×100 Pan-Apochromat oil lenses. The fluorescent dyes were imaged sequentially in frame-interlace mode to eliminate cross talk between channels. Images shown are representative of at least three experiments, with multiple images taken per slide. Cells were homogenized in a RIPA lysis buffer (20 mmol/L Tris, 50 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 1% sodium deoxycholate, 1% TX-100, and 0.1% SDS, pH 7.4), containing a proteinase inhibitor cocktail (1:100, Sigma) and phosphatase inhibitor cocktails 1 and 2 (both at 1:200, Sigma). Lysates were then cleared by centrifugation (20 minutes at 14,000 × g), diluted with 2× SDS sample buffer, and boiled. SDS polyacrylamide gel electrophoresis and immunoblotting were conducted by standard protocols with 10 to 20 μg protein per lane. Proteins of interest were visualized after their transfer onto nitrocellulose membranes using appropriate primary and horseradish peroxidase-conjugated secondary antibodies. Monoclonal and polyclonal primary antibodies were diluted to a final concentration of 0.5 to 1 μg/ml. Results shown are representative immunoblots of three independent experiments. Protein expression was quantified by densitometric analysis of immunoblot images using UN-SCAN-IT digitizing software (Silk Scientific, Orem, UT). Epithelial cells cultured in T75 flasks were scraped into RIPA buffer modified by the increased concentration of NaCl (250 mmol/L) and EGTA (5 mmol/L) and supplemented with 0.1 mmol/L phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, protease inhibitor cocktail (Sigma), 1 mmol/L dithiothreitol, and 5 mmol/L MgATP. After 10 minutes incubation on ice, the samples were centrifuged (10,000 × g, 10 minutes) and the supernatant was separated by SDS polyacrylamide gel electrophoresis. The Coomassie Blue stained bands near the 205 kDa molecular size marker were excised, destained, reduced and alkylated, digested with trypsin, and submitted to the National Heart Lung and Blood Institute Proteomics Core Facility for analysis by liquid chromatography tandem mass spectroscopy. Peptide numbers for each of the NM II heavy chain isoforms were counted and the percent contribution to total amount of myosin II heavy chain peptides was calculated. siRNA-mediated knock-down of NM IIA, was performed using either isoform-specific siRNA SmartPools (Dharmacon, Lafayette, CO) or the individual siRNA duplex-2 (5′-GGCCAAACCUGCCGAAUAAUU-3′) obtained from the same vendor. Cyclophilin B siRNA SmartPool or the individual cyclophilin B siRNA (5′-UCACCGUAGAUGCUCUUUCUU-3′) were used as controls. SK-CO15 cells were transfected using the DharmaFect 1 transfection reagent (Dharmacon) in Opti-MEM I medium (Invitrogen) according to manufacturer's protocol with a final siRNA concentration of 100 nmol/L. A plasmid encoding full-length enhanced green fluorescent protein (EGFP)-tagged NM IIA32Wei Q Adelstein RS Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells.Mol Biol Cell. 2000; 11: 3617-3627Crossref PubMed Scopus (153) Google Scholar was obtained from Addgene (Cambridge, MA). Control EGFP-C3 vector was provided by Dr. Kevin Bourzac (Oregon Health Science University, Portland OR). COS-7 cells were transfected with EGFP-NM IIA or control plasmid using Lipofectamine 2000 and analyzed at 48 hours post-transfection. The calpain activity assay was obtained from Biovision Research Products (Mount View, CA) and performed according to the manufacturer's protocol. Briefly, SK-CO15 cells were transfected with either NM IIA or control siRNAs, and 80 to 84 hours post-transfection, cells were trypsinized and counted. An equivalent number of cells (2 × 106) was pelleted, and the pellets were resuspended in supplied extraction buffer and incubated on ice for 20 minutes. After a brief centrifugation (10,000 × g, 1 minute), the obtained lysates were transferred to a Costar flat-bottomed black polystyrene 96-well assay plate, mixed with a fluorogenic calpain substrate and incubated for 1 hour at 37°C. The fluorescence intensity at 400 nm excitation and 505 nm emission wavelengths was measured using a Fluostar plate reader (BMG Labtechnologies, Durham, NC). Numerical values from individual experiments were pooled and expressed as mean ± SEM throughout. Obtained numbers were compared by a single-tailed Student's t-test, with statistical significance assumed at P < 0.05. By using reverse transcription-PCR, immunoblotting, and immunofluorescence analyses we recently demonstrated that human colonic epithelial cells express NM IIA, IIB, and IIC heavy chains.15Ivanov AI Bachar M Babbin BA Adelstein RS Nusrat A Parkos CA A unique role for nonmuscle myosin heavy chain IIA in regulation of epithelial apical junctions.PLoS ONE. 2007; 2: e658Crossref PubMed Scopus (129) Google Scholar However, these analyses did not allow a direct quantitative comparison of the levels of each NM II isoform. Such quantification is important because NM IIA, IIB, and IIC might be functionally redundant, and the most highly expressed isoform could therefore play the most significant physiological role.21Bao J Jana SS Adelstein RS Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis.J Biol Chem. 2005; 280: 19594-19599Crossref PubMed Scopus (90) Google Scholar, 22Bao J
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