Cells, cell culture, plasmids and transfection
HEK (human embryonic kidney) 293 cells were cultured at 37 °C and 5% CO2 in Dulbecco’s Modification of Eagle’s Medium (DMEM) (Mediatech, Manassas, VA), supplemented with 10% fetal bovine serum (FBS) (Hyclone Logan, UT), 2mM glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen-GIBCO, Grand Island, NY).
The full length wild-type human non-muscle myosin IIA heavy chain plasmid pEGFP-myosin IIA and its truncated mutant pEGFP-myosin II ARF (1666–1961)[22
] were generous gifts from Dr. Masayuki Takahashi (Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, 060–0810 Japan). Using standard cloning techniques, truncated mutants encoding the head portion of myosin IIA linked to EGFP (pEGFP1-843) and the rod part of myosin IIA linked to EGFP (pEGFP844-1665) were generated from the pEGFP-myosin IIA plasmid that was used as a template. The vectors pcDNA3.1V5HGAL and pcDNA3.1V5 HGAL delta C encoding amino acids 1–118 of the HGAL protein were previously reported.[13
Polyfect transfection reagent (Qiagen, Valencia, CA) was used for transfection of plasmids into HEK 293 cells according to the manufacturer’s instructions. Briefly, cells were plated at 2 × 106 cells in 100-mm culture dishes (Nalge Nunc International, Rochester, NY) in 10 mL DMEM and grown overnight at 37°C and 5% CO2. Plasmid DNA (8μg), diluted and mixed in 300 μl of serum-free DMEM was incubated for 5 min at room temperature. Polyfect transfection reagent was added to the mixture, which was incubated for additional 10 min at room temperature and then added to the cells in a drop-wise manner. The cells were incubated for 48 h before proceeding with further experiments.
Antibodies, Western blot analysis and immunoprecipitation
Mouse monoclonal anti-HGAL antibody was generated in our laboratory, as reported previously[9
]. Mouse monoclonal anti GFP antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and mouse monoclonal anti V5 antibody was from Invitrogen Inc. (Carlsbad, CA) Western blotting and immunoprecipitation were performed as previously reported[24
]. Briefly, whole-cell extracts were preparedby lysing 5 × 106
cells in radioimmunoprecipitation assay (RIPA)buffer (1x phosphate-buffered saline, 1% Nonidet P-40 [NP-40],0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS],10 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 100 mM sodium orthovanadate) on ice for 30 min. Cell lysate was centrifuged at 14,000 g
for 10 minutes at 4°C. Protein concentration of lysates was determined using Coomassie protein assay reagent (Thermo Scientific, Rockford, IL) and samples were adjusted to equal protein concentration. For immunoprecipitation (IP), 400 μg of protein was precipitated for 1 to 2 hours with the indicated antibody at 4°C with rotation. Protein G-agarose (Invitrogen, CA) or TrueBlotTM anti-Mouse Ig IP Beads (eBioscinece, CA) were added and the mixture was rotated for an additional 1 hour. Precipitated complexes were washed four times in RIPA buffer, boiled in Laemmli buffer (2 x concentrate: 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, 0.125 M Tris HCl (pH 6.8)), separated on 12% SDS-PAGE gel and immunoblotted with the indicated antibodies.
