RNA extraction and RT-PCRs
Mouse RNA was extracted with TRIZOL (Invitrogen) according to the manufacturer’s instructions. Adult human total heart RNA was obtained from Agilent Technologies. cDNA pools were prepared using Moloney murine leukemia virus reverse transcriptase (Promega).
Murine cDNAs were amplified with DNA polymerase (Phusion; Finnzymes) and splicing-specific (E25-27 forward, 5′-ACCGATGACGAGGATGAAGCTGAG-3′, and reverse, 5′-CTCTCTGACTGGGCACTTGGGTGG-3′; and ΔE25 forward, 5′-ATCACTGACTCTGGCAAGTTC-3′, and reverse, 5′-CTCTCTGACTGGGCACTTGGGTGG-3′) or splicing-independent (E24-27 forward, 5′-CTCATTTTTACTGTTTATGGGCCATCC-3′, and reverse, 5′-CTCTCTGACTGGGCACTTGGGTGG-3′) primer sets. Amplification of glycerine aldehyde 3-phosphate dehydrogenase (forward, 5′-CTCAAGATTGTCAGCAATGCATCC-3′, and reverse, 5′-CCAGTGGATGCAGGGATGATGTTC-3′) was used to normalize the amount of cDNA.
Preparation of transfection constructs
The FHOD3-FH2 domain was amplified from adult human heart cDNA using 5′-GAAGATCTGCCACCATGGTTCCTCCTCCTCCAGTG-3′ forward and 5′-CCCCGCGGCGGCTGGCTTGGGGGC-3′ reverse primers and cloned into the BglII and SacII restriction sites of a pEGFP-C1 vector (Takara Bio Inc.). Constructs containing or excluding the alternative T(D/E)5XE exon were selected after sequencing. CK2 receptor residues were mutated to alanine and aspartic acid by standard site-directed mutagenesis using 5′-GACCAGAGGGAAGATGATCGCCGATACTGATGAGGAGGAGG-3′ (T1474A), 5′-GGGAAGATGATCACCGATGCTGATGAGGAGGAGGAAGTTG-3′ (T1476A), 5′-CAGAGGGAAGATGATCGCCGATGCTGATGAGGAGGAGGAAG-3′ (T1474A + T1476A), and 5′-CAGAGGGAAGATGATCGATGATGATGATGAGGAGGAGGAAG-3′ (T1474D + T1476D) primers. FH2-GST constructs were created by subcloning the respective fragments into the BglII and SacII restriction sites of a pGEX-2TK vector (GE Healthcare).
Full-length FHOD3 was amplified from human heart cDNA with LA-taq polymerase (Takara Bio Inc.) using 5′-ATAACCGGTGCCACCATGGCCACGCTGGCTTGCC-3′ forward and 5′-TCCCCGCGGCAGCTGCAACTCCGAGG-3′ reverse primers, digested with AgeI and SacII restriction enzymes, and cloned into the BspEI and SacII restriction sites of a pEGFP-C1 or HA-C1 vector. Splice isoforms were selected after sequencing, and phosphomutants were created as described earlier for the FH2 domain constructs. Diaphanous autoregulation domain (DAD) deletion constructs were amplified from full-length FHOD3, and the respective splice isoforms and mutant constructs were amplified by using the full-length forward and the FH2 reverse oligos and cloned as described for the full-length constructs.
The FHOD1-FH2 domain was amplified from adult human skeletal muscle cDNA using 5′-GGAATTCGACAGCTCAGCCCTCCCC-3′ forward and 5′-GGGGTACCGGCGATTGTGTGTGGTG-3′ reverse primers and cloned into the EcoRI and KpnI restriction sites of a pEGFP-C2 vector. mDia1 was amplified from adult human heart cDNA using 5′-CCGCTCGAGCCACCATGGAGCCGCCCGGCGG-3′ forward and 5′-GGGGTACCGTGCTTGCACGGCCAACC-3′ reverse primers and cloned into the XhoI and KpnI restriction sites of a HA-C1 vector. All p62 constructs were a gift from M. Gautel (King’s College London, London, England, UK).
