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Originally published In Press as doi:10.1074/jbc.M502767200 on April 11, 2005

J. Biol. Chem., Vol. 280, Issue 25, 23741-23747, June 24, 2005
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Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells*

Wayne Chan{ddagger}, German Calderon{ddagger}, Amy L. Swift{ddagger}, Jamie Moseley{ddagger}, Shaohua Li{ddagger}, Hiroshi Hosoya§, Irwin M. Arias{ddagger}, and Daniel F. Ortiz{ddagger}||

From the {ddagger}Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, the §Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan, and the Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, March 14, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Localization of MDR1 (ABCB1), MDR2 (MDR3 in humans) (ABCB4), and BSEP/SPGP (ABCB11) in the plasma membrane of hepatocytes is restricted to the apical domain, where the transporters mediate ATP-dependent secretion of essential biliary constituents. BSEP transports bile salts (1), MDR1 secretes small cationic hydrophobic drugs (2), and MDR2 mediates phospholipid transfer into bile (3, 4). Genetic defects in BSEP produce progressive familial intrahepatic cholestasis type II (PFIC II) (5), and mutations in MDR3 cause PFIC III (6). Targeting of the transporters to the canalicular membrane is indispensable to their function. BSEP (7) and MDR3 (8) mutations that affect delivery to the apical membrane are associated with PFICII and cholestasis of pregnancy, respectively.

Newly synthesized BSEP is processed (9) in the early secretory pathway, conveyed from the trans-Golgi network to a RAB11a-containing endosomal compartment from which it cycles constitutively to the canalicular membrane (10). BSEP abundance in the apical membrane is regulated by cAMP, taurocholate (11–13), and changes in osmolarity (14). Delivery and cycling of BSEP to the canalicular membrane of hepatocytes and WifB cells requires microtubules (10, 11, 13). Imaging studies suggest that BSEP removal from the apical membrane of WifB cells is actin-dependent (10). In Madin-Darby canine kidney (MDCK)1 cells, BSEP internalization from the apical membrane is mediated by clathrin and regulated by HAX-1 (15), which directly binds BSEP and interacts with cortactin, an actin-binding protein that participates in clathrin-mediated endocytosis (16, 17). Little is known regarding proteins required for BSEP delivery to the apical membrane. A yeast two-hybrid screen identified myosin II regulatory light chain, MLC2, as a BSEP binding partner. Expression of a dominant negative MLC2 mutant reduced apical membrane BSEP levels, and pharmacological inhibition of myosin II impeded delivery of newly synthesized transporter to the apical membrane in MDCK cells. These findings indicate that myosin-II is required for BSEP trafficking to the apical membrane in polarized epithelial cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Antibodies—Cytotrap yeast two-hybrid system and vectors were purchased from Stratagene (La Jolla, CA), pEGFP-N1 and pEYFP-N2 vectors from Clontech (Palo Alto, CA), and pMAL-p2x from New England Biolabs (Beverly, MA). Monomeric pDsRed-N1 vector was a gift from Dr. Roger Tsien. Easytag NEG-772 [35S] methionine and cysteine protein labeling mix were from PerkinElmer Life Sciences (Boston, MA). c219 monoclonal antibody was obtained from Signet (Dedham, MA); fluorescently labeled secondary antibodies were from Jackson Immunochemicals (West Grove, PA) and Molecular Probes (Eugene, OR). Goat anti-MLC2 antibody (MLCA-20) and rabbit antiphospho MLC (MLCP) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-non-muscle myosin II heavy chain BT561 antiserum was from Biomedical Technologies (Stoughton, MA). Anti-FLAG M5 monoclonal antibody was from Sigma, and anti-MBP rabbit antibodies were from New England Biolabs. LVT90/Tu41 anti-BSEP antibodies were prepared (12). Streptavidin-agarose beads, sulfo-NHS-LC-LC-biotin, and dithiobis(succinimidylpropionate) were purchased from Pierce. Blebbistatin was obtained from Calbiochem. All other reagents were from Sigma.

