Myosin light chain proteins cooperatively promote sarcomere growth in fast-twitch muscle

Fig. 1. The mylpfa gene is expressed more abundantly than mylpfb in fast-twitch muscle. a Images illustrating myofibril and sarcomere structure. The myofibril bundle width (m.b. width) represents axis of measurements for myofibril bundle width. b Illustration of Mylpf localization within a thick filament. c–c”’ In situ hybridization chain reaction (HCR ISH) imaged in somites from the mid-yolk tube region of a 36 hpf embryo. Shown as a single channel for mylpfa (c), mylpfb (c’) or the slow muscle marker myl10 (c”), and as a merged image (c”’). d Plot showing the brightness of mylpfa and mylpfb in the HCR ISH images (n = 3 to 4 fish per gene per timepoint). e Amount of mylpfa and mylpfb transcript per µg of RNA at 24, 36, 48, and 72 hpf. Data are presented as copy number per µg of total RNA measured by qPCR using a standard curve–based normalization method. f The ratio of mylpfa to mylpfb transcript abundance calculated from the absolute copy numbers shown in (e) (n = 3 pools per genotype per timepoint). g Image of a western blot showing Mylpfb and Mylpfa protein abundance at these timepoints. Examples of Mylpfa and Mylpfb bands quantified in GelBox are shown beneath the image. The “bl”…

Fig. 2. Zebrafish mylpfa is necessary for fast-twitch myofibril formation. a–d Zoomed region of somite muscle, with myofibril bundles marked using six1b:LifeAct-mTurquoise2 transgene (LifeAct-mTq) at 2 dpf and the same animal at 6 dpf in wild-type (a, b) or mylpfa−/− mutant siblings (c, d). a’–d’ Cross-sectional views of whole somites from the same animals. e Myofibril bundle widths measured using the LifeAct images in the sagittal plane. Olive-colored lines connect the same individuals through time (n = 16 WT/het, n = 11 mylpfa−/−). f Plot of signal periodicity shows that the mylpfa−/− mutant has less LifeAct periodicity in sarcomeric intervals (gray bars). Lightly colored regions indicate 95% bootstrap confidence intervals. g This readout is simplified as a “sarcomeric fraction”, which shows the fraction of signal in sarcomeric (1.85 µm) or half-sarcomeric (0.9 µm) lengths out of the total periodicity; further explanation is provided in Fig. S4. The sarcomeric fraction remains constant through development but differs by genotype (n = 11 WT/het, 8 mylpfa−/−). h Somite area, measured from the cross-sectional views. The mylpfa−/− mutant somite is smaller than in wild-type or heterozygous…

Fig. 3. Mutations in mylpfa and mylpfb cause strong loss of function. a Illustration of mylpfa and mylpfb gene structure. Color code: 5′ UTR (brown), coding sequence (purple), 3′ UTR (gray), and frameshift locations (green arrowheads). b Chromatogram showing the gRNA target in wild-type sequence (top) and the 5 bp mylpfboz39 lesion sequenced from a homozygous mutant (bottom). c Illustration of Mylpfa and Mylpfb proteins, with arrowheads marking frameshift locations. d, e Quantitative RT-PCR results from the mylpfa;mylpfb mutant series showing mylpfa expression per genotype (gen.) normalized to the average mylpfa abundance in mylpfa+/+ genotypes. Result shown as individual genotypes (d) or as a merger of mylpfa genotypes (e). f, g Quantitative RT-PCR results for mylpfb expression, normalized to the average mylpfb abundance in mylpfb+/+ genotypes, shown for individual genotypes (f) or as a merger of mylpfb genotypes (g) (n = 4 per genotype for d–g). h Predicted protein structure generated using Robetta. Color code indicates the heavy chain (purple), the peptides lost after mylpfboz39 frameshift (black), and those retained even in the mutant (green). i Western blot showing MyHC, Mylpfb, and…

