Dependence of myosin filament structure on intracellular calcium concentration in skeletal muscle

Figures

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  Figure S1.. Schematic of the x-ray experimental setup. The bundles were horizontally mounted between the levers of a force transducer (FT) and a motor (MT) in a thermoregulated multidrop mechanical apparatus that allowed activation using a temperature jump technique (Linari et al., 2007). The positions of the bundle (vertical dashed line) and of the x-rays path were fixed, while the trough could move horizontally and vertically (x and z axis, respectively). (A–C) The bundle (red) was kept in a preactivating solution at low temperature (1°C, LT) for 2 min (A), then transferred to the activating solution at 1°C by horizontal shift of the trough (B), in which little force was developed. When force became steady (within 10 s), the bundle was transferred to an activating solution at 25°C (HT) and, following full force development, the trough was lowered (∆z) so that the bundle was transferred to air in a narrow aluminum-delimited chamber for the x-ray exposure (C, and xz sections and view along the x axis from the detector on the right). Exposure in air avoids x-ray absorption by the solution drops. (D) The bundle was then relaxed in a relaxing solution. The detector in C would be at 5 m from the…

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  Figure 1.. Dependence of force and RLC orientation on calcium concentration in the steady state. (A) Force at different pCa normalized by the maximum force at pCa 4.7, obtained with a conventional preactivation protocol at 25°C (orange circles, T0 = 267 ± 10 kPa; mean ± SE, n = 5 fibers; data from Fusi et al. [2016]) or using the temperature jump protocol (black circles; T0 = 286 ± 25 kPa; mean ± SE, n = 3 fibers). Data from single psoas muscle fibers; sarcomere length, 2.40 µm; temperature, 25°C; 5% dextran. (B) <P2> for the RLC E-helix probe in the same experiments as A. Lines in A and B are fits of the Hill equation to the average <P2> at each pCa. (C) <P2> for the RLC E-helix probe at pCa 9, 25°C in the steady state (orange line) and after a temperature jump to 25°C (black line) at time zero. (D) Force at different pCa following temperature jumps to 25°C; data points from individual fiber bundles are identified by different symbols. The line represents the Hill equation for the mean parameters in Table 1 obtained from four fiber bundles.

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  Figure 2.. Equatorial x-ray reflections. (A) Equatorial intensity distributions at different calcium concentrations (average of two to four bundles). pCa 9, red; pCa 7, orange; pCa 6.5, green; pCa 6.2, blue; and pCa 4.5, violet. (B and C) Ratio of equatorial intensities (I11/I10, B) and spacing of 1,0 reflection (d10, C) following a temperature jump to 25°C. Different magenta symbols identify individual fiber bundles. The magenta line represents the Hill equation for the mean parameters in Table 1 obtained from four fiber bundles. Horizontal magenta lines and shading (mean ± SD, n = 4 bundles) denote steady-state values at pCa 9. Black lines are the Hill fit to force from Fig. 1 D.

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  Figure 3.. First myosin layer line. (A) Intensity distributions parallel to the meridional axis at different calcium concentrations (average of two to four bundles). pCa 9, red; pCa 7, orange; pCa 6.5, green; pCa 6.2, blue; pCa 4.5, violet. (B) Intensity of the first myosin layer line (IML1) was normalized by the value at pCa 7, following a temperature jump to 25°C. (C) Square root of IML1 (AML1). Magenta symbols and lines as in Fig. 2. Black lines are the Hill fit to force from Fig. 1 D.

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  Figure 4.. Effect of calcium concentration on the meridional profiles. Meridional intensity distributions at 25°C (average of two to four bundles) in relaxing solution (pCa 9, red) and after a temperature jump to 25°C pCa 7 (orange), pCa 6.5 (green), pCa 6.2 (blue), and saturating calcium concentration (pCa 4.5, violet).

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  Figure S2.. Intensity of M6 reflection (IM6) following a temperature jump to 25°C and normalized by the value at pCa 7. Data are corrected by the cross-meridional width. Due to the lower signal-to-noise, the meridional intensity distributions of the M6 reflection were averaged from two to four bundles before determining its intensity at each pCa. Horizontal magenta line denotes steady-state value at pCa 9. Black line is the Hill fit to force from Fig. 1 D.

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  Figure 5.. Spacing of M6 reflection (SM6) following a temperature jump to 25°C. Meridional intensity profiles were averaged from n = 4 bundles, except points at pCa 5.6 and 4.5, n = 2 bundles. Hill equation fit is shown as a magenta line. Horizontal magenta line denotes steady-state value at pCa 9. Black line is the Hill fit to force from Fig. 1 D.

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  Figure 6.. M3 reflection. (A) Meridional intensity distributions corrected by the cross-meridional width (average of two to four bundles). Red, pCa 9; orange, pCa 7; yellow, pCa 6.7; light green, pCa 6.6; dark green, pCa 6.5; cyan, pCa 6.3; blue, pCa 6.2; Bordeaux, pCa 5.6; and violet, pCa 4.5. Vertical dashed lines mark reciprocal spacings 1/(14.8 nm), 1/(14.6 nm), 1/(14.4 nm), and 1/(14.2 nm). (B and C) Intensity (IM3, B) was corrected by its cross-meridional width and normalized by the value at pCa 7, and spacing (SM3, C) following a temperature jump to 25°C. Magenta symbols and lines as in Fig. 2. Black lines are the Hill fit to force from Fig. 1 D.

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  Figure S3.. M3 component peaks and star peak. (A) Square root of the M3 intensity. (B) Fractional intensity of low-angle peak (LM3 = Ila peak/IM3). (C) Intensity of star peak (Istar). (D and E) Spacing (D) and intensity (E) of the star (cyan) and M3 component peaks (green, la; purple, ma; orange, ha) following a temperature jump to 25°C. Different symbols identify individual fiber bundles. Horizontal lines and shading (mean ± SD, n = 4 bundles) denote steady-state values at pCa 9. Note that the la and star peak are not visible at pCa 9. Black lines are the Hill fit to force from Fig. 1 D.

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  Figure 7.. Forbidden reflections. (A) Intensity of M1 reflection (IM1). (B) Intensity of M2H reflection (IM2H). (C) Intensity of M2L reflection (IM2L), all following a temperature jump to 25°C and normalized by the value at pCa 7. Magenta symbols and lines as in Fig. 2. Black lines are the Hill fit to force from Fig. 1 D.

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  Figure 8.. Intensity of T1 reflection (IT1) following a temperature jump to 25°C and normalized by the value at pCa 7. Magenta symbols and lines as in Fig. 2. Black line is the Hill fit to force from Fig. 1 D.