Plasmids—Plasmids used in yeast two-hybrid analyses and GST fusion proteins have been described (15). The pMP47–5 plasmid isolated in the yeast two-hybrid screen contains a full-length rat Mlc2 cDNA (GenBankTM accession X05566 [GenBank] ). Mlc2 cDNA was excised from pMP47–5 with XbaI and XhoI and transferred to pFLAG-CMV to generate pMlc2-FLAG. Mlc2 cDNA excised with EcoR1 and SalI was subcloned into pMAL-p2x to produce pMAL-Mlc2. cDNA inserts from pBsep-EYFP, pMlc2-WT-GFP, and pMlc2-AA-GFP (18) were transferred to pmDsRed-N1, which encodes a monomeric DsRed fluorescent protein (19), to generate pBsep-DsRed, pMLC-WT-DsRed, and pMLC-AA-DsRed.

Yeast Two-hybrid Screen—A PCR DNA fragment coding for amino acids 653–741 of rat Bsep was cloned into pSOS vector (Stratagene) to generate bait plasmid pSL49. A liver cDNA library was generated in the pMyr vector from rat liver poly(A)+RNA using the Superscript system (Invitrogen). Library plasmid DNA was co-transformed into temperature-sensitive cdc25h yeast with pSOS bait plasmids. Yeast grown for 24–48 h at 25 °C were replica-plated on galactose plates and incubated at the non-permissive temperature of 37 °C for 3–5 days. Individual colonies that grew at 37 °C were picked and expanded in liquid medium at 25 °C. Washed cells were spotted on galactose or glucose solid media and incubated at 37 °C.

GST Pulldowns—GSH-agarose beads (Amersham Biosciences) were used to purify GST, GST-MDR2, and GST-BSEP fusion proteins from BL-21 Escherichia coli harboring plasmids pGEX5–3x, pSL27, or pSL25 (15). For binding to GST-MDR2, 5 mg of protein from a rat liver homogenate 2000 x g pellet were solubilized for 30 min in binding buffers containing 1% Triton X-100, protease inhibitors (1 µg/ml pepstatin, 1 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml benzamidine, 0.1 µl/ml aprotinin), 50 mM KPO4, and either 250, 350, or 450 mM NaCl and centrifuged at 13,000 x g. Supernatants were incubated for 2–3 h with 10 µg of GST or GST-MDR2 proteins bound to GSH-Sepharose beads. The beads were washed four times with the corresponding binding buffers, and bound proteins were eluted with SDS loading buffer for immunoblot analyses with anti-MLC2 antibodies. Maltose-binding protein (MBP) and MBP-MLC2 were purified from TB-1 E. coli according to the manufacturer's instructions. GSH-Sepharose beads coated with 1 µg of GST or GST-BSEP were incubated for 2 h with 100 ng of MBP or MBP-MLC2 in PBS 0.1% Triton X-100. The beads were washed three times with PBS 0.1% Triton X-100. Bound proteins were separated by SDS-PAGE and immunoblotted with anti-MBP antibodies (New England Biolabs).

Immunofluorescence Microscopy and Culture and Transfection of Mammalian Cells—Rat liver cryosections were prepared and immunostained (20). Semi-thin sections were fixed in cold methanol and incubated with MLC2-P and c219 antibodies or normal rabbit serum and normal mouse IgG. Primary antibodies were labeled with Alexa-488 anti-rabbit IgG and Alexa-594 anti-mouse IgG secondary antibodies. WifB9 and HepG2 cells were cultured on poly-lysine-coated glass coverslips, which were washed in PBS, fixed in methanol, and blocked for 1 h in 3% bovine serum albumin and 3% normal donkey serum in Dulbecco's PBS. Cells were then incubated 1–2 h with goat anti-MLC-A20 and mouse anti-MDR1 (c219) antibodies diluted in IF buffer (3% bovine serum albumin in Dulbecco's PBS). Coverslips were washed ten times in PBS, blocked for 30 min with IF buffer, and incubated with Texas red-labeled donkey anti-goat and fluorescein-labeled donkey anti-mouse antibodies for 45 min. Coverslips were washed ten times in PBS and mounted using Prolong anti-quench mounting medium. Mounted tissue sections and cells were viewed with a Hammamatsu digital camera on an Axiovert 10 epifluorescence microscope. MDCKII cells (gift from Dr. Enrique Rodriguez-Boulan) were grown in transwells (Corning) and transfected using Lipofectamine 2000 (Invitrogen). Transwells were washed with PBS and fixed for 10 min in ice-cold methanol. Filters were excised, washed ten times with PBS, and mounted on glass slides. Fluorescent proteins were viewed with a Leica TRS2 laser confocal microscope.

