Myosin essential light chain 1sa decelerates actin and thin filament gliding on β-myosin molecules

Figures

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  Figure S1.. Determination of resting sarcomere length by laser diffraction. (A) Schematic drawing. (B) Photograph of the setup.

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  Figure 1.. Gliding velocities of actin filaments on ventricular and M. soleus β-/slow myosin. (A) On a BSA-coated assay chamber surface M. soleus β-/slow myosin molecules purified from native tissue moved actin filaments with 0.279 µm/s (±0.011 µm/s SE, n = 19 assay chambers), while native tissue derived ventricular β-/slow myosin moved actin filaments with 0.872 µm/s (±0.047 µm/s SE, n = 21). (B) On a nitrocellulose-coated assay chamber surface immobilized M. soleus β-/slow myosin molecules moved actin filaments with 0.101 µm/s (±0.007 µm/s SE, n = 7, 2 mM MgATP, T = 23°C). Under the same conditions, ventricular β-/slow myosin moved actin filaments with 0.200 µm/s (±0.010 µm/s SE, n = 6). Data points represent mean gliding velocities of individual assay chambers, while bars represent mean values ± SE. Data could be described by normal distributions (intrinsic curves).

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  Figure S2.. Gliding velocities of actin filaments on ventricular and M. soleus β-/slow myosin purified from the same animal or the same M. soleus tissue. (A) On BSA-coated coverslips M. soleus myosin molecules moved actin filaments with 0.263 µm/s (±0.005 µm/s SE, n = 5 assay chambers, left) or 0.273 µm/s (±0.005 µm/s SE, n = 3 assay chambers, right) significantly slower than ventricular myosin purified from the same animals (1.004 ± 0.062 µm/s SE, n = 6 assay chambers and 1.077 ± 0.016 µm/s SE, n = 3 assay chambers, respectively). No significant differences were detected between M. soleus and ventricular myosin preparations from both animals (P = 0.3036 and P = 0.3379, respectively). (B) From one animal, M. soleus myosin was extracted in two preparations, and no significant difference regarding gliding velocity was observed (0.282 ± 0.019 µm/s SE, n = 10 assay chambers; 0.269 ± 0.011 µm/s SE, n = 4 assay chambers; P = 0.5926). Data points represent mean gliding velocities of individual assay chambers, while bars represent mean values ± SE.

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  Figure 2.. Gliding velocities of actin filaments and native ventricular and M. soleus thin filaments on native tissue purified ventricular and M. soleus β-/slow myosin on a nitrocellulose-coated assay chamber surface. (A) Movement of F-actin (0.101 ± 0.007 µm/s SE, n = 7 assay chambers) and of M. soleus native thin filaments (0.082 ± 0.004 µm/s SE, n = 21) on M. soleus myosin. (B) Movement of F-actin on ventricular β-myosin molecules (0.200 ± 0.010 µm/s SE, n = 6) was significantly slower than that of thin filaments prepared from ventricular myofibrils (0.604 ± 0.029 µm/s SE, n = 28). (C) Native thin filaments purified from M. soleus were translocated significantly slower by M. soleus myosin (0.082 ± 0.004 µm/s SE, n = 21, T = 23°C) than by ventricular myosin (0.247 ± 0.016 µm/s SE, n = 21). (D) M. soleus myosin molecules moved cardiac thin filaments significantly slower (0.247 ± 0.009 µm/s SE, n = 19) than ventricular myosin molecules (0.604 ± 0.029 µm/s SE, n = 28). Data points represent mean gliding velocities of individual assay chambers, while bars represent mean values ± SE. Data could be described by normal distributions (intrinsic curves).

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  Figure S3.. SDS-PAGE analyses of actin and muscle thin filament preparations. (A) Rabbit back muscle actin (#1610374; Colormarker Biorad, Precision Plus Protein Dual Color Standard) and chicken actin preparations (#26614; Thermo Fisher Scientific, PageRuler Unstained Protein Ladder). (B) Ventricular (left) and M. soleus (right) muscle native thin filament composition. Source data are available for this figure: SourceData FS3.

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  Figure 3.. SDS-PAGE and Western blot analyses of native tissue purified ventricular and M. soleus myosin heavy and light chain composition. (A) 8% (top) and 6.5% (bottom) SDS-PAGE analyses of MyHCs in ventricular and M. soleus myosin. Rabbit M. psoas myosin containing mainly MyHC-IIb and atrial myosin containing both α- and β-MyHC served as references. No α-MyHC could be detected in myosin purified from ventricular tissue, while M. soleus myosin contained traces of MyHC-IIa (faint upper band). (B) 15% SDS-PAGE analysis of MLCs in atrial, ventricular, and M. soleus myosin preparations. Atrial myosin contained the ELC MLC1a and RLC MLC2a, while ventricular myosin contained ELC MLC1sb/v and RLC MLC2s/v. In addition to the latter two light chains, M. soleus myosin contained the ∼0.7 kD larger ELC MLC1sa. (C) Western blot analysis of MLCs in atrial and ventricular myosin preparations. Both atrial and ventricular ELCs MLC1a and MLC1s/v were immunolabeled by the antibody anti-MYL4 (top), and no signal of MLC1a was detected in ventricular myosin preparations. The atrial RLC (MLC2a) was labeled with an MLC2a specific antibody (bottom) and was absent in ventricular myosin samples. Source data are…

