Materials
Antibodies
Monoclonal mouse anti-skeletal α-actin (clone AC-1-20.4), anti-ß-actin (clone AC15), mouse anti-sarcomeric α-actinin, rabbit anti-all actins (clone C11) antibodies were obtained from Sigma-Aldrich (Munich, Germany). Donkey anti-mouse Alexa Fluor® 488 or 568 and anti-rabbit Alexa Fluor® 488 or 568 antibodies were from Molecular Probes (Eugene, Oregon; USA). Goat anti-HA (clone Y-11) antibody was obtained from Santa Cruz Biotechnology (Dallas, Texas, USA); mouse anti-cardiac α-actin was purchased from Progen Biotechnik GmbH (Heidelberg, Germany). Monoclonal anti-myomesin (clone B4) antibody (Grove et al., 1984) was a kind gift from Dr. E. Ehler (King’s College London, London, U.K.).
Clones:
The pcDNA3.1/NT-GFP-TOPO®-WT-α-cardiac actin and the mutants p.A295S, p.R312K, and p.E361G were donated from Dr. Cora-Ann Schoenenberger (University Basel, Switzerland). The p.R312H mutant was generated by site-directed mutagenesis from the p.R312K variant. Since the biochemical properties of the p.R312K variant were found to be very similar to wt c-actin, the results of its analysis are included in the Supplementary Information. The c-α-actin containing plasmids served as templates for cloning the c-α-actin variants into p3xHA-C1 plasmid. The p3xHA-C1 plasmid was a kind gift from Dr. T. Engel (Leibniz-Institut für Arteriosklerosis, Münster University, Germany), who deleted cDNA of EGFP from pEGFP-C1 plasmid (Clontech) and instead cloned into this plasmid the cDNA of a three times repeated hemagglutinin-tag (HA). The primers used for amplifying the actin cDNAs were as follows: 5’-GTTATGTGTGACGACGAGGAGACC-3’ and 5’-ATTGCCCTTTTAGAAGCATTTGCG-3’. PCR inserts were cloned into p3xHA-C1 using XbaI and XhoI sites.
The deletion construct of human gelsolin G4-6 was kindly supplied by Dr. A.G. Weeds (MRC-LMB, Cambidge, UK) and subcloned from shuttle vector pKN172 into the cold-shock expression plasmid pCOLD II (Takara Bio Inc., Kusatsu, Japan) using the restriction sites for BamHI and HindIII enzymes obtained from Fermentas (Vilnius, Lithuania). The pCOLD II plasmid provides a His-Tag sequence for affinity chromatography, which was fused to the N-terminus of G4-6and subsequently used to affinity-purify the c-α-actins [39,42]. Arp2/3 complex isolated from Acanthameba castellani was kindly supplied by Prof. M. Barber (San Francisco, California, USA) and mDia3-FH2 by Prof. Alfred Wittinghofer (MPI, Dortmund, Germany).
Reagents
Pyrene-maleinimide was obtained from Sigma Aldrich (Munich, Germany). All reagents were of analytical grade.
Protein expression and purification
Rabbit skeletal muscle and bovine c-α-actin were prepared from dried acetone powder and human cardiac muscle wt α-actin and its mutants were expressed in the baculovirus/Sf21-system [2,39] as detailed in the Supplementary Information. Preprations of myosin subfragment 1 (myosin-S1) from skeletal muscle and bovine cardiac muscle [58], of cardiac tropomyosin (cTm) [4,21] and troponin complex (cTn) [1,10,12], the N-terminal C0C2 fragment of human cardiac MyBP-C [51], and the gelsolin deletion mutant G4-6 [42] were performed with modifications of published procedures as detailed in the Supplementary Information.
Analytical procedures
ATPase assay
Stimulation of the ATPase activity of ß-cardiac or skeletal muscle myosin-S1 activated by human wt cardiac α-actin or the cardiomyopathy inducing mutants was performed at 25 °C using a modified version of NADH-coupled assay according to [35] in a buffer containing 40 mM HEPES, pH 7.4, 25 mM KCl, 2 mM MgCl2, 0.5 mM DTT, 0.2 mM NADH and an ATP regeneration system consisting of 0.05 mg/ml pyruvate kinase, 0.5 mM PEP, and 0.02 mg/ml LDH. The reaction was started by addition of myosin-S1 to a final concentration of 1 µM. NADH oxidation was followed by measuring the decrease in absorption at 340 nm (ε = 6220 M1 cm− 1) [48,61] using a spectrophotometer (DU 800, Beckman Coulter, Krefeld, Germany). The ATPase rates were determined by linear curve fitting and repeated at least three times for each condition with at least two different c-actin variant purifications.