Preparation of Membrane Fractions, Immunoblot Analysis, and Immunoprecipitation—Preparation of rat liver subcellular fractions, immunoblotting, and immunoprecipitations from rat liver fractions were performed as indicated (20). Enrichment of marker proteins was determined by immunoblot analyses, except for lysosomes and mitochondria, which were measured by biochemical enzyme assay. Rat liver lysosomes (21) and mitochondria (22) were a gift from Dr. Ana Maria Cuervo. MDCK cells cultured in six-well plates were transfected with pBsep-DsRed, or pmDsRed, pMlc2-FLAG, or pFLAG-CMV. Two days later cells were washed twice with PBS and incubated for 30 min at room temperature with 100 µM dithiobissuccinimidylpropionate. Cells were washed with 50 mM glycine in PBS and lysed for 1 h in 2x PBS 1% Triton X-100, 10 mM glycine. Homogenates were prepared by douncing 10 strokes in a glass homogenizer and centrifuging for 30 min at 10,000 x g. Supernatants were incubated for 1 h with protein G-Sepharose beads, which were removed by centrifugation. Supernatants were incubated with 10 µl of Tu41 antisera or normal rabbit serum overnight. Antibodies were recovered with 50 µl of protein G-Sepharose beads, which were washed four times with 2x PBS 0.25% Triton X-100. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted.



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  		FIG. 1.
Interaction of MLC2 with BSEP, MDR1, and MDR2 in yeast. A, diagram outlining the structure of MDR-type proteins. Hatched bars represent the twelve transmembrane helices (TM), shaded boxes indicate the two nucleotide binding domains (NBD), and the black box represents the linker region (Link). B, yeast two-hybrid interaction of MLC2 with different bait chimeras. The pM47–5 plasmid, which contains the full-length Mlc2 cDNA in pMYR, was transformed into yeast with pSOS plasmids carrying rat BSEP (pSL47), MDR2 (pSL49), and MDR1A (pSL67) linker domains. Yeast grown in liquid medium at 25 °C were spotted on galactose (gal) or glucose (glu) solid medium and incubated at 37 °C, which is a non-permissive temperature for growth of the cdc25h temperature-sensitive mutant. Prey expression is under control of the Gal4 promoter, and no growth is expected on glucose medium. Controls included empty pSOS vector and pSOS containing 40 amino acids from the amino terminus (pSL55) or 30 residues of the carboxyl terminus of MDR2 (pSL56). pSOS bait plasmids were also transformed into yeast in the company of the empty pMyr vector. C, mapping of the MLC2 binding region in the BSEP linker domain. Two hybrid analyses were done with constructs expressing truncated versions of the BSEP linker opposite the pMP47–5 plasmid, which expresses full-length MLC2. The thick boxes represent the 90-amino acid BSEP linker. Plus and minus signs designate the relative growth of yeast at 37 °C. All deletion constructs were co-transformed into yeast with the pMYR empty vector to test for false positive growth. D, mapping of the MLC2 binding region in the MDR2 linker.

 
Biotinylation and Pulse-Chase Metabolic Labeling—Cell surface biotinylation and pulse-chase labeling of proteins in MDCK cells were performed as described (20, 23) for the time periods indicated. Blebbistatin was dissolved in Me2SO to produce a 10-mM stock solution that was stored under nitrogen. For myosin II inhibition, transwells were preincubated for 30 min before the pulse in medium containing 100 µM Blebbistatin or Me2SO. Inhibitor was maintained throughout the pulse and chase periods.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Linker Domains of MDR1, MDR2, and BSEP Interact with MLC2 in Yeast—A yeast two-hybrid screen of a rat liver cDNA library was performed using the BSEP linker domain as bait. The linker resides immediately downstream of the first nucleotide binding domain and connects the homologous halves of BSEP (Fig. 1A) (24). The screen identified a full-length cDNA that encodes the rat non-muscle regulatory myosin II light chain MLC2 as a BSEP interacting protein. Mlc2 cDNAs were isolated on three separate occasions.