Newborn rat hearts were digested using the neonatal cardiomyocyte isolation system (Worthington Biochemical Corp.) according to the manufacturer and cultured basically as described previously (Lange et al., 2002
). Cells were plated onto collagen-coated dishes (PureCol; Inamed Biomaterials) with a density of 0.4 × 106
cells per 35-mm culture dish in plating medium (68% DME [Sigma-Aldrich], 16% medium M199 [Sigma-Aldrich], 10% horse serum [Sigma-Aldrich], 5% fetal calf serum [PAA], 4 mM glutamine [Sigma-Aldrich], and 1% penicillin–streptomycin [Sigma-Aldrich]). After 24 h in culture, the cells were transfected for 6 h with ~1 µg of plasmid DNA per 35-mm dish with Escort III (Sigma-Aldrich) or jetPRIME (Polyplus-transfection via Source BioScience) in antibiotics-free medium and then changed to maintenance medium (78% DME, 20% medium M199, 4% horse serum, 1% penicillin–streptomycin, 4 mM glutamine, and 0.1 mM phenylephrine [Sigma-Aldrich]). After 48 h, cells were fixed with 4% paraformaldehyde (Agar Scientific) in PBS for 10 min. For long-term maintenance, the cells were kept in long-term maintenance medium (20% medium M199, 75% DBSSK [116 mM NaCl, 1 mM NaH2
, 0.8 mM MgSO4
, 5.5 mM glucose, 32.1 mM NaHCO3
, and 1.8 mM CaCl2
, pH 7.2], 4% horse serum, and 4 mM glutamine). Freshly isolated ARCs were donated by F. Cuello (King’s College London, London, England, UK). For fixation, cells were incubated with 4% paraformaldehyde (Agar Scientific) in PBS for 10 min. To permeabilize the membrane, cells were incubated for 5 min with 0.2% Triton X-100/PBS and then washed three times in PBS. Primary and secondary antibodies were diluted in antibody dilution buffer (1% BSA, 20 mM Tris-base, 155 mM NaCl, 2 mM EGTA, and 2 mM MgCl2
, pH 7.5). Blocking reagent (MAXblock; Active Motif) was used before stainings with the FHOD3 antiserum according to the manufacturer’s instructions. The cells were incubated in the primary antibody solution for 1 h at RT (overnight at 4°C for ARCs) and then washed three times in PBS, followed by incubation with the secondary antibody solution for 1 h at RT (5 h at RT for ARCs). For triple-immunofluorescence staining, Cy2/Alexa Fluor 488–, Cy3-, and Cy5/Alexa Fluor 633–conjugated secondary antibodies and phalloidin (Jackson ImmunoResearch Laboratories, Inc.), as well as DAPI (Sigma-Aldrich), were used. Cells were mounted in 0.1 M Tris-HCl/glycerol (3:7) and 50 mg/ml N
-propyl-gallate, pH 9.5.
For immunohistochemistry, tissues were cut on a cryostat (Jung CM1800; Leica) into 12-µm sections, mounted on poly-l-lysine–coated microscope slides, fixed with precooled acetone for 5 min at −20°C, and stained as described for the NRCs.
Human heart samples were obtained from the heart tissue bank set up by C. dos Remedios at the University of Sydney (Hospital Research Ethical Committee approval 09-2009-12146). Human materials were used in accordance with the ethical guidelines of King’s College London (College Research Ethical Committee 04/05-74) and the current UK law.