DNase I inhibition assay
The DNase I inhibition assay was performed as described [34]. The DNase test solution contained 50 µg/ml salmon sperm DNA (Sigma-Aldrich D1626) in 10 mM Tris-HCl, pH 8.0, 1 mM MgCl2 and 0.1 mM CaCl2. To determine the endonuclease activity of DNase I, a 10 µl pre-incubation reaction containing 3.2 µM DNase I from bovine pancreas (Sigma-Aldrich DN25) and zero to 6.4 µM of G-actin was prepared in G-buffer and incubated at room temperature for 20 min. Aliquots of the samples were added to 0.8 ml of 50 µg/ml DNA-solution and the absorbance was immediately monitored at 260 nm for 10 min using the Beckman DU 800 spectrophotometer. From the ratio of the initial linear rates of DNase I activity were determined and expressed as Kunitz units (KU/min; 1 KU = ΔOD 260 nm of 0.001).
Gel electrophoresis
Polyacrylamide gel electrophoresis in the presence of SDS (SDS-Page) was performed as given [30]. Native gel electrophoresis was performed on 10% polyacrylamide gels without SDS and run as described previously [47].
Immunoblotting:
Cells were lysed 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) and vortexed for 30 s and frozen until use. After thawing, the extracts were vortexed again and centrifuged at 20,817x g at 4 °C for 5 min. The protein concentration was estimated according to Bradford [7]. 30 µg of protein extracts were separated on 12.5 % SDS-Page gels. Proteins were transferred to nitrocellulose membrane using a wet blotter [24,59]. Subsequently the membranes were blocked for 1 h in Tris-buffered saline with 1% Tween-20 (TBS-T) containing 5 % non-fat milk powder (blocking solution) and then incubated overnight at 4 °C with primary antibody diluted in blocking solution (goat anti-HA 1:500, mouse anti-α-cardiac actin 1:200, mouse anti-α-actinin 1:2,000, mouse anti-GFP at 1:2,500 and rabbit anti-actin C11 at 1:1,000 dilution). After three washing steps with TBS-T for 15 min at room temperature, the nitrocellulose sheets were incubated with secondary antibodies linked to horse radish peroxidase (HRP) diluted in blocking solution (1:2,000) directed against either mouse or rabbit or goat for 1 h at RT. The nitrocellulose membranes were developed with the help of enhanced chemiluminescence (ECL) system (GE Amersham, Stepstone, UK). Occasionally membranes were subsequently stripped, re-blotted and immunostained for total actin.
Actin polymerization assays:
Actin polymerization rates were determined by the increase in fluorescence caused by incorporation of pyrene-labelled actin into actin filaments [28,39]. Pyrene-labelled actin was pre-cleared by dialysis against G-buffer (10 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 7 mM β-mercaptoethanol, 1 mM ATP) and centrifugation at 100,000xg for 30 min. In these tests we used pyrene-labelled skeletal muscle actin that was added to the c-actins at a ratio of 20:1 (0.25 to 5 c-α-actin). Since pyrene-labelled skeletal-actin on its own at 0.25 µM did not show significant polymerization, i.e. increase in fluorescence. Therefore, we assume that the increase in fluorescence observed after mixing it with globular c-α-actin in G-buffer was solely due to the polymerization of the c-α-actins. Polymerization was induced by addition of 2 mM MgCl2 and 0.1 M KCl. Though The increase of pyrene fluorescence with excitation wavelength of 365 nm was monitored at 385 nm using a Schimadzu RF5001PC spectrofluorometer.
Critical concentration
To determine the critical concentration of c-α-actin polymerization the varying concentrations of the c-α-actins supplemented with 5% pyrene-actin were polymerized in the presence of 2 mM MgCl2 and 0.1 M KCl overnight. The actin concentrations varied from 0.1 to 10 µM. The steady-state fluorescence of polymerized actin was plotted versus monomeric actin concentration and the critical concentration was calculated form the intersections with the abscissa.
Determination of the Ca 2+ -dependence of Tm movement on cardiac F-actins:
The Ca2+-dependence of pyrene-labelled cTm movement on polymerized c-actin variants was determined by the increase of the eximer pyrene-fluorescence at excitation and emission wavelengths of 340 nm and 480 nm, respectively, using an Infinite 200 microplate reader (Tecan, Männedorf, Switzerland). Thin filaments were reconstituted from each c-actin variant with pyrene-labelled cTm and reconstituted cTn complex, myosin-S1, and N-cMyBP-C each added at a 1:6 molar ratio to actin subunits. Distinct free Ca2+-concentrations in the presence of 1 mM ATP were generated in black 96-well plates [10]. Fluorescence intensities were corrected for background fluorescence and normalized to Fmax = 1 and Fmin = 0. Nine experiments were performed for each c-α-actin variant and condition. The data were fitted using a normalized Hill equation (Sigma Plot, Systat Software, Erkrath, Germany).