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  		FIG. 2.
MLC2 binds GST-MDR2 and GST-BSEP fusion proteins. A, proteins extracted from rat liver homogenates by agarose beads adsorbed with GST alone (G) or with GST-MDR2 (M) (GST fused to 79 amino acids of the MDR2 linker domain) were separated by SDS-PAGE. Immunoblotting revealed that GST-MDR2 extracted MLC2 from liver homogenates but GST alone did not (lower panel). Because myosin II complexes precipitate out of solution at salt concentrations below 200 mM, GST beads were incubated with liver extracts and washed in the presence of 250, 350, and 450 mM NaCl. Coomassie staining of the nylon membrane (upper panel) indicated that equivalent amounts of GST and GST-MDR2 were used in the pulldown assays. A rat liver homogenate (tot) lane was included as control. B, a maltose-binding protein-MLC2 chimera (MBP-MLC2) binds GST-BSEP fusion protein (GST fused to 90 amino acids of the BSEP linker domain). Agarose beads adsorbed with GST-BSEP, or GST alone, were incubated with MBP-MLC2 (lc)orMBP alone (mb) purified from E. coli. Proteins bound to washed beads were separated by SDS-PAGE. Immunoblotting with anti-MBP antibodies indicated that MBP-MLC2 binds GST-BSEP but not GST alone (lower panel). MBP alone does not bind GST-BSEP or GST. Purified MBP-MLC2 and MBP were loaded in the first two lanes. Coomassie staining of the immunoblot membrane confirmed that equivalent amounts of GST and GST-BSEP were used in the assay (upper panel).

 
Plasmid pM47–5, which contains the full-length Mlc2 cDNA, permitted growth at 37 °C of yeast that harbored plasmids expressing BSEP (pSL47), MDR2 (pSL49), and MDR1a linkers (pSL 67) (Fig. 1B). It did not enable growth of yeast carrying the empty pSOS vector, pSOS-Coll (a negative control that expresses amino acids 148–357 of murine type IV collagenase), or pSOS fusions that contain the amino-terminal 40 amino acids of MDR2 (PSL55) or the carboxyl-terminal 35 residues of MDR2 (pSL56). Thus, MLC2 interacts with the linker domains of three MDR subfamily members. Two-hybrid analyses of pSL47 and pSL49 deletion derivatives revealed that MLC2 interacted in yeast with portions of the MDR2 and BSEP linkers that are proximal to the amino-terminal nucleotide binding domains (Fig. 1, C and D). The minimal MLC2 binding region was 32 amino acids in BSEP and 24 residues in MDR2.

GST-MDR2 and GST-BSEP Fusion Proteins Bind MLC2— GSH-agarose beads adsorbed with a chimeric GST protein containing 79 amino acids of the MDR2 linker, or with GST alone, were incubated with rat liver homogenates. GST-MDR2 beads effectively extracted MLC2 from liver homogenates, whereas GST alone did not (Fig. 2A). The interaction was robust, and GST-MDR2 was capable of binding MLC2 in the presence of 0.5 M salt. GSH-agarose beads coated with GST-BSEP, which contains 90 amino acids of the BSEP linker, were incubated with purified MBP or an MBP-MLC2 chimera. Immunoblots of bound proteins revealed that MBP-MLC2 interacted with GST-BSEP, but not with GST alone (Fig. 2B). MBP alone did not bind GST-BSEP or GST alone. These experiments, together with the yeast two-hybrid results, indicate that MDR2 and BSEP linker domains interact directly with MLC2.



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  		FIG. 3.
MLC2 co-immunoprecipitates with BSEP from transfected cell extracts. MDCK cells were co-transfected with BSEP-DsRed (Bsep) or empty pDsRed vector and FLAG-tagged MLC2 (Mlc-Fl), or empty pCMV-FLAG vector. Proteins were immunoprecipitated with anti-BSEP antisera (Tu41) or an equivalent amount of normal rabbit serum (nl). Immunoblots of immunoprecipitates (I.P.) with anti-BSEP (top panel) or anti-FLAG tag (middle panel) antibodies revealed that MLC2-FLAG co-immunoprecipitated with BSEP. Normal rabbit serum did not immunoprecipitate BSEP or MLC2, and MLC2 was not immunoprecipitated by Tu41 in the absence of BSEP. Immunoblot of cell extracts (Total) confirmed that equivalent amounts of MLC2-FLAG were present in material used for immunoprecipitation.