COS-1 and HeLa cells
COS-1 or HeLa cells were cultured in maintenance medium (10% fetal calf serum, 1% penicillin–streptomycin, and 4 mM glutamine in DME) at 37°C in 5% CO2. Cells were passaged at 70–80% confluency by standard trypsinization. For transfection, cells were plated onto 35-mm dishes and grown to 50–70% confluency. COS-1 and HeLa cells were transfected with 1 µg of plasmid DNA using transfection reagent (Escort IV; Sigma-Aldrich) or FuGENE HD (Roche), respectively. After 48 h, cells were fixed for immunocytochemistry as described for the NRCs or lysed with 200 mM NaCl, 10 mM Tris, 0.5% NP-40, 1 mM DTT, and protease inhibitor (Complete, Mini; Roche), pH 7.4, for pull-down assays and coimmunoprecipitations. For phosphorylation-sensitive experiments, 0.05 µM calyculin A, 0.2 mM NaVO4, and 30 mM sodium pyrophosphate were added to the lysis buffer.
All confocal microscopy was performed at RT on a laser-scanning upright confocal microscope (LSM510; Carl Zeiss, Inc.) with solid-state (diode) laser excitation at 405 nm and emission at 420–480 nm, Ar laser excitation at 488 nm and emission at 505–530 nm, or HeNe laser excitation at 563 and 633 nm and emission at 560–615 nm and >650 nm using LCI Plan-Neofluar 25×/0.8 NA, Plan-Apochromat 63×/1.4 NA, or Plan-Neofluar 100×/1.3 NA oil immersion objectives.
Bacterial protein expression
Expression plasmids were transformed into the Escherichia coli expression strain BL21-RIL (EMD). Bacteria were incubated until the OD600 reached 0.6, and protein expression was induced with 0.4 mM IPTG. After 3 h, bacterial cells were harvested by centrifugation, and cell pellets were frozen in liquid nitrogen. Cells were lysed with lysozyme and DNase in 10 mM Tris, 200 mM NaCl, and 1 mM DTT, pH 7.4. GST fusion proteins were purified with glutathione Sepharose 4 fast flow beads (GE Healthcare) according to the manufacturer’s instructions.
GST pull-down assays and coimmunoprecipitations
The amount of GST fusion proteins bound to glutathione beads was normalized by SDS-PAGE and Coomassie staining. Equal amounts of fusion proteins were then added to lysates of COS-1 cells, previously transfected with GFP fusion constructs as described earlier. Unmodified GST peptides were used as a control. Samples were incubated for 90 min at 4°C on a shaking platform before beads were centrifuged and washed with PBS. Supernatant was collected in new tubes. SDS samples of beads and COS-1 lysates were prepared and applied to SDS-PAGE and immunoblotting as described previously (Lange et al., 2002
). For coimmunoprecipitation assays, lysates were incubated with anti-HA or anti-GFP antibodies for 4 h at 4°C before addition of BSA-blocked protein G–Sepharose (Sigma-Aldrich) for 4 h. Beads were separated by centrifugation, washed with PBS, boiled in SDS buffer, and applied to SDS-PAGE and immunoblotting. For tissue immunoprecipitation and in cyto phosphorylation assays, the antibodies were covalently cross-linked to protein G–Sepharose using dimethylpimelimidate as a coupling agent. Samples were prepared as described earlier and applied to SDS-PAGE and immunoblotting. After incubation with the primary antibody, blots were incubated with immunoprecipitation detection reagent (Clean-Blot; Thermo Fisher Scientific) instead of the secondary antibody for detection of the bands with ECL.
CK2 phosphorylation assays and CK2 inhibitor experiments
For in vitro phosphorylation assays, 2.5 µg of purified GST fusion proteins were incubated with 50 U CK2 (New England Biolabs, Inc.) in 20 mM Tris, 50 mM KCl, 10 mM MgCl2, and 25 µM ATP, pH 7.5, for 30 min at 30°C and subjected to SDS-PAGE and immunoblotting. For in cyto experiments, COS-1 cells or NRCs were grown for 24 h and then treated with 10 µM of the CK2 Inhibitor DMAT (InSolution; Merck Chemicals) for 24 h before cell lysis or fixation and antibody staining as described earlier.
