MTLn3 and MTC are cell lines derived from the same 13762NF rat mammary adenocarcinoma and were cultured according to the method of Segall et al. (1996)
. CEFs were isolated from 12-d chicken embryos and cultured as described by Kislauskis et al. (1993)
Construction of Expression Vectors for Glutathione S-Transferase (GST) Fusion Proteins
Constructs for expressing GST fusion proteins were made using a vector, pGEX-KG (Guan and Dixon, 1991
). Each truncate was obtained by PCR using a pair of primers corresponding to each end of the desired sequence and inserted into the vector. For example, to make a construct to express GST–403–456 fusion protein (containing amino acid residues 403–456 of Dictyostelium
EF1α), the fragment was obtained using primers GCG GAA TTC TAC CAA TGT GTG TTG AAT CA and CG CGA AGC TTA TTT CTT CTT TGA TGG AGC AGC and was inserted into the vector at Eco
RI and Hin
dIII sites. For GST-408-462 (containing amino acid residues 408–462 of rat EF1α), the fragment was obtained using primers CGG AAT TCA AAT GAA GCC CAT GTG TGT TGA G and CGA AGC TTC ATT TAG CCT TCT GAG CTT T and was inserted into the vector at Eco
RI and Hin
dIII sites. This strategy was used to make all of the GST fusion protein expression constructs that were used in this study. All of the expression constructs were validated by DNA sequencing.
Construction of Expression Vectors for EF1α-GFP Fusion Proteins
Constructs for expressing full-length or truncated EF1α as a green fluorescence protein (GFP) fusion protein were made by using an expression vector, pGL-1, originally from Life Technologies (Gaithersburg, MD). To express fusion proteins with GFP fused to the C terminus of EF1α or its truncate, this vector was modified by Dr. Jeffrey Segall (Albert Einstein College of Medicine, New York) by replacing a stop codon with a HindIII site 5′ of the GFP-coding sequence, resulting in pGL-H3. A cDNA sequence encoding rat EF1α was amplified from an expression construct for GST-EF1α fusion protein (a gift from Dr. Richard Stanley, Albert Einstein College of Medicine) by PCR using primers TCA GGA ATT CGA TTC AAA GCA AAA ATG and CTC GTC GAC CTT TAG CCT TCT GAG C for the full-length EF1α and primers TCA GGA ATT CGA TTC AAA GCA AAA ATG and GTT GAG CTC GCT TGC CAG GGA CCA T for truncate rat EF1α containing amino acid residues 1–408. The full-length and truncated rat EF1α sequences were cloned into the pGL-H3 at sites of EcoRI/SalI and EcoRI/SacI, respectively. The rat EF1α domain III (amino acids 330–462) truncate and EBS of yeast Bni1p were obtained similarly by PCR and cloned into the pEGFP-C1 expression vector (CLONTECH, Palo Alto, CA). These constructs were transformed into Escherichia coli cell XL1-Blue (Stratagene, La Jolla, CA) and selected with 100 μg/ml ampicillin or 25 μg/ml kanamycin. Colonies were screened by PCR with the corresponding pair of primers for each construct. Positive colonies were selected and the correct inserts were confirmed by DNA sequencing.
Transfection and Screening for GFP-positive Cells
Transfection of MTLn3 and MTC cells was performed by using LipofectAMINE according to the protocol of Life Technologies. The rat tumor cell lines were plated onto dishes (MaTTek, Ashland, MA) such that each dish contained 100,000 cells 24 h before the transfection (for 50–80% confluence at transfection). The constructs for expression, EF1α-GFP or EF1αΔC-GFP, were cotransfected with vector pSV7 containing the neomycin-resistant gene (kindly provided by Dr. Fishman, Albert Einstein College of Medicine) at a ratio of 10:1 at different doses ranging from 0.125 to 1.0 μg of total DNA per dish. Antibiotic resistance selection was started 72 h after transfection by using 0.8 mg/ml Geneticin (G418). Surviving cells were subsequently screened using fluorescence microscopy. Colonies that emitted green fluorescent light were marked and loop cloned with glass cloning cylinders (Sigma, St. Louis, MO) into Petri dishes, and this procedure was repeated until a stable green fluorescent light-emitting population was obtained. The GFP-EF1α-domain III (domain III) truncate was transiently expressed using LipofectAMINE Plus (Life Technologies) with 10–40% transfection efficiency. The expression of the corresponding GFP fusion proteins were validated by Western blots using antibodies against GFP (CLONTECH) and EF1α (Upstate Biotechnologies, Lake Placid, NY).
