Actinin

To construct mammalian expression plasmids, the respective genes of FPs were PCR-amplified as AgeI-NotI fragments and swapped with a gene encoding EGFP in the pEGFP-N1 plasmid (Clontech). IFP2.0-N1 and mIFP-N1 plasmids were acquired from Addgene (#54785 and #54620, respectively).
For protein tagging and labelling of intracellular structures study, miRFPs were amplified, digested with restriction enzymes and then swapped with mTagBFP2 either as C- (for α-tubulin and clathrin) or N-terminal fusions (for keratin, α-actinin, LifeAct, EB3, myosin, vimentin, clathrin, LAMP1, zyxin, H2B and mitochondrial signal) as previously described50 (link). C-terminal fusions (SGGGG)_n linker was increased to 30 amino acids. N-terminal fusions linker length was left unchanged.
To create an IκBα reporter plasmid (CMV-IκBα-miRFP703), we used a CMV-IκBα-FLuc plasmid kindly provided by S. Achilefu and D. Piwnica-Worms. A FLuc gene was replaced with one of the miRFP genes. Kozak sequence was deleted in the CMV-IκBα-miRFP703 and CMV-miRFP control plasmids.
miSplit670 and miSplit709 reporter plasmids, which are pC4-RHE-PAS, pC4EN-F1-mGAF670 and pC4EN-F1-mGAF709, were constructed from an iSplit plasmids25 (link) by swapping either PAS or GAF domains. A linker -ggggsggggs- was left unchanged. Where appropriate, an NLS sequence in the pC4EN-F1 plasmid was deleted by site-directed mutagenesis.
For mRNA labelling, a CMV-PAS-MCP plasmid was constructed as follows. PAS-ggggsggggs- without STOP codon was amplified as a single fragment and inserted into the C1 vector backbone using AgeI and KpnI sites, MCP was amplified from an ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC plasmid (Addgene, #52985) and inserted at KpnI and BamHI sites. The cmv-PCP-mGAF670 and cmv-PCP-mGAF709 plasmids were constructed as follows. A PCP without STOP codon was amplified from an ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC plasmid and then inserted into the C1 vector backbone using AgeI and EcoRI restriction sites. A -ggggsggggs-miGAF was amplified as a single fragment and inserted using EcoRI and KpnI sites. A phage-cmv-cfp-12xMBS-PBS was obtained by swapping a 12xMBS-PBS fragment from a Pcr4-12xMBS-PBS (Addgene, #52984) with 24xMS2 in a phage-cmv-cfp-24xms2 plasmid (Addgene, #40651). An ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC, a phage-cmv-cfp-24xMS2 and a Pcr4-12xMBS-PBS plasmids were gifts from B. Wu and R. Singer.
Plasmids encoding several green-red Fucci cell cycle reporters were provided by A. Miyawaki. The mKO2 and mAG genes fused with hCdt1(30–120), hCdt1(1/100), hGem(1/110) and hGem(1/60) sequences in the pCSII-EF-MCS plasmids were swapped with the miRFP709 or miRFP670v1 genes.

IPSc-derived cardiomyocytes were cultured on glass slides coated with Synthemax II-SC (Corning) in RPMI-1640/B-27 with Y27632 (5 μM) media for 2 days. Cells were fixed with 4% formaldehyde for 15 min at room temperature. Fixed cells were blocked in antibody buffer (5% BSA, 0.1% Tween-20 in PBS) for 1 h at room temperature. Following blocking, cells were incubated overnight at 4 °C with cardiac troponin-T antibody (abcam, ab45932, 1:200), α-Actinin antibody (Sigma, A7811, 1:800), and/or Connexin 43 (Cell Signaling Technology, 1:100) in antibody buffer. After the overnight incubation, cells were washed three times in antibody buffer. Following washing, cells were incubated with Alexa-Fluor conjugated secondary antibodies (Thermo Fisher Scientific, Alexa 488 Donkey anti-Rabbit IgG and Alexa 594 Goat anti-Ms IgG1) at a 1:1,000 dilution in antibody buffer for 1 h at room temperature. Nuclei were stained by DAPI at 1 μg/ml and Wheat Germ Agglutinin (Thermo Fisher Scientific, W21404) was used to stain cell membrane for 10 min at room temperature in antibody buffer. Following washing in PBS to remove unbound complexes, sarcomeres were analyzed using a Zeiss LSM510 confocal microscope. Images were processed with Zeiss software (Axiovision Rel4.8 and Zen Blue). Circularity measurements were made by comparing cardiomyocyte length to width and were expressed as a circularity index whereby circularity index = width/length^{15 (link)}. Sarcomere distance measurements were made with ImageJ^{15 (link)}. All measurements were made with a double-blinding method.

To test for putative G-actin sequestering functions of YARS1, 16 µM muscle actin (Cytoskeleton^TM) in General Actin Buffer was incubated with α-actinin and BSA (both controls at 2 µM final concentration) and 2 µM YARS1 proteins for 30 min at room temperature. All proteins were also tested alone, in the absence of G-actin. After the 30 min incubation, Actin Polymerization Buffer (Cytoskeleton^TM) was added to each tube with additional incubation at room temperature for 30 more minutes. All tubes were spun at 150,000 g for 1.5 h at 24 °C. Separation of supernatants and pellets, gel electrophoresis, and staining were identical to the ones described in the other pelleting assays.

Recombinant GST-tagged PLS3 (2 µg) was co-incubated with recombinant His-YARS1 proteins (2 µg) or IgG beads (GE Healthcare) in PBS with 0.05% Tween-20 at pH 7.4 for 1 h on a rotary shaker at 4 °C. YARS1 was immunoprecipitated with YARS antibody (H00008565-M02, Abnova). The input and IP fractions were analyzed via Western blotting using the YARS1 antibody and rabbit polyclonal PLS3 antibody (ab137585). Following the Dynabeads manufacturer’s instructions, recombinant His-YARS1 proteins were preincubated individually with GST-PLS3 or α-actinin (Cytoskeleton) in the presence of prewashed magnetic beads (Dynabeads) for 20 min at room temperature with constant agitation. The binding/washing buffer was 50 mM NaP, pH 8.0, 300 mM NaCl 0,01% Tween-20. Beads were collected via magnet, the supernatant was discarded, and the beads were washed four times. His-YARS1 was eluted with the low pH elution buffer (150 mM Imidazole, 50 mM NaP pH 8.0, 300 mM NaCl, 0.01% Tween-20) and the eluate was mixed with protein sample buffer. The input and pull-down fractions were analyzed with SDS-PAGE. The gels were stained with Coomassie R250 dye-based reagent (Pierce).