Cooperativity of myosin II motors in the non-regulated and regulated thin filaments investigated with high-speed AFM

Figures

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  Figure 1.. The kinetics of double-headed myosin motors bound to non-regulated actin filaments. (a) Diagram illustrating the approach used to study cooperative behavior of HMM molecules bound between two parallel filaments. (b) Structural model of actin–myosin complex for the experimental design used in the study; actin–myosin complex in rigor conditions (PDB accession no. 1M8Q) with upper and lower heads bound between two parallel actin filaments and attached by their S2 fragments (PDB accession no. 2FXO). (c) Kinetics model of actin–myosin interaction and approach used to calculate the backward and forward myosin displacements; the green dots indicate the center of mass of the head. KT and K-AD are the constants of the ATP binding and ADP release; Kw and Kws are the constants of the weak biding and weak-to-strong transition, respectively. (d and e) Representative time course of binomial binding (d) and head displacements (e) calculated for the individual HMM molecules (M1–M4) at the given time for upper and lower heads of each HMM molecule in the presence of 2 μM ATP. (f) The backward and forward myosin displacements in the F-actin–HMM complex (6,589 events, ∼43 HMM molecules); data sets…

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  Figure S1.. Successive HS-AFM images of F-actin–HMM complex in the presence of 0.2 μM ATP. (a) Double-headed HMM motors bound between two actin filaments in ∼37 nm distance to form a stable structure with up to eight individual myosin heads (four HMM molecules), attached by their S2 regions. (b) HMM heads between two actin filaments highlighted by red colors, S2 region of the HMM molecules highlighted by green color. (c) High-contrast images from a to highlight the connected S2 regions between HMM molecules. Numbers indicate the time in seconds, scan rate: 14.4 fps, scale bar, 30 nm.

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  Figure S2.. Arrangement of myosin heads between two parallel filaments. (a and b) Cross-sections of myosin upper and lower heads bound between two parallel cTFs (left, red profiles) and F-actin (right, blue profiles), scale bars, 30 nm. (c and d) The measured distance between two parallel cTFs (left) and F-actin (right) averaged in d. The difference in distance between non-regulated F-actin and regulated cTFs (P = 0.001, unpaired t test) did not affect the HMM binding and displacement analysis (Fig. 1 f and Fig. 2 c). (e and f) Two types of HMM binding between parallel cTFs or actin filaments were observed; the upper and lower heads bound towards the similar direction (right HS-AFM images in e and f) or the upper and lower heads bound towards the opposite directions (left HS-AFM images in e and f). The captured snapshots in left (e) and left (f) panels indicate the rare situation occurred due to head displacement on the filament in the presence of ATP. HMM heads between two actin filaments highlighted by red colors, S2 region of the HMM molecules highlighted by green color. The corresponding polarity of the filaments is shown. Scale bars, 30 nm.

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  Figure 2.. The kinetics of double-headed myosin motors bound to regulated cTFs. (a and b) Representative time course of binomial binding (a) and heads displacements (b) calculated for the individual HMM molecules (M1–M4) at the given time for upper and lower heads of each HMM molecule in the presence of 0.5 μM ATP and high Ca2+ concentrations. (c) The backward and forward myosin displacements in the cTFs–HMM complex (5,911 events, ∼35 HMM molecules); data sets were fitted by sum of two Gaussians (r2 = 0.99). The two peaks for backward displacement were −1.7 ± 0.27 and −4.2 ± 1.2 nm; the two peaks for forward displacement were 1.7 ± 0.29 and 3.9 ± 0.64 nm.

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  Figure 3.. Frequency distribution of one HMM head or two HMM heads bound between two filaments in the presence of ATP. (a–c) Distribution of one (1) and two (2) HMM heads in F-actin–HMM complex in the presence of 0.2 μM ATP (319 binding events), 2.0 μM ATP (488 binding events), or 10 μM ATP (1,072 binding events). (d–f) Distribution of one (1) and two (2) HMM heads in cTFs–HMM complex in the presence of 0.5 μM ATP and low Ca2+ (702 binding events), 0.5 μM ATP and high Ca2+ concentration (545 binding events) and 2.0 μM ATP, high Ca2+ concentrations (440 binding events). The 0 indicates the temporarily no bindings of HMM heads to the binding sites along the length of the filaments in analyzed datasets. HMM heads are indicated by white arrows (one head bound) and red arrows (two heads bound). Scale bars, 30 nm; z-scales indicated for each HS-AFM image. Fa, F-actin.