Transfection of CEF
Transfection of CEF for transient expression of GFP, GFP-EF1α-domain III, was performed with the Effectene Transfection Reagent kit from Qiagen (Valencia, CA) according to the manufacturer's recommended procedure. Plasmic DNA (1 μg) was used for each coverslip with CEFs in MEM without removing serum.
Immunofluorescence Staining and Fluorescence In Situ Hybridization (FISH)
To study β-actin mRNA localization in CEFs, FISH was performed as described by Kislauskis et al. (1994)
. For double probing of protein localization and mRNA, cells were first subjected to the normal process of indirect immunofluorescence staining using RNase-free reagents and RNase inhibitor. After secondary antibody binding and washes, the samples were fixed again using 3.7% paraformaldehyde to preserve the antibodies, followed by the normal FISH process. Both signals of FISH and immunostaining are compromised in such double-probed samples compared with either FISH or immunostaining alone. However, the general pattern of distribution of mRNA and protein in double-probed samples is comparable to that for single probing. Nevertheless, samples for either FISH or immunostaining were usually processed along with the double-probed sample to ensure the correct interpretation of signals from both mRNA and protein.
Cell Permeabilization and Fixation
The transfected MTLn3 cells were plated onto MatTek dishes precoated with rat tail collagen I (Collaborative Research, Bedford, MA) and cultured overnight. The cells were serum starved by incubation for 3 h in medium supplemented with 12 mM HEPES, pH 7.0, and 0.35% bovine serum albumin (BSA; MEMH) and then stimulated with 5 nM epidermal growth factor (EGF) to induce the extension of lamellipodia. To assess the actin cytoskeleton localization of EF1α-GFP fusion proteins, these cells were briefly permeabilized after ~4 min of EGF stimulation to remove soluble cytoplasmic proteins with 100 μl of permeabilization buffer (20 mM 1,4-piperazinediethanesulfonic acid [PIPES], pH 6.5, with 30 mM KOH, 4 mM MgCl, and 10 mM EGTA, pH 6.5 with 20 mM KOH and 0.025% saponin). After 10 s of permeabilization at 37°C, the dishes were gently flooded with 2 ml of fixation buffer containing 3.7% formaldehyde, 5 mM PIPES, 1.1 mM Na2HPO4, 0.4 mM KH2PO4, 137 mM NaCl, 5 mM KCl, 4.0 mM NaHCO3, 2 mM MgCl2, 2 mM EGTA, and 5.5 mM glucose. After 5 min of fixation at 37°C, aldehyde autofluorescence was quenched with 0.1 M glycine for 10 min. The cells were blocked with 1% BSA and 1% fetal calf serum in TBS (20 mM Tris-HCl, pH 8.0, and 150 mM NaCl) and then incubated with rhodamine-conjugated phalloidin (50 nM plus 100 nM nonlabeled phalloidin) for 2 h or overnight. The transfected MTC cells were treated similarly except that they were not EGF stimulated before saponin permeabilization.
All samples were viewed with an Olympus (Tokyo, Japan) microscope equipped with a cooled CCD camera. Optical sections were obtained by deconvolution using IPLab computer software (Scanalytics, Fairfax, VA). Image processing was performed with NIH Image (version 1.6, National Institutes of Health, Bethesda, MD) and Photoshop (version 5.0; Adobe Systems, Mountain View, CA) software.