Expression and purification of recombinant HGAL protein
For expression and purification of Trx-HGAL fusion protein, BL21 (DE3) cells were transformed with pTRX-HGAL plasmid. Individual clones were grown at 37 °C in liquid broth containing 100 μg/ml ampicillin until A600 reached 0.6, followed by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration 0.1 mM) for 3 h. The bacteria were collected and re-suspended in a sonication buffer (10mM Tris–HCl, 150 mM NaCl, pH8.0) and lysed by sonication on ice. Following 20 min centrifugation at 12,000 g, the supernatant was applied to Ni2+ chelating Sepharose column (50/40 cm, Amersham–Pharmacia Biotech; Sunnyvale, CA) pre-equilibrated with starting buffer (50mM Tris–HCl, 150 mM NaCl, and 10mM imidazole, pH8.0), washed with washing buffer (50mM Tris–HCl, 150 mM NaCl, and 80mM imidazole, pH7.0) and fusion protein was eluted using elution buffer (50mM Tris–HCl, 150 mM NaCl, and 200 mM imidazole, pH7.0). The buffer of the eluate was changed to the strong anion ion exchange chromatography starting buffer (50 mM Tris–HCl, 20 mM NaCl, pH8.0) and the solution was applied to Q.F.F. sepharose column (16/20, GE Healthcare Bio-Science Corp; Piscataway, NJ). The proteins were eluted from the column with linear gradient of 1M NaCl in starting buffer at 5 ml/min constant speed for 100 min. Fractions containing the Trx-Src I fusion protein were pooled together.
Rabbit skeletal muscle myosin
Rabbit skeletal muscle myosin II was isolated and purified as described in Greenberg et al[25
]. Briefly, minced rabbit skeletal muscle was extracted in 3 volumes of ice cold Guba Straub buffer (0.1 M KH2
, 0.05 M K2
, and 0.3 M KCl, pH 6.5) with constant stirring. The muscle mince was pelleted by centrifugation at 12,000 × g for 30 minutes at 4°C. The myosin containing supernatant was decanted and the myosin was precipitated using 13 volumes of ice-cold 1mM EDTA. Precipitated myosin was collected by serial centrifugation at 8,000 × g for 10 minutes at 4°C. The final spin was for 10 minutes at 12,000 × g. The supernatant was removed and discarded. The myosin pellets were then dissolved in cold 20 mM MOPS, pH 7, 1 mM DTT buffer and KCl to a final concentration of 0.5 M. Resuspended myosin was then ultracentrifuged at 125,000 × g for 1.5 h at 4°C. The myosin containing supernatant was then removed and precipitated a second time using 14 volumes of ice cold water. Myosin was collected by centrifugation at 4,500 × g for 10 minutes at 4°C. Following centrifugation the supernatant was removed and discarded. Myosin pellets were left with a minimal volume of 1mM DTT/water and stored overnight on ice. The following morning myosin pellets were resuspended as previously in 20 mM MOPS, pH 7, 1 mM DTT and 0.5 M KCl and ultracentrifuged again. The supernatant containing myosin was removed, mixed with glycerol in a 1:1 by volume ratio and stored at −20°C until needed for experiments.
Rabbit skeletal muscle actin was prepared according to Pardee and Spudich[26
] with modifications. Briefly, rabbit skeletal acetone powder was extracted with a G-actin buffer consisting of 2 mM Tris-HCl (pH 8.0), 0.2 mM Na2
ATP, 0.5 mM β-mercaptoethanol, 0.2 mM CaCl2
, and 0.0005% NaN3
at a ratio of 20 ml/g for 30 min with stirring on ice. The extract was centrifuged at 7,000 × g
at 4 °C for 1 h to clarify and the tissue pellet was discarded. The supernatant was removed and adjusted to a final concentration of 40 mM KCl, 2 mM MgCl2
and 1 mM Na2
ATP (pH 8.0). The F-actin was allowed to polymerize for 2 h at 4°C. The KCl concentration was then increased again very slowly to a final concentration of 0.6 M and the solution was stirred slowly on ice for 30 min. This step was necessary to remove possible traces of tropomyosin-troponin. The F-actin was then collected by ultracentrifugation at 200,000 × g
at 4 °C for 1.5 h. The supernatant was discarded and the F-actin pellets were re-dissolved in a buffer consisting of 10 mM Mops (pH 7.0) and 40 mM KCl.