Fig. 4. mylpfa and mylpfb are required for myosin heavy chain localization in fast-twitch muscle of young myofibers. a–d Immunolabel of fast muscle fibers at 26 hpf shown as orthogonal views of Z-stacks of wild-type (a), mylpfb−/− (b), mylpfa−/− (c), and mylpfa−;mylpfb−/− double mutant zebrafish (d). a’–d’ Zoomed sagittal views of the same animals. e Box plots showing Spearman’s correlation between the F-actin and MyHC label for the genotypes examined (n = 6 wild-type, 13 mylpfb−/−, 9 mylpfa+/−;mylpfb+/−, 7 mylpfa−/−, 11 mylpfa−/−;mylpfb−/−). f Plots showing the width of myofibril bundles in each animal (n = 8 to 13 per genotype). g Overview of protocol to measure image brightness from edge-to-center. The fast-twitch muscle cells are (1) manually outlined, (2) segmented using these outlines, then (3) the outlines are used to determine edges when calculating the brightness of each channel from edge to center of the drawn cells. h–j Localization at 26 hpf of the F-actin marker phalloidin (h), the muscle nuclei label anti-Rbfox1l (i), and MyHC antibody A4.1025 (j). Data is plotted as percent of total image brightness from edge to center of segments. For each genotype, the mean is shown with…

Fig. 5. Levels of myofibril formation correspond to dosages predicted by mylpfa and mylpfb loss of function. a Scatterplot of myofibril bundle widths in all genotypes tested at 26 hpf (N = 49), with the X-axis representing Mylpf dosage as predicted by a 7:1 ratio between Mylpfa and Mylpfb. b–d Scatterplots of myofibril bundle widths in all genotypes tested at 48 hpf (N = 52), 72 hpf (N = 35), and 6 dpf (N = 47), with the X-axis representing Mylpf doses predicted by a 2:1 ratio between Mylpfa and Mylpfb. The 26 hpf width data is also shown in Fig. 4f. e Cross-sectional area measurements of these somites at 6 dpf. The somite sizes do not differ significantly between WT and any other genotype (N = 47). f Examples of fast-twitch muscle structure, labeled with phalloidin, in the nine genotypes arising from mylpfa+/−;mylpfb+/− incross, viewed at high magnification. g Fast-twitch myofibril bundles at 48 hpf, labeled for the myonuclei (Rbfox1l, blue), F-actin (phalloidin, red), and MyHC (A4.1025, green). h The colocalization between F-actin and MyHC label (Spearman’s correlation) corresponds well with modeled protein ratios (2 Mylpfa to 1 Mylpfb) (N = 64). i, j Graph showing how the sarcomeric…

Fig. 6. The mylpfa−/− single mutant and the mylpfa−/−;mylpfb−/− double mutant animals do not swim at high speed but increase the frequency of slower movements. a–c Escape response in wild-type or in mylpfa−/− animals, showing images captured 1/60th of a second apart (a, b). Blue line shows maximal tail bend, quantified in (c) (n = 9 WT/het, 7 mylpfa−/−). d Box plot of distances traveled when fish were imaged 25-min daily from 2 to 6 dpf (N = 41 WT, 37 mylpfa−/−). e, f A logarithmic plot of the proportion of time that select genotypes swim at different speeds, with solid lines for the mean and shaded bootstrap confidence intervals shown for each genotype (e). The inset region highlights differences in the mylpfa−/− mutant behaviors that become apparent by 5 dpf (e’). These differences are more striking when normalized to wild-type siblings (f) (N = 58 WT, 57 mylpfa−/−). g, h Box plots show total distance traveled (g) or highest movement speed (h) of the mylpfa;mylpfb knockout series at 6 dpf (N = 290 for g, h). i A logarithmic plot of the proportion of time that select genotypes swim at different speeds, with solid lines for the mean and shaded bootstrap 95% confidence intervals shown for…

Fig. 7. Expression of either mylpfb-GFP or mylpfa-GFP can rescue mylpfa−/− myofibrils to a degree proportionate with GFP abundance. a Schematic illustrating the Tg(mylpfa:mylpfa-GFP)mai102 and Tg(mylpfa:mylpfb-GFP)mai103 transgenes. b Box plot showing the variation of fluorescence brightness in the mylpfa-GFP transgenic animals and their non-transgenic siblings (N = 35 non-transgenic, 58 mylpfa-GFP). The animals imaged for (b) were pooled into groups of 10 and run on acrylamide gels for western blotting. c Western blot of Mylpf protein from the wild-type fish and their Mylpfa-GFP+ siblings at 3 dpf shows that the transgene produces one thin band that is shifted upwards by the addition of GFP. An empty well is denoted as bl. d Plot showing quantification of the Mylpfa and Mylpfa-GFP bands, normalized to Mylpfa (n = 4 Non-transgenic pools, 7 Mylpfa-GFP pools, for a total of 11 pools with native Mylpfa). e Correlation plot showing that average fluorescence intensity from these pools corresponds precisely with band intensity in the western blots (N = 11). Linear regression is indicated with a black line and 95% confidence for fit of regression is shown with gray shading. f Confocal slices…