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  Figure S4.. Sequence comparison of MLC1s isoforms. RefSeq accession nos. for MYL3 (MLC1sb/v) NP_000249 and for MYL6B (MLC1sa) NP_002466 were taken from NCBI. MYL3 (MLC1sb/v): 21,932.05 D, 195 amino acids; MYL6B (MLC1sa): 22,763.98 D, 208 amino acids. Red dashes indicate charge relevant changes and additional lysine residues within the N-terminal extensions.

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  Figure 4.. Phosphorylation levels of native tissue-purified M. soleus and ventricular myosin RLCs. Compared to a phosphorylation to protein fluorescence (Pro-Q/SyproRuby) ratio of the positive control (pPS; 45 kD band of the PeppermintStick phosphoprotein standard) of 3.00 ± 0.362 (SE, n = 4), RLCs of M. soleus (MLC2s) and ventricular (MLC2v) myosin showed much lower and comparable ratios of 0.23 ± 0.017 (SE, n = 5) and 0.21 ± 0.018 (SE, n = 5), respectively. The unphosphorylated control protein (upPS; 116 kD band of the PeppermintStick phosphoprotein standard) had a Pro-Q/SyproRuby fluorescence ratio of 0.28 ± 0.094 (SE, n = 4). Neither D/S ratios of M. soleus and ventricular RLCs were significantly different nor ratios of RLCs and of the unphosphorytated control protein. Bars represent mean values ± SE.

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  Figure S5.. Electrophoretic analysis of MLC composition and phosphorylation. (A–C) Imperial protein stain (Thermo Fisher Scientific) SDS-PAGE of the MLCs of skeletal M. psoas and M. soleus, as well as cardiac ventricular and atrial tissue, (B) SyproRuby, PS = PeppermintStick, and (C) SyproRuby color corrected (−25 contrast, 0.22 gamma correction), (D) ProQ, and (E) ProQ color corrected (−25 contrast, 0.22 gamma correction). Protein bands representing the RLCs are found at a molecular weight of ∼19 kD. Source data are available for this figure: SourceData FS5.

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  Figure 5.. MLC composition and actin filament gliding velocity of myosin extracted from individual type-I M. soleus fibers. (A) Example of MLC SDS-PAGE analysis of individual type-I M. soleus fibers. For each fiber, the respective relative content of MLC1sa (upper band) versus MLC1sb/v (lower band) was determined by densitometric analysis of the SyproRuby-stained 15% SDS-PAGE gel. Note that all tested M. soleus fibers contained both ELC isoforms; the majority showed a higher content of MLC1sa, which was not detected in the control ventricular myosin. (B) Mean actin gliding velocity of 19 individual M. soleus fibers as a function of the ELC MLC1sa/MLC1sb/v ratio. A decrease of actin gliding velocity with increasing MLC1sa/MLC1sb/v ratio is reflected by a linear regression to the velocity data with a slope of −0.0383 ± 0.007 (black line). Inset: On a BSA-coated assay chamber surface myosin extracted from individual type-I M. soleus fibers with a lower MLC1sa/MLC1sb ratio (<40% MLC1sa) moved actin filaments with 0.387 µm/s (±0. 014 µm/s SE, n = 3 muscle fibers representing light grey squares in large plot). Actin gliding on myosin prepared from M. soleus fibers containing >60% MLC1sa was…

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  Figure S6.. MLC composition and actin filament gliding velocity on nitrocellulose-coated coverslips of myosin extracted from individual type-I M. soleus fibers as a function of the ELC [MLC1sa]/[MLC1sb/v] ratio. (A) MLC SDS-PAGE analysis of individual type-I M. soleus fibers. For each fiber, the respective relative content of MLC1sa (upper band) versus MLC1sb/v (lower band) was determined by densitometric analysis of the Coomassie-stained 15% SDS-PAGE gel. (B) Mean actin gliding velocity of six individual M. soleus fibers as a function of the ELC MLC1sa/MLC1sb/v ratio. A decrease of actin gliding velocity with increasing MLC1sa/MLC1sb/v ratio is reflected by a linear regression to the velocity data with a slope of −0.0392 ± 0.008 (black line). Data points represent mean gliding velocities of individual muscle fibers ± SE. Source data are available for this figure: SourceData FS6.