 



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  		FIG. 4.
MLC2 is enriched in the apical domain of hepatocytes and Wif-B9 and HepG2 cells. Immunofluorescence microscopy of rat liver tissue sections (liver), Wif-B9 (WifB), and HepG2 (HepG2) cells. A, rat liver sections were stained with c219 antibody (green), which detected MDR1 and MDR2 P-glycoproteins in the canalicular membrane and pericanalicular vesicles. MLC2 was labeled with P-MLC antibody (red), which recognizes phosphorylated MLC2. MLC2 was concentrated in the apical domain but was detected at multiple loci in hepatocytes. Superimposition of the images reveals MDR1 and MDR2 colocalization with phosphorylated MLC2 in the apical domain (Merge). B, WifB9 cells were stained with c219 antibody and the MLC-A20 antibody, which recognizes MLC2. Endogenously expressed MDR1 resided primarily in the canalicular membrane. MLC2 displayed some staining in the canalicular membrane, but most of the signal was adjacent to the apical membrane. C, HepG2 cells were immunostained in the same manner as WifB9 cells and exhibited similar distribution patterns for endogenously expressed MDR1 and MLC2.

 
MLC2 Co-precipitates with BSEP from Mammalian Cell Extracts—BSEP was immunoprecipitated from MDCK cells cotransfected with plasmids expressing BSEP-DsRed and MLC2 tagged with the FLAG peptide. Immunoblot analysis revealed that immunoprecipitates contained MLC2 in addition to BSEP (Fig. 3). MLC2 was not immunoprecipitated from cells that did not express BSEP, indicating that anti-BSEP Tu41 antibody does not cross-react with MLC2. Co-immunoprecipitation indicates that MLC2 and BSEP interact in mammalian cells.



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  		FIG. 5.
Myosin II is enriched in canalicular membrane vesicles and co-immunoprecipitates with BSEP or MDR1 and MDR2. A, immunoblots of purified rat liver fractions reveal that MLC2 and myosin II heavy chain (MHC) are enriched in canalicular membrane vesicles. Equal amounts of protein (20 µg) from each subcellular fraction were immunoblotted and stained for MDR1 and MDR2 (C219 antibody), BSEP (Tu41 antisera), MLC2 (MLC-A20 antibody), or Myosin II heavy chain (BT561 serum). Subcellular fractions tested were: gol, Golgi vesicles; cmv, canalicular membrane vesicles; mic, endoplasmic reticulum microsomal fractions; ccv, clathrin-coated vesicles; smv, sinusoidal membrane vesicles; mit, mitochondria; lys, lysosomes; tot, total liver homogenate. B, immunoblots of proteins immunoprecipitated from rat liver CMV (CMV) or a 3,500 x g supernatant (3.5k sup) that contains myosin II but is devoid of detectable BSEP, MDR1, or MDR2. Immunoprecipitates obtained with anti-BSEP Tu41 (41) or normal rabbit IgG (NL) were immunoblotted and stained with either Tu41 or anti-Myosin II heavy chain (MHC) antibodies. Tu41 immunoprecipitates BSEP from CMV but not from the 3.5k Sup fraction (top panel). MHC is found in CMV, but not 3.5k sup, immunoprecipitates (bottom panel), indicating that Tu41 does not immunoprecipitate MHC in the absence of BSEP. C, immunoprecipitates obtained with c219 antibody, which recognizes MDR1 and MDR2, or isotype-matched normal mouse IgG2a were immunoblotted and stained with c219 or anti-MHC antibodies. As in panel B, MHC was immunoprecipitated with MDR1 and MDR2 from CMV but not from the 3.5 k sup, which contains myosin II but no MDR1 or MDR2.

 
MLC2 Is Enriched in the Canalicular Domain of Hepatocytes, WifB9, and HepG2 cells—Immunofluorescence microscopy of rat liver sections revealed that MLC2 was enriched in the apical domain of hepatocytes. Colocalization of MLC2 and MDR1/2 fluorescence was most pronounced in pericanalicular vesicles rather than in the canalicular membrane (Fig. 4A). The hepatoma-derived WifB9 (25) and HepG2 (26, 27) cell lines polarize in culture to form canalicular structures between cells that are functionally and structurally analogous to the bile canaliculus of hepatocytes. MLC2 is enriched in the apical domain of WifB9 (Fig. 4B) and HepG2 (Fig. 4C) cells. In both hepatocytes and cell lines, MLC2 colocalized with MDR1 in abundant pericanalicular vesicles. Close proximity of these vesicles to the canalicular membrane prevented resolution as to whether MLC2 and the transporters colocalize in the canalicular membrane.