Actin Cosedimentation Assay
Actin cosedimentation was performed in sedimentation buffer containing 2 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol (DTT), 15 mM KCl, 25 mM PIPES, and 2 mM EGTA (pH to 6.6 with 45 mM KOH). Results of testing indicated that actin isolated from Dictyostelium and rabbit muscle behaves similarly in actin cosedimentation assays (for native and GST fusion of Dictyostelium and rat EF1α, recombinant Dictyostelium EF1α domains I, II, and III). Therefore, rabbit actin was used in all actin-binding assays presented in this report. The tested proteins were mixed without (as control) or with preformed actin filaments and incubated at room temperature for 1 h. The mixtures were then centrifuged in an Airfuge (Beckman Coulter, Fullerton, CA) at 100,000 × g for 20 min. The amount of actin and tested protein in the supernatants and pellets were measured by SDS-PAGE and densitometry using Molecular Dynamics laser scanner and Image Quant software (Amersham Pharmacia Biotech, Piscataway, NJ). Most of the recombinant EF1α truncates are very soluble and showed only trace amounts in the pellet fraction after centrifugation in the absence of F-actin except truncate GST–49–221, which was fairly soluble but showed slightly more in the pellet than other truncates. The amount of the truncates in the pellet fraction in the absence of F-actin was subtracted from that in the presence of F-actin as the net binding. For measurement of β-actin mRNA binding to EF1α/F-actin bundles, EF1α or other actin-binding proteins were allowed to interact with F-actin at room temperature for 1 h. Biotin-labeled β-actin mRNA was then added and incubated for 1 h before being subjected to low-speed centrifugation at 50,000 × g for 2 min. Fractions of the reaction mixture before centrifugation and the supernatant after centrifugation were measured for biotin-labeled mRNA using the modified enzyme-linked immunosorbent assay (ELISA) as described in this report. Under the same conditions, the F-actin–bundling activity of rat liver EF1α, α-actinin (Cytoskeleton, Denver, CO), aprotinin (Sigma), fascin (gift from Dr. Fumio Matsumora), and fimbrin (gift from Dr. Paul Matsudaira) was confirmed. All of these tested proteins showed a single band on SDS-PAGE gel and pelleted with F-actin after low-speed centrifugation.
actin and EF1α, rabbit skeletal muscle actin, GST fusion proteins of Dictyostelium
and rat EF1α, and corresponding truncations were prepared as described previously (Liu et al., 1996b
). All of the GST fusion proteins were clarified by centrifugation at 320,000 × g
at 4°C for 30 min and stored in storage buffer (10 mM PIPES, 0.05% NaN3
, 0.1 mM EDTA, 1 mM DTT, and 25% glycerol, pH 7.0) on ice for the short term or under liquid nitrogen for the long term.
Affinity Purification of Domain-specific Antibodies
A recombinant fusion protein consisting of a Dictyostelium
EF1α C-terminal actin-binding sequence (amino acid residues 403–456), dihydro-folate reductase, and a 6× His-tag was expressed in bacteria using an expression vector, pQE-40, from Qiagen. The fusion protein was purified using a Ni- nitrilotriacetic acid agarose bead affinity column. The fusion protein was injected into rabbits and the antiserum obtained was purified on a GST-EF1α truncate (amino acids 403–456) fusion protein affinity column using a standard procedure described previously (Bresnick et al., 1991
Immunoprecipitation of Native Dictyostelium EF1α by Anti–C-Terminal Antibody
The affinity-purified C-terminal actin-binding site antibody was first incubated with protein-A beads (Amersham Pharmacia Biotech) in phosphate-buffered saline (PBS) containing 20 mM NaPO4, 300 mM NaCl, 0.1% of Tween 20, and 1 mg/ml BSA, pH7.5. Preimmune serum was used as a control. After 45 min at room temperature, the beads were washed four times with PBS and then were incubated with 0.5 μM Dictyostelium EF1α. After a 1-h incubation at room temperature, the reaction mixture was briefly centrifuged and the supernatant was obtained for SDS-PAGE. The beads were washed three times with PBS containing BSA and two times with PBS in the absence of BSA. The beads were finally boiled with gel sample buffer for SDS-PAGE. Because the heavy chain of rabbit immunoglobulin (Ig) G comigrates with Dictyostelium EF1α on SDS-PAGE gel, the amount of coprecipitated EF1α was detected by Western blotting using a chicken anti-rat EF1α peptide antibody that cross-reacts with Dictyostelium EF1α.
In Vitro Transcription of mRNA
A 1.8-kb full-length cDNA sequence of chicken β-actin was inserted into a pcDNA3 vector (Invitrogen, Carlsbad, CA) at the HindIII and XbaI sites. To linearize this plasmid for in vitro transcription, it was digested with XbaI, BamHI, or HindIII for full-length β-actin mRNA, the antisense mRNA of β-actin 3′-UTR, or the antisense mRNA of β-actin, respectively. The digested DNAs were separated by electrophoresis on a 1% agarose gel followed by purification using the Qiagen DNA extraction kit. These linearized cDNA sequences were then used as templates for in vitro transcription with an SP6 or T7 Maxiscript kit following the manufacturer's recommendations (Ambion, Austin, TX). mRNA transcripts were labeled with either biotin-16-UTP (Boehringer Mannheim, Indianapolis, IN) or [32P]α-CTP (Amersham Pharmacia Biotech). After biotin-labeling, unincorporated nucleotides were removed by using ProbeQuant G50 MicroColumns (Amersham Pharmacia Biotech). 32P-labeled RNA was purified on a 6% denaturing polyacrylamide gel containing 42% (wt/vol) urea. The position of the labeled RNA was identified by brief exposure of the gel to x-ray film. The band containing the labeled RNA was excised, and the RNA was eluted with elution buffer (1% SDS and 1 M ammonium acetate) overnight at 37°C. The RNA was then precipitated and washed with ethanol and redissolved in water.