Fig. 8. Expression of GFP-fused human MYLPF is sufficient to restore fast-twitch myofibril formation in the mylpfa−/− mutant. a Schematic illustrating the Tg(mylpfa:MYLPF-GFP)mai104 transgene. b Scatterplot showing correlations between GFP brightness and the fast-twitch myofibril bundle width from an intercross of mylpfa−/+ heterozygotes segregating this transgene (N = 65). c–e Examples of confocal images of mylpfa−/− mutant animals that have no GFP expression (c), dim GFP expression (d), and bright GFP expression (e). Quantification of these individuals is shown in (b). f Scatterplot of m.b. width data from all mylpfa-GFP, mylpfb-GFP, and MYLPF-GFP transgenic experiments in this study (Fig. S12), normalized to the mean wild-type value per experiment (N = 179). We find similar slopes in the mylpfa−/− mutant animals bearing these transgenes, suggesting that these three Mylpf genes act with equivalent efficiency. None of these three transgenic lines significantly influence the widths of myofibrils in wild-type (mylpfa+/+) animals. Linear regression is indicated with a solid line and confidence for fit of regression shown with shaded areas, color-coded by group. Details on statistics and…

Fig. 9. A MYLPF protein variant found in Distal Arthrogryposis fails to rescue the mylpfa−/− mutant. a Predicted MYLPF structure, with pink color and filled space highlighting the position of Glycine at residue 163. b Illustration of a construct that expresses GFP-fused human MYLPF or the G163S variant MYLPF. c, d Representative images of mylpfa−/− mutants that were injected with plasmid expressing MYLPF-GFP protein (c) or the G163S variant (d), resulting in mosaic GFP expression. Asterisks indicate balls of highly disordered GFP fluorescence signal, which are only seen in the G163S MYLPF protein variant. e Scatterplot showing correlates between myofibril width and GFP fluorescence for both injected constructs in the mylpfa−/− mutant and their WT/het siblings (N = 31 animals). f Scatterplot showing the correspondence between F-actin sarcomeric fraction and GFP fluorescence in the mylpfa−/− mutant and their WT/het siblings (N = 31 animals). Linear regression is indicated with a solid line and confidence for fit of regression shown with shaded areas, color coded by group (e, f). g Box plots showing the degree to which wild-type MYLPF-GFP or the G163S MYLPF-GFP localizes to a sarcomeric…

Fig. 10. Modeling the impact of Mylpf on muscle structure. Scatterplot graphs, with Y-axis showing the myofibril bundle (m.b.) widths scaled to the non-transgenic wild-type level per experiment, and the X-axis showing the Mylpf dosage as calculated using formulas shown in Fig. S12. a A plot of data from the mylpfa;mylpfb knockout series shows strong overlap at each timepoint examined (1, 2, 3, and 6 dpf) using datapoints presented in Fig. 5b–d (N = 183). The Hill regression model shows a high correlate across timepoints. b A plot of the transgenic data, excluding GFP < 10 (N = 90), alongside the most severe Mylpf loss of function alleles from the knockout lines (mylpfa−/−;mylpfb+/− and mylpfa−/−;mylpfb−/−, gray dots, N = 28). This dataset also produces a Hill regression with a high R2, high Hill slope and moderate EC50. Asterisks on Hs and EC50 values reflect the fit of the dataset in b to the regression from (a), with significance determined using an extra-sum-of-squares F-test. Asterisks, *** indicates P < 0.001. c Examples of three potential types of phenotypic responses to protein dose. Mylpf appears to have a sigmoidal response curve with a steep slope at inflection and an EC50 value…