Myosin II Co-immunoprecipitates with BSEP and MDR1/2 from Rat Liver CMV—Immunoblot analyses of rat liver subcellular fractions indicated that BSEP, MDR1, MDR2, MLC2, and myosin II heavy chain were enriched in canalicular membrane vesicles (CMV) (Fig. 5A). BSEP or MDR1/2 were immunoprecipitated from CMV using Tu41 or c219 antibodies, respectively. Immunoprecipitates were examined for subunits of myosin II, which is a heterohexamer composed of two heavy chains, two essential light chains, and two regulatory light chains. Immunoprecipitation of the transporters also precipitated myosin heavy chain (Fig. 5, B and C). MLC2 detection in immunoprecipitates was obscured by IgG light chains, which co-migrated with MLC2 in SDS-PAGE gels. Tu41 and c219 antibodies did not precipitate myosin heavy chain from liver fractions that contained myosin II but were devoid of BSEP, indicating that anti-BSEP and anti-MDR1 antibodies do not cross-react with myosin II. These data indicate that BSEP, MDR1, and MDR2 transporters interact with the myosin II holoenzyme in hepatocytes.

Expression of a Non-phosphorylatable MLC2 Mutant Decreases Apical Membrane BSEP in MDCK Cells—MLC2 phosphorylation at threonine 18 and serine 19 residues positively regulates myosin II activity (28). Plasmids expressing a non-phosphorylatable MLC2 mutant (MLC-AA-GFP), in which Thr-18 and Ser-19 were replaced by alanine, or wild-type MLC2 (MLC-WT-GFP) fused to GFP, were cotransfected into MDCK cells with constructs expressing BSEP-DsRed. Confocal fluorescence microscopy of polarized MDCK monolayers revealed that MLC-WT-GFP was concentrated in punctate structures that were enriched in the apical domain and colocalized with apical BSEP-DsRed. The MLC-AA-GFP mutant was dispersed in the cytoplasm and did not display the apical punctate distribution. Intracellular BSEP-DsRed fluorescence was markedly increased in cells expressing MLC-AA-GFP when compared with controls expressing MLC-WT-GFP (Fig. 6).



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  		FIG. 6.
BSEP distribution in MDCK cells expressing wild-type MLC2 or a non-phosphorylatable MLC2-AA mutant. Confocal fluorescence micrographs of polarized MDCK cells co-transfected with plasmids expressing BSEP-DsRed and MLC-WT-GFP (top panels) or MLC-AA-GFP (bottom panels). XZ vertical cross-section images of MDCK monolayers reveal that more BSEP-DsRed resides within cells expressing MLC-AA-GFP than in controls transfected with MLC-WT-GFP, in which BSEP displays a predominantly apical localization. MLC-WT-GFP exhibits an apical punctate distribution, which is not seen with MLC-AA-GFP, and colocalizes more extensively with BSEP-DsRed than does the non-phosphorylatable mutant.

 
Biotinylation of surface proteins in transfected MDCK cells revealed that plasma membrane BSEP-EYFP resides exclusively in the apical domain (15). Apical BSEP-EYFP levels in cells co-transfected with MLC-AA-GFP were less than half (45 ± 6%) of those observed in controls expressing MLC-WT-GFP (Fig. 7, A and B). Expression of MLC-AA-GFP did not significantly affect BSEP-EYFP expression levels, and biotinylation analyses indicated that MLC-AA-GFP did not induce mistargeting of BSEP-EYFP to the basolateral membrane (not shown). Expression of mutant MLC-AA-GFP did not affect transepithelial permeability of MDCK monolayers to mannitol (Fig. 7C), indicating that tight junction integrity is not affected by expression of the dominant negative mutant.