Yeast two-hybrid assay
FHOD3-FH2 (including or excluding the alternative T(D/E)5XE exon) was amplified by PCR and cloned into the NcoI and BamHI sites of a modified pLexA vector (provided by M. Gautel). Yeast two-hybrid screening procedures and β-galactosidase filter assays were performed as described by the manufacturer. The plasmid was transferred into Saccharomyces cerevisiae reporter strains (NMY51; Dualsystems), which were then transformed with an adult human cardiac cDNA library (Takara Bio Inc.). 105 primary transformants were screened by plating on selection plates lacking the amino acids histidine, tryptophan, and leucine. NMY51 clones were transferred to more restricted selection plates, additionally lacking adenine for further exclusion of false-positive interactions. Library plasmids from positive clones were isolated, transformed into E. coli XL1-blue for amplification of the plasmid, and sequenced. For further verification of the interaction, identified clones were retransformed into the yeast together with the empty pLexA plasmid or the corresponding FHOD3-pLexA constructs and grown on selection plates lacking histidine, tryptophane, and leucine and containing 3-AT in concentrations between 0.5 and 5 mM.
Oligonucleotides for shRNA were designed to target 22 nucleotide sequences in the FH1 and FH2 domains of Rattus norvegicus FHOD3
with ends compatible to BglII and XhoI restriction sites. Oligos (FHOD3-shRNA forward, 5′-GATCCCCATAGACCAGTTGGAGAACAATATTCAAGAGATATTGTTCTCCAACTGGTCTATTTTTTGGAAC-3′, and reverse, 5′-TCGAGTTCCAAAAAATAGACCAGTTGGAGAACAATATCTCTTGAATATTGTTCTCCAACTGGTCTATGGG-3′; and control shRNA forward, 5′-GATCCCCAGAAGGTTCCAAAGGTTAAAGATTCAAGAGATCTTTAACCTTTGGAACCTTCTTTTTTGGAAC-3′, and reverse, 5′-TCGAGTTCCAAAAAAGAAGGTTCCAAAGGTTAAAGATCTCTTGAATCTTTAACCTTTGGAACCTTCTGGG-3′) were obtained from Sigma-Aldrich. 2 nmol of forward and reverse oligonucleotides were diluted to 50 µl of 30 mM Hepes, 100 mM potassium acetate, and 2 mM magnesium acetate, pH 7.4, incubated for 4 min at 98°C and 10 min at 70°C, and then slowly cooled down to 4°C. Annealed oligos were ligated into the BglII and XhoI restriction sites of a modified cDNA 3.1 vector, which was constructed by inserting the H1 expression cassette from pSUPER (Brummelkamp et al., 2002
; donated by R. Agami, Netherlands Cancer Institute, Amsterdam, Netherlands), as well as an EGFP gene under the control of a cytomegalovirus promoter (Fukuzawa et al., 2008
). Successful cloning was confirmed by restriction digest and sequencing. shRNA constructs were used to transiently transfect COS-1 cells and NRCs as described earlier.
NRCs were cultured and transfected as described earlier. Cells were then grown for 5 d in long-term maintenance medium containing 1 µg/ml verapamil and then treated with 20 µM latrunculin B (VWR International) in long-term maintenance medium containing verapamil overnight. Latrunculin was washed out, and cells were incubated for 30 min or 2 h in maintenance medium containing verapamil before fixation and antibody staining as described earlier.
COS-1 cells were cultured and transfected as described earlier. 24 h after transfection, 1 µg/µl cycloheximide was added to the cells to inhibit protein synthesis. Cells were harvested at different time points between 0 and 72 h. Lysates were normalized by total protein content and applied to immunoblotting as described earlier.