Labeling of F-actin with pyrene
For fluorescence assays rabbit skeletal actin was labeled with Pyrene lodoacetamide (PIA) by the method of Cooper et al[27
]. Briefly 20–40 μM F-actin was incubated at room temperature, in the dark, for 16 hours with a 1.5 molar excess of N-(1-pyrene) iodoacetamide (Invitrogen, Molecular Probes) in a buffer containing 10 mM MOPS, pH 7.0 and 40 mM KCl. Then the reaction was quenched with 1mM DTT and the preparation was centrifuged at 200,000 × g for 1.5 hours. Then F-actin was dialyzed against G-actin buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2
, 0.2 mM ATP) to remove excess of pyrene and DTT and polymerized overnight to form F-actin. The resulting molar ratio of pyrene/F-actin was 0.8 determined using the molar extinction coefficient, ε344
Actin cosedimentation assay
Non-muscle human actin (Cytoskeleton Inc, Denver, CO) was diluted to 1mg/ml in a buffer containing 5 mM Tris pH 8, 0.2 mM CaCl2, 0.2 mM ATP and 0.5 mM DTT, centrifuged at 40,000 × g for 10 minutes at 4°C. The supernatant was used to induce actin polymerization by adding 50 mM KCl, 1 mM ATP and 2 mM MgCl2. The polymerization occurred at room temperature for 1 hour. Recombinant HGAL protein was spun down at 100,000 × g in Beckman ultracentrifuge for 20 minutes at 22°C. HGAL protein in the supernatant (0.01mM and 0.02mM) and F-actin (0.05mM) were incubated in the buffer containing 10 mM Tris pH 7.0, 1 mM ATP, 0.2 mM DTT, 1 mM EGTA, 0.1 mM CaCl2 and 2 mM MgCl2 for 1 hour at room temperature, then spun at 100,000 × g at 22°C. The supernatants were carefully removed and 5 X Laemmli SDS-PAGE sample buffer was added; 1 X Laemmli SDS-PAGE sample buffer was added to the pellets. The pellets and supernatants were analyzed following their separation by SDS-PAGE and Coomassie Blue staining of the gel.
Myosin-HGAL cosedimentation assay
Rabbit skeletal muscle myosin was precipitated with 13 volumes of ice cold 1mM DTT. Myosin was then collected by centrifugation at 8,000 × g for 10 minutes. The pellet was resuspended in a buffer containing 0.4M KCl, 1mM DTT, and 10mM MOPS at pH 7 and dialyzed overnight. Myosin was diluted at 1:11 ratio with 1mM DTT and put on ice for one hour. Recombinant HGAL protein was spun down at 100,000 × g in a Beckman ultracentrifuge for 20 minutes at 22°C. HGAL in the supernatant (0.01mM and 0.02mM) and myosin (0.05mM) were mixed and incubated on ice for 30 minutes and then spun down at 100,000 × g in Beckman ultracentrifuge for 30 minutes at 22°C. The supernatants and pellets were carefully collected and analyzed following their separation by SDS-PAGE and Coomassie Blue staining.
Actin-activated myosin ATPase activity in the presence or absence of HGAL
The kinetics of the actomyosin interaction in the presence or absence of HGAL protein was measured using actin-activated myosin ATPase assays. Rabbit skeletal muscle myosin at the concentration of 1.9 μM was titrated with the increasing concentrations of rabbit skeletal F-actin (in μM): 0.1, 1, 2, 5, 10, 15, 20 and 25. The assays were performed it triplicates on 96-well microplates in a 120-μl reaction volume containing 25 mM imidazole (pH 7.0), 4 mM MgCl2
, 1 mM EGTA and 1 mM DTT. The final salt (KCl) concentration was 0.107 M. The reactions were initiated with the addition of 2.5 mM ATP with mixing in a Jitterbug incubator shaker (Boekel) and allowed to proceed for 5 minutes at 30 °C and then terminated by the addition of 4% trichloroacetic acid. Samples were then centrifuged at 3720 × g
for 20 minutes and 50 μl of supernatants were transferred to the microplate for determination of inorganic phosphate by the Fiske–Subbarow method[28
]. Data were analyzed using the Michaelis–Menten equation, yielding the Vmax
parameters. In control experiments, pyrene-actin was titrated with myosin containing either HGAL-buffer with no HGAL added or BSA (bovine serum albumin) included in each titration point with myosin.