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  		FIG. 7.
Non-phosphorylatable MLC-AA-GFP decreases BSEP abundance in the apical membrane of MDCK cells but does not affect transepithelial permeability. A, immunoblot of surface-biotinylated proteins isolated from MDCK cells transfected with plasmids expressing BSEP-EYFP and MLC-WT-GFP or MLC-AA-GFP. Apical surface proteins were labeled by addition of biotinylation reagent to upper transwell chambers. Total extracts (Total) and apical membrane proteins (Apical), which were isolated by adsorption to streptavidin-agarose, were immunoblotted with the anti-BSEP Tu41 antibody. B, densitometric analyses of immunoblots indicated that MLC-AA-GFP expression was accompanied by a 8 ± 5% decrease in total BSEP levels and by a 67 ± 12% decrease in apical membrane BSEP-EYFP when compared with cells expressing MLC-WT-GFP (values are relative to the mean of MLC-WT-GFP measurements in each of three experiments done in triplicate, n = 9, means ± S.E. p values are from a Student's t test; **, p <0.005). C, MLC-AA-GFP expression does not increase transepithelial mannitol permeability of MDCK monolayers. 3H-Mannitol was added to the lower chamber of transwells containing confluent MDCK cells transfected with MLC-WT-GFP, MLC-AA-GFP, or GFP alone. After 1 h of incubation, 3H-mannitol levels in the upper chamber were measured. Values are expressed as percentage of mannitol that has migrated to the upper chamber relative to the total amount added to the lower chamber. No significant differences were detected among cultures expressing MLC-WT-GFP, MLC-AA-GFP, or GFP (n = 9, three experiments performed in triplicate).

 



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  		FIG. 8.
Blebbistatin inhibition of myosin II blocks BSEP delivery to the apical membrane. Top, time course of metabolically labeled apical membrane BSEP in transfected MDCK cells incubated with 100 µM Blebbistatin (Blebstn +) or with Me2SO (–). Cells were pulse labeled, chased for the time periods indicated, and apical surface proteins were biotinylated. BSEP-EYFP was immunoprecipitated from cellular extracts, and aliquots separated by SDS-PAGE were visualized by phosphorimaging (Total). Biotinylated BSEP-EYFP was extracted from immunoprecipitates by adsorption to avidin beads and was visualized and quantified by phosphorimaging (Apical). Bottom, apical BSEP-EYFP values from cells incubated with Blebbistatin (black boxes) were significantly different from controls (gray boxes) after 2, 3, and 4 h of chase (**, p <0.005, n = 3). Values are relative to apical levels in control cells after 2 h of chase. Total labeled BSEP-EYFP values were not significantly different in Blebbistatin-treated cells and controls.

 
Myosin II Inhibition Impairs BSEP Delivery to the Apical Membrane—Myosin II activity is specifically inhibited by Blebbistatin (29), which is membrane permeable. Pulse-chase metabolic labeling was performed in transfected MDCK cells treated with 100 µM Blebbistatin and in controls. Total cellular levels of metabolically labeled BSEP were not significantly different in Blebbistatin-treated cells and controls (Fig. 8, Total), indicating that Blebbistatin did not affect BSEP-EYFP translation or processing. Delivery of newly synthesized BSEP-EYFP to the apical membrane was determined by immunoprecipitation of surface-biotinylated proteins. Metabolically labeled BSEP-EYFP was detected in the apical membrane of MDCK cells after a 1-h chase, and its levels peaked at 3–4 h (15). In Blebbistatin-treated cells, apical membrane levels of radioactively labeled BSEP were reduced by 72 ± 14, 61 ± 15, and 60 ± 11% after 2, 3, and 4 h of chase, respectively, when compared with the corresponding controls (Fig. 8, Apical). These data indicate that myosin II inhibition impairs delivery of newly synthesized BSEP to the apical membrane of MDCK cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The MLC2 gene product was identified as a binding partner for BSEP, MDR1, and MDR2 in a yeast two-hybrid screen. Interactions were confirmed in vitro by GST pulldown assays and in mammalian cells by co-immunoprecipitation. MLC2 encodes a 20-kDa regulatory light chain subunit of the non-muscle myosin II holoenzyme, which consists of two heavy chains, two regulatory light chains, and two essential light chains (30). The regulatory light chains are an integral part of the complex and have no known function separate from the myosin II heavy chains in cells. Accordingly, immunoprecipitation of BSEP or MDR1 and MDR2 co-precipitated the myosin II heavy chain, indicating that the transporters interact with MLC2 as part of the myosin II holoenzyme.