Quantification of actin polymerization activity
HeLa cells were transfected as described earlier, changed to starvation medium (0.1% fetal calf serum, 1% penicillin–streptomycin, and 4 mM glutamine in DMEM) 24 h after transfection, and fixed as described earlier after culturing for another 24 h. Cells were stained and imaged as described earlier. Activation of actin polymerization was analyzed with CellProfiler Image Analysis software (Carpenter et al., 2006
) essentially by measuring the phalloidin pixel intensity and calculating the ratio between transfected and untransfected cells. Presented values are the means between three separate experiments with 9–12 quantified images per construct per experiment.
For production of a polyclonal rabbit (pRb) anti-FHOD3 antibody, the N-terminal 339 amino acids of FHOD3 were amplified from human cDNA with 5′-GGAATTCCGCCACCATGGCCACGCTGGCTTGCC-3′ forward and 5′-CGGGATCCCGCCCGGCACCCACTGGG-3′ reverse primers and cloned into the EcoRI and BamHI restriction sites of a pET-3b (EMD) vector. The His6 fusion protein was expressed as described earlier and purified with 1 ml high performance columns (HisTrap; GE Healthcare) on an HPLC system (Äkta; GE Healthcare).
Purified His6 fusion proteins were dialyzed against PBS and concentrated to a final concentration of >2 µg/µl. Purity was tested by SDS-PAGE and Coomassie staining. The purified His6 fusion protein was sent for the immunization of rabbits to BioScience. Anti-FHOD3 immunsera were affinity purified with antigen-coupled Sepharose columns.
The monoclonal mouse anti–sarcomeric α-actinin (clone EA-53) antibody, as well as the pRb anti–all actin antibody, was obtained from Sigma-Aldrich. The monoclonal mouse anti-GFP (mixture of clones 7.1 and 13.1), as well as the monoclonal rat anti-HA antibody (clone 3F10), was purchased from Roche. The monoclonal mouse anti–phospho-threonine (clone 4H4; donated by G. Fruhwirth, King’s College London, London, England, UK) and the pRb LC3B antibodies were obtained from Cell Signaling Technology, and the monoclonal mouse anti-p62 antibody was obtained from Abcam. The monoclonal mouse anti–cardiac actin antibody (clone Ac1-20.4.2) was obtained from Progen. The polyclonal goat anti-GST antibody was obtained from GE Healthcare. The monoclonal mouse anti-myomesin (clone B4) was a gift from J.-C. Perriard’s Laboratory (Institute of Cell Biology, Swiss Federal Institute of Technology Zürich Hönggerberg, Zürich, Switzerland). The pRb antibodies anti–myosin-binding protein C and anti-p62 were gifts from M. Gautel. Horseradish peroxidase–conjugated anti–mouse Igs were purchased from Dako. Horseradish peroxidase–conjugated anti–rabbit and anti–goat Igs were purchased from EMD. Cy2-conjugated anti–rabbit Igs, Cy3-conjugated anti–mouse Igs, and Cy5-conjugated anti–rabbit and anti–mouse Igs were all purchased from Jackson ImmunoResearch Laboratories, Inc.
Original digital images obtained either from the confocal microscope or by scanning of x-ray films using a scanner (Perfection 4870 Photo; Epson) were assembled to the figures and labeled using Photoshop or Illustrator (Adobe). Only linear contrast adjustments were used and were always applied to the entire image.
Online supplemental material
Fig. S1 shows a modified diagram of the gene structure of human (Homo sapiens
), chimpanzee (Pan troglodytes
), and mouse (M. musculus
as annotated on Ensembl. Fig. S2 shows that all FHOD3 isoforms colocalize with F-actin and affect actin polymerization to a comparable extent. Fig. S3 shows the characterization of the affinity-purified polyclonal antibody against FHOD3. Fig. S4 depicts that the muscle FHOD3 isoform shows increased stability in COS-1 cells compared with FHOD3 lacking the T(D/E)5
XE exon and with mutants that cannot be phosphorylated. Fig. S5 shows that knockdown of FHOD3 in NRCs leads to myofibril disassembly. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201005060/DC1