Gel Mobility Shift Assay
Gel mobility shift was used to detect the RNA-protein interaction. Labeled chicken β-actin mRNA (10,000 cpm of 32P) was incubated with buffer or 5 μg of rabbit muscle actin or rat liver EF1α at room temperature for 20 min in 20 μl of reaction buffer (10 mM Tris-HCl, pH 7.4, 300 mM KCl, 1 mM DTT, 5 mM MgCl2, and 5% glycerol). To minimize nonspecific RNA-protein binding, heparin was added to a final concentration of 5 mg/ml and incubated for a further 10 min at room temperature. Samples were loaded on 3% native gel for electrophoresis. The gel was dried, and the position of the mRNA was identified by autoradiography.
ELISA to Detect the mRNA-EF1α Interaction
Because the ELISA assay can be used to analyze a large number of samples objectively and quickly, we used this assay for most of the characterization of the binding of EF1α to mRNA. The EF1α-mRNA interaction was analyzed by using a modified ELISA assay on a 96-well plate. Briefly, tested proteins were allowed to interact with F-actin to form complexes. The protein samples were then diluted and quickly distributed into the wells of the plate and incubated overnight at 4°C in assay buffer containing 100 mM KCl, 50 mM Tris-HCl, and 10 mM MgCl2, pH 7.4. These proteins were bound tightly and nonspecifically to the surface of the wells through charge and hydrophobic interactions. The wells were then washed five times with assay buffer and blocked with 1% BSA in assay buffer containing 0.05% Tween 20 for 30 min at room temperature. mRNA was added and incubated for 1 h at room temperature. After five washes with assay buffer, horseradish peroxidase-conjugated streptavidin (1:5000) was added and incubated for 30 min followed by 10 washes. Peroxidase substrate TMB (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added for color development for 10 min before the reaction was stopped by adding stop solution. The samples were read at a wavelength of 450 nm using an MRX Revelation Microplate Reader with Revelation software, version 4.0 (DYNEX Technologies, Chantilly, VA).
The effect of anti–C-terminal actin-binding site antibody (anti–403–456) on EF1α's cross-linking of actin filaments was analyzed using right angle light scattering as described previously (Liu et al., 1996b
). Briefly, rabbit muscle G-actin was allowed to polymerize for 2 h at room temperature before it was used. Native Dictyostelium
EF1α was incubated with IgGs for 1 h before being added to the F-actin solution in the cuvette, while the light scattering was being measured at 600 nm.
Electrophoresis and Western Blots
SDS-PAGE was performed according to the method of Laemmli (1970)
. Western blotting was performed according to the manufacturer's protocol using an ECL kit (Amersham Pharmacia Biotech).
Estimation of Binding Affinity
To estimate the binding affinity of EF1α truncates to F-actin, constant amounts of GST-EF1α recombinant proteins were allowed to interact with various amounts of F-actin. To measure the binding affinity of EF1α for mRNA, a constant amount of EF1α was used to interact with various amounts of biotin labeled β-actin mRNA. To estimate the apparent Kd
, the binding titration data were graphed with Origin software (version 4.1, RockWare, Golden, CO) and then curve fitted by nonlinear least squares to a bimolecular binding isotherm according to the following expression (Hulme and Birdsall, 1992
where AG is the concentration of bound ligand/receptor, At is the total concentration of ligand, and Gt is the total concentration of the receptor.
In addition, we have also used the GraphPad Prism software package (version 3.0; GraphPad Software, San Diego, CA) to analyze and curve fit the data for one- and two-site binding. The equations for one- and two-site binding are: Y = Bmax × X/(Kd + X) and Y = Bmax1 × X/(Kd1) + Bmax2 × X/(Kd2 + X), where Y is the total concentration of the receptor, X is the bound ligand, and Bmax is the maximal binding of the ligand.