The effect of HGAL on the binding of myosin to F-actin
We used a fluorescence-based assay to examine the effect of HGAL on the binding of rabbit skeletal muscle myosin to pyrene-labeled F-actin. The measurements were performed in a 2ml cuvette in a buffer containing 10 mM MOPS, 0.4M KCl, pH 7 using a JASCO FP-6500 Fluorometer (Jasco, USA). Pyrene-labeled F-actin at 0.5 μM, was titrated with the increasing concentrations of rabbit skeletal muscle myosin in the presence or absence of HGAL protein. Fluorescence was recorded at 408 nm with excitation wavelength of 340 nm. The data were collected using the computer program Felix (Photon Technology International) and fitted to a nonlinear binding model yielding apparent dissociation constants Kd
The effect of HGAL on actin polymerization
Actin polymerization assay was done using the Actin Polymerication Biochem Kit (Cytoskeleton Inc, Denver, CO) according to the manufacturer’s instructions. Briefly, pyrene-actin was added into the G-actin buffer (5 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, pH 8.0) and mixed with HGAL or just HGAL buffer (10 mM MOPS, 40 mM KCl, 1mM DTT, pH 7.0). The samples were read on a Spectra Max Gemini EM fluorescence microplate reader (Molecular Devices) at 60 second interval for 120 cycles with excitation at 368 nm and emission at 430nm.
In vitro motility assay
To examine the effect of HGAL on the interaction of actin and myosin at the molecular level we have utilized the in vitro
motility assays, as described previously. Briefly, [14
] a flow chamber was formed between a nitrocellulose coated coverslip and a standard glass slide with 3M double stick tape at the boundaries. Rabbit skeletal muscle myosin was introduced into the chamber in high salt buffer (300 mM KCl, 25 mM imidazole, 1 mM EGTA, 4 mM MgCl2
, 1 mM DTT) and allowed to bind to the nitrocellulose surface for 2 minutes. Sixtyμl of 0.5 mg/ml BSA in high salt buffer was then flowed through the chamber to remove any unbound myosin and block the remaining surface sites to avoid nonspecific attachment of actin filaments or HGAL. After blocking, 60μl of low salt buffer (25 mM KCl, 25 mM imidazole, 1mM EGTA, 4 mM MgCl2
, 1 mM DTT) was flowed through the chamber to remove any unbound BSA. Tetramethylrhodamine isothiocyanate (TRITC) labeled F-actin filaments (~5 nM) in low salt buffer were added and allowed to bind to the myosin in the absence of ATP. Unbound actin was removed by washing with low salt buffer and movement initiated by the addition of low salt buffer with 1mM ATP, scavenger (glucose oxidase, catalase, and dextrose) and 0.5% methylcellulose added. For some experiments 30 μl of 400 μg/ml HGAL was incubated for 5 minutes after the BSA blocking step and allowed to bind to the surface immobilized myosin.
Data was collected for several different loading concentrations of myosin. Filament movement was observed at 25oC with an ICCD camera model IC200 (PTI, Birmingham, NJ). Video sequences were captured using Scion image and an AG-5 image grabber (Scion Corp, Frederick, MD). The average velocity for a given filament was determined from the distance traveled by the filament between 10–12 consecutive video images taken at 1 second intervals using Retrac, the freeware program written by Dr. Nick Carter. 15–25 filaments from each video segment were averaged and at least two video segments were obtained per flow cell.
Data are expressed as the average of n experiments ± SEM (standard error of the mean). Comparisons between groups were performed using an unpaired Student’s t-test (Sigma Plot 11; Systat Software, Inc., San Jose, CA, USA). The significance was defined as p< 0.05.