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  Figure 4.. Probability of myosin heads binding to the non-regulated actin filaments. (a and b) Successive HS-AFM images of F-actin–HMM complexes, where HMM heads bind between two actin filaments in the presence of 0.2 μM ATP (a, scan area: 150 × 90 nm2, 80 × 48 pixels2) or 2 μM ATP (b, scan area: 150 × 75 nm2, 80 × 40 pixels2). Dashed color boxes indicate upper and/or lower HMM heads bound between two actin filaments. Numbers on each frame show the time in seconds. Related to Videos 1, 2, and 3. Scale bars, 30 nm. (c–e) Probabilities of the individual HMM head binding to the four or five binding sites on the non-regulated actin filaments in the presence of 0.2 μM ATP (c, 1,235 events), 2 μM ATP (d, 567 events) and 10 μM ATP (e, 934 events). The average fractional occupancies by HMM heads for all of the sites for each of the ATP conditions showed a decrease in the occupancy with time (right panels in c–e, n = 4). (f) Frequency distributions for the number of myosin heads attached to four neighboring binding sites along a given actin filament or thin filament over time (n = 8 experiments, ∼35 HMM molecules, 898 events). Mean number (±95% CI) of attached myosin heads relative to the four…

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  Figure 5.. Probability of myosin heads binding to the regulated cTFs. (a and c) Fitting the simulated HS-AFM images (Amyot and Flechsig, 2020) of the cTFs at Ca2+- free (a) and Ca2+-bound (c) states into the cTFs molecular structures, scale bar, 2.5 nm. PDB accession nos. 6KN7 and 6KN8 for Ca2+ free and high Ca2+ states, respectively. (b and d) Representative HS-AFM snapshots of HMM molecules bound between two cTFs in the presence of 0.5 μM ATP and either low Ca2+ (b, scan area: 200 × 200 nm2, 120 × 120 pixels2) or high Ca2+ (d, scan area: 200 × 200 nm2, 120 × 120 pixels2) concentrations at the indicated times. Scale bars, 30 nm. Note that the number of bound heads increase with time in d. (e–g) Probabilities of the HMM heads binding to the regulated cTFs in the presence of 0.5 μM ATP and low Ca2+ (492 events) or 0.5 μM ATP and high Ca2+ concentrations (2,830 events; f) or 2 μM ATP and high Ca2+ concentration (1,839 events; g) with the corresponding average fractional occupancy versus time shown in the right panels. (h) Frequency distributions for the number of myosin heads attached to four binding sites along all studied thin filaments over time in the activating conditions (n = 7…

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  Figure S3.. Distribution of the bound heads in the F-actin–HMM and cTFs–HMM complexes. (a–h) Kymograph images of the F-actin–HMM (a–d) and cTFs–HMM (e–h) complexes at the different ATP concentrations. Scan area and scan rates of the top HS-AFM images for F-actin–HMM complex: (a) 150 × 75 nm2, 80 × 40 pixels2, 10 fps; (b) 150 × 90 nm2, 80 × 48 pixels2, 20 fps; (c) 200 × 120 nm2, 80 × 40 pixels2, 10 fps; (d) 200 × 120 nm2, 80 × 40 pixels2, 12.5 fps. Horizontal scale bars, 30 nm; vertical scale bars in kymographs, 20 ms. Scan area and scan rates of the top HS-AFM images for cTFs–HMM complex at the activating conditions: (e) 120 × 120 nm2, 200 × 200 pixels2, 6.7 fps; (f) 120 × 120 nm2, 200 × 200 pixels2, 2 fps; (g) 150 × 75 nm2, 80 × 40 pixels2, 6.7 fps; (h) 200 × 200 nm2, 120 × 120 pixels2, 2 fps. Horizontal scale bars, 30 nm; vertical scale bars in kymographs, 50 ms (e and f) and 100 ms (g and h). The dashed yellow line indicated the initial position to create a kymograph image.

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  Figure S4.. Probability of binding of myosin heads to the non-regulated or regulated filaments in rigor conditions or in the presence of ATP-γ-S. (a–c) HS-AFM images of HMM molecules bound between two actin filaments in the presence of ATP-γ-S (a), HMM molecules bound between two actin filaments in the absence of Ca2+ and ATP (b) and HMM molecules bound between two cardiac thin filaments in the absence of Ca2+ and ATP (c). Scale bars, 30 nm. (d–f) Probabilities of binding of the HMM heads in the F-actin–HMM complex in the presence of ATP-γ-S (d, 1,145 events), in the absence of Ca2+ and ATP (e, 1,408 events) and in the cTFs–HMM complex in the absence of Ca2+ and ATP (f, 663 events). Individual data points are shown for each HMM molecules (M1–M4) as mean values ± 95% CI, n = 3. (g–i) Corresponding fractional occupancies of the binding sites by HMM heads for the conditions shown in d–f.

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  Figure S5.. Cooperative probability of the myosin heads bound to the non-regulatory F-actin. The independent experiments showing different patterns of the cooperative probabilities between the one HMM molecule Mn and the next molecule Mn+1 in the F-actin–HMM complex. The linear regression fittings showed in the solid lines and the dashed curves represent 95% CI.

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  Figure S6.. Cooperative probability of the myosin heads bound to the regulatory cardiac TFs. The independent experiments showing different patterns of the cooperative probabilities between the one HMM molecule Mn and the next molecule Mn+1 in the cTFs–HMM complex. The observed differences, most likely related to the diverse population of activated/non-activated segments across the thin filament length that led to different degree of positive cooperativity of myosin bindings. The linear regression fittings showed in the solid lines and the dashed curves represent 95% CI.