Generation of recombinant adenoviruses
For the generation of recombinant adenoviruses (Ad) the AdEasy™ kit (Qbiogene) was applied [37,39]. DNA sequences encoding wt- and the mutants A295S-, R312H-, and E361G- c-α-actins fused at the N-terminus to a HA-tag were amplified by PCR with the primers: 5’ATCATGGATTACCCATACGATGTTC-3’ and 5’-ATCGCCCTTTTAGAAGCATTTGCG-3’. As templates served p3xHA-C1 plasmids encoding wt and the mutant cardiac α-actins. The EcoRV site was used to clone PCR inserts into pAdTrack-CMV shuttle plasmid. Electro-competent bacteria E.coli BJ5183 were simultaneously transformed with the shuttle plasmid linearized with the help of PmeI and adenoviral AdEasy-1 DNA backbone. Following homologous recombination in bacteria, clones were screened by restriction with the PacI enzyme that in the case of positive clones resulted in two 33 kb and 4.5 kb fragments. Lipofectamine™ (Invitrogen) reagent was used to transfect HEK293 cells with linearized pAdEasy-1 construct encoding wild type and the mutant cardiac α-actins. Since pAdEasy-1 lacks of E1 and E3 genes critical for successful packaging of adenoviruses, it was crucial to generate adenoviral particles in HEK293 cells, which contain these two genes. The adenoviral DNA encoded additionally EGFP enabling tracking the generation of viral particles. After two to three weeks, the cells were lysed liberating viral particles. HEK293 cells were twice re-infected with recombinant adenoviruses in order to obtain higher amounts of viral particles. For more details concerning the structure of recombinant adenoviral DNA and the steps of recombinant adenoviruses generation see [22]. The correctness of DNA constructs was verified by sequencing.
Cell culture and immunohistological procedures
Cells
HeLa, C2C12 were from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) and MDCK cells (Marvin-Darby canine cells) were kindly supplied by Prof. Anna Starzinski-Powitz (Frankfurt, Germany). The cells were propagated in DMEM medium containing 0.5% glucose, 1% penicillin/streptomycin, 1% glutamine, 0.5% sodium pyruvate, and 10% fetal calf serum. Cells were cultured in 25 cm2 flasks (Falcon®, Becton Dickinson GmbH, Heidelberg, Germany) at 37 °C in 5% CO2 and 90% humidified air and split weekly, using 0.25% trypsin/0.05% EDTA solution.
Cardiomyocytes from 1–5 days old rats were isolated following the modified protocol described in [16,37] as detailed in the Supplementary Information.
Cell transfection
The cells were seeded on glass coverslips in 6-well plates (3 x 105 cells/well) and transfected with the help of MATra-A reagent (Iba, Munich, Germany) with 3 µg of DNA encoding either for GFP-actins or HA-actins. For Western blot analysis the cells were seeded in 6 cm plastic Petri dishes and transfected with 5 µg DNA. 24 h or 48 h after transfection the cells on coverslips were fixed with 4% formaldehyde (FA) or harvested in Petri dishes in lysis buffer using a rubber policeman.
NRCs were infected with 20 µl of adenoviruses added to 2 ml of medium following the procedure described [39]. 72 h after infection the cells were either fixed with warm (37 °C) 4% formaldehyde (FA) for immunocytochemistry or harvested with the help of rubber policeman for Western blotting. For controls, cells were infected with only EGFP-encoding viruses.
Confocal microscopy
Control cells, transfected cells and those infected with adenoviruses were fixed with warm (37 °C) 4% FA for 20 min at RT and permeabilized with 0.1% Triton X-100 in PBS for 6 min. For staining with anti-cardiac α-actin-antibody, we additionally fixed the cells with ice-cold methanol for 6 min at 4 °C. After fixation the coverslips or plastic dishes were blocked for 30 min with 3% BSA in PBS. All antibodies were diluted in PBS containing 3% BSA. The cells were immunostained either with goat anti-HA IgGs, or with monoclonal antibodies directed against anti-β-actin, anti-c-α-actin, anti-sarcomeric α-actinin, and anti-myomesin. The secondary IgGs were conjugated either with Alexa Fluor® 488 or Alexa Fluor® 568. In the case of double immunostaining, when the goat anti-HA antibody was applied, donkey anti-mouse IgGs were used in order to avoid cross-reactivity. F-actin was visualized by staining with TRITC-conjugated phalloidin (Sigma-Aldrich). The nuclei were visualized with the help of Hoechst 33342 (Riedel-de-Haen). The coverslips or plastic dishes were washed several times with PBS for 5 min. After all incubations and washing steps the cells were mounted in DAKO cytomatic fluorescent mounting medium. Immunofluorescence microscopy was performed using a Zeiss LSM 800 laser-scanning microscope (Jena, Germany). For documentation at least 5 cells were photographed from three independent experiments and a representative image is presented. Co-localization analysis was performed by using ZEN 2007 software (Carl Zeiss Vision GmbH, Goettingen, Germany) and confirmed when the Pearson’s correlation coefficient was > 0.3.
