A combined experimental and computational analysis of mantATP turnover in skinned muscle fibers

Figures

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  Fig. 1.. Experimental scheme and model. (A) Simulated amount of bounded mantATP in a single sarcomere under different hypotheses for the cooperativity between myosin motors. Model 0: noncooperative thermodynamically equilibrated motors. Model 1: two populations of motors with diverse thermodynamic equilibrium. Model 2: only intradimer cooperativity with a forbidden state (free head in SRX with blocked head in DRX; see below). Model 3: full model with both intradimer and intermolecular cooperativity as detailed in panel B. (B) Kinetic diagram relating the transition rates to the motors state in each dimer (Upper panel, intradimer cooperativity) and along the filament (Lower panel, intermolecular cooperativity). Intradimer cooperativity is simulated preventing a dimer configuration with a free motor in the SRX state (SRX states are indicated in purple and have ATPase rate of 0.005 s−1) and a blocked motor in DRX state (DRX states are indicated in green and have ATPase rate of 0.05 s−1). (C) Schematic representation of a fluorescence decay trace: fast diffusing free mantATP (yellow), a combination of mantATP binding nonspecifically to structural elements and nonmyosin ATPases, as well as…

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  Fig. 2.. Myosin extraction on ghosted fiber and mantATP fluorescence emission patterns. (A) Control skinned fiber and (B) ghosted fibers incubated with 250 µM mantATP (blue is mantATP, red is Phalloidin, magnification 63×). (C) Plot profiles of a control fiber showing the alternance of A-bands (mantATP, blue) and I-bands (phalloidin, red) of five sequential sarcomeres. (D) Plot profiles of a ghosted fiber showing that the remaining mantATP signal left is located within the I-band (mantATP, blue, and phalloidin, red). (E) Normalized fluorescence intensities of A-band and I-band of control (CTRL) and ghosted (GHOSTED) fibers in 250 µM mantATP (n = 14 control, n = 13 ghosted fibers, mean ± SEM). (F) Intensity profile obtained from averaged single sarcomeres in control and ghosted fibers, normalized for background intensity, A-band is highlighted by the gray area (n = 14, mean ± SEM).

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  Fig. 3.. Mass spectrometry analysis of extracted protein and ghosted fibers. Normalized protein amounts in extract and in the ghosted fiber, proteins are divided into thick filament (A), thin filament (B), z-disk (C), and ATPases (D). Individual intensities are normalized for the sum of extract and corresponding ghosted fiber intensity, statistical analysis is ordinary two-way ANOVA and Šidák multiple comparison test (n = 7, mean ± SEM).

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  Fig. 4.. Kinetics of the NSP in mantATP chasing of skinned muscle fibers. (A) ATP consumption rates of control and ghosted fibers, with and without 20 µM para-aminoblebbistatin (Ablebb) (n = 3 to 6, mean ± SEM). (B) Chasing traces of control fibers and ghosted fibers incubated with 20 µM para-aminoblebbistatin (Ablebb) (n = 6 to 8, mean ± SEM). (C) values of time constant TDRX related to curves in panel B. (D) Simulations of a mantATP chasing experiment on ghosted fibers without the nonspecific buffer (light orange) and with the nonspecific buffer (orange), compared to the experimental data (black curve). The presence of the buffer improves the fitting at higher times (fitting parameters kON = 107 M−1 s−1 kA = 5.6 104 M−1).

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  Fig. 5.. Simulated decay of mantATP concentration. (A) Example of the simulated decays of the four components of mantATP inside the fibers: free, nonspecifically bound, bound to DRX, and bound to SRX (continuous lines, blue, red, green, and purple, respectively). Dashed-dot lines represent the estimated decays of SRX and DRX from a three exponential fitting (same colors). (B) Absolute SRX-DRX populations imposed on the model (straight lines, purple, and green, respectively) and their estimation through a two (light colors dashed lines) or a three (dark colors dashed lines) exponential fitting at imposed NSP = 60%. (C) Relative populations [SRX/(SRX+DRX) and DRX/(SRX+DRX)] and estimated parameters without (dashed lines) and with (dotted lines) the independent information about NSP [estimated DRX = 1−PSRX/(1−NSP)]. The gray area located at approximately 37% in the SRX population axis indicates the area in which NSP correction performs better than three exponential fitting to estimate populations.

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  Fig. 6.. mantATP chasing and NADH coupled ATPase-mantATPase of control and piperine incubated fibers. (A) Exponential decay traces of chasing on skinned fibers incubated with DMSO (control) and 50 µM piperine. Histograms are related to estimation of PDRX and PSRX populations by using the triple exponential fitting (darker bars) and correcting the values for the NSP (lighter bars), and histograms related to time constants TDRX and TSRX (n = 8, mean ± SEM). (B) Resting skinned fibers ATPase is measured by NADH double-reaction assay on control skinned fibers and incubated with 50 µM piperine using mantATP and normal ATP. Data are analyzed using an identify outliers ROUT method Q = 0.5%, Kolmogorov–Smirnov normality test, ordinary one-way ANOVA, and Tukey’s multiple comparison test (n = 16 to 27, mean ± SEM).