MLC2 is phosphorylated on threonine 18 and serine 19 by myosin light chain kinase, p21 kinase, or RHO kinase and dephosphorylated by myosin light chain phosphatase (28, 30). MLC2 phosphorylation regulates activity of the myosin II complex by influencing its ability to interact with actin and stimulating multimerization of heavy chains (31, 32). A non-phosphorylatable form of MLC2, in which threonine 18 and serine 19 have been replaced by alanine residues, supplants MLC2 on the holoenzyme and inhibits localized myosin II activation by cellular kinases (18). Expression of this dominant negative MLC2 mutant inhibits oocyte fertilization (33), chromaffin granule secretion (34), and cell migration after wounding (18). Confocal fluorescence microscopy and cell surface biotinylation of polarized MDCK cells revealed that non-phosphorylatable MLC2 increased intracellular BSEP abundance and significantly reduced apical membrane BSEP levels. To elucidate how myosin II influences BSEP distribution, myosin II was inhibited by Blebbistatin, and trafficking of metabolically labeled proteins to the apical membrane was analyzed by pulse-chase and cell surface biotinylation. Blebbistatin is a specific membrane-permeable inhibitor that stabilizes myosin II in an actin-detached state (35, 36) and inhibits muscle and non-muscle myosin II activities with IC50s ranging from 0.05 to 5 µM. It does not affect activity of non-conventional myosins I, V, X, or XV at 100 µM (37). Pulse-chase studies indicated that myosin II inhibition did not affect BSEP synthesis or post-translational modification. Analyses of metabolically labeled surface-biotinylated proteins revealed that Blebbistatin significantly impaired appearance of newly synthesized BSEP in the apical surface of MDCK cells, confirming that myosin II plays an important role in delivery of the transporter to the apical membrane.

Non-muscle myosin II is required for cell motility, cytokinesis, and morphology maintenance in many cell types. In hepatocytes, myosin II is enriched in the apical actin network and MLC2 phosphorylation is coincident with canalicular contraction (38, 39). There are several possible mechanisms by which myosin II could participate in BSEP trafficking. Myosin II is associated with membranes of the Golgi and trans-Golgi networks (40, 41) and is required for formation and release of specific vesicle subtypes in these compartments (42, 43). Thus, myosin II inhibition could impair BSEP delivery to the apical membrane by blocking its departure from Golgi and/or trans-Golgi networks. This mechanism is unlikely because inhibition of myosin II with non-phosphorylatable MLC2 did not result in BSEP accumulation in Golgi.

The myosin II motor also participates in vesicle transport. Anti-myosin II antibodies and inhibitors of MLC phosphorylation prevented movement of myosin II-containing vesicles on actin filaments in vitro (44, 45). Inhibition of myosin II activity or MLC phosphorylation suppressed insulin secretion in pancreatic islet cells (46), granule exocytosis in chromaffin cells (34), and mobilization of synaptic vesicles in presynaptic neurons (47, 48). Thus, myosin II may facilitate BSEP vesicular transport to the canalicular membrane. BSEP trafficking to the apical domain requires microtubules (10, 11, 13). However, microtubules do not contact the canalicular membrane but rather abut the cortical actin sheath that surrounds the canaliculus (49, 50). Immunofluorescence microscopy revealed that myosin II is enriched in the apical domain of hepatocytes, WifB9, and HepG2 cells and colocalizes with endogenously expressed ABC transporters in pericanalicular vesicles. Delivery of rhodopsin (51) and Trk receptor (52) to their target membranes requires direct interactions with light chains of the dynein motor complex. Similarly, BSEP association with the myosin II regulatory light chain may enable vesicles carrying the transporter to negotiate the dense, multilayered actin network that envelops the canaliculus.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants RO1DK35652 and T32DK07542 (to I. M. A.) and R01 DK060719 (to D. F. O.) and by a pilot grant (to D. F. O.) from the Center for Gastroenterology Research on Absorptive and Secretory Processes (GRASP) at Tufts University and the New England Medical Center, supported by National Institutes of Health Grant P30 DK34928. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 617-636-3828; Fax: 617-636-0445; E-mail: daniel.ortiz{at}tufts.edu.

1 The abbreviations used are: MDCK, Madin-Darby kidney cells; BSEP, bile salt export protein; ABC, ATP binding cassette; CMV, canalicular membrane vesicles; DsRed, monomeric Discosoma sp. Red fluorescent protein; EYFP, enhanced yellow fluorescent protein; GSH, glutathione; GST, glutathione S-transferase; MBP, maltose-binding protein; MDR, multidrug resistance protein; Mlc2, myosin II regulatory light chain; MHC, myosin II heavy chain; PBS, phosphate-buffered saline; WT, wild-type. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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