Control cells, transfected cells and those infected with adenoviruses were fixed with warm (37 °C) 4% FA for 20 min at RT and permeabilized with 0.1% Triton X-100 in PBS for 6 min. For staining with anti-cardiac α-actin-antibody, we additionally fixed the cells with ice-cold methanol for 6 min at 4 °C. After fixation the coverslips or plastic dishes were blocked for 30 min with 3% BSA in PBS. All antibodies were diluted in PBS containing 3% BSA. The cells were immunostained either with goat anti-HA IgGs, or with monoclonal antibodies directed against anti-β-actin, anti-c-α-actin, anti-sarcomeric α-actinin, and anti-myomesin. The secondary IgGs were conjugated either with Alexa Fluor® 488 or Alexa Fluor® 568. In the case of double immunostaining, when the goat anti-HA antibody was applied, donkey anti-mouse IgGs were used in order to avoid cross-reactivity. F-actin was visualized by staining with TRITC-conjugated phalloidin (Sigma-Aldrich). The nuclei were visualized with the help of Hoechst 33342 (Riedel-de-Haen). The coverslips or plastic dishes were washed several times with PBS for 5 min. After all incubations and washing steps the cells were mounted in DAKO cytomatic fluorescent mounting medium. Immunofluorescence microscopy was performed using a Zeiss LSM 800 laser-scanning microscope (Jena, Germany). For documentation at least 5 cells were photographed from three independent experiments and a representative image is presented. Co-localization analysis was performed by using ZEN 2007 software (Carl Zeiss Vision GmbH, Goettingen, Germany) and confirmed when the Pearson’s correlation coefficient was > 0.3.
Control cells, transfected cells and those infected with adenoviruses were fixed with warm (37 °C) 4% FA for 20 min at RT and permeabilized with 0.1% Triton X-100 in PBS for 6 min. For staining with anti-cardiac α-actin-antibody, we additionally fixed the cells with ice-cold methanol for 6 min at 4 °C. After fixation the coverslips or plastic dishes were blocked for 30 min with 3% BSA in PBS. All antibodies were diluted in PBS containing 3% BSA. The cells were immunostained either with goat anti-HA IgGs, or with monoclonal antibodies directed against anti-β-actin, anti-c-α-actin, anti-sarcomeric α-actinin, and anti-myomesin. The secondary IgGs were conjugated either with Alexa Fluor® 488 or Alexa Fluor® 568. In the case of double immunostaining, when the goat anti-HA antibody was applied, donkey anti-mouse IgGs were used in order to avoid cross-reactivity. F-actin was visualized by staining with TRITC-conjugated phalloidin (Sigma-Aldrich). The nuclei were visualized with the help of Hoechst 33342 (Riedel-de-Haen). The coverslips or plastic dishes were washed several times with PBS for 5 min. After all incubations and washing steps the cells were mounted in DAKO cytomatic fluorescent mounting medium. Immunofluorescence microscopy was performed using a Zeiss LSM 800 laser-scanning microscope (Jena, Germany). For documentation at least 5 cells were photographed from three independent experiments and a representative image is presented. Co-localization analysis was performed by using ZEN 2007 software (Carl Zeiss Vision GmbH, Goettingen, Germany) and confirmed when the Pearson’s correlation coefficient was > 0.3.
Electron microscopy
For negative staining F-actin samples were diluted to 0.1 mg/ml and adsorbed to freshly glow-discharged carbon-coated copper grids (200 mesh) for 45 sec. Negative staining with 1.0% uranyl acetate was performed as described [26,48]. The samples were examined in a Zeiss electron microscope EM923 (SESAM) run at 150 kV fitted with a TemCamF416 camera (Tietz Video and Image Processing Systems, Gauting, Germany).
Data evaluation
DNA sequences were analysed in DNAstar Lasergene software (DNASTAR Inc., Madison, Wisconsin, USA). Densitometric analysis of bands was performed with the help of the Ultra Quant 6.0 software (Thermo Fisher Scientific, Schwerte, Germany). Graphs were plotted in Excel 2007 (Microsoft®) or in Origin 8.5 (OriginLab). In both the myosin-S1 ATPase activity and Ca2+-dependence of cTm movement assays the data are given as mean values (± SEM, standard error of the mean). In addition, the significance of the Ca2+-dependency data were analysed by the Student´s t-test and the R-square (R2) analysis using Sigma Plot Software (Systat, Erkrath, Germany).