The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle

Figures

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  Figure 1.. Longitudinal and cross-sectional organization of the cardiac muscle sarcomere. (A) Illustrative representation of myosin and actin, which form interdigitating filaments organized within a sarcomere. MyBP-C is bound within the ∼387-nm-long C-zones of the 1.57-µm bipolar myosin filaments (Zoghbi, et al., 2008). (B) Schematic representation of MyBP-C with 11 Ig-like (yellow) and Fn-like (red) domains. The location of the Myc-tag for Alexa Fluor (magenta star), two internal hinges, four phosphorylation sites, and the actin- and myosin-binding regions are underlined. (C) Illustrative representations of a longitudinal view of part of a single myosin thick filament showing potential interactions of MyBP-C with myosin and actin. (D) Illustrative representations of cross-sectional views of myosin and actin filaments organized within a hexagonal lattice. Note: each myosin filament (blue) is surrounded by six actin filaments (green). (E and F) Negative-stain EM images of longitudinal (E) and transverse (F) cryo-sections of mouse papillary muscle sarcomeres from an active and relaxed preparation, respectively. Note: MyBP-C is not visible in the micrograph. Scale bars, 500 nm (E) and 250 nm…

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  Figure S1.. Single ATP turnover measurements from multicellular ventricular preparations from wild-type and transgenic mice expressing myc-tag–labeled MyBP-C. (A) The lifetime of nucleotide turnover associated with fast phase of fluorescence decay. (B) The lifetime of nucleotide turnover associated with the slow (SRX) phase of fluorescence decay. (C) The amplitude of the slow phase of fluorescence decay, indicating the fraction of myosin molecules that adopted the SRX state. n = 4 wild-type mice (14 technical replicates) and n = 2 myc-tag–labeled MyBP-C mice (12 technical replicates). Error bars indicate ±SEM (P > 0.4).

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  Figure S2.. Longitudinal and cross-sectional views of mouse cardiac papillary muscle. (A and B) Negative-stain EM images of 100-nm-thick cryo-sections, longitudinal (A) and transverse (B), of mouse papillary muscle from active and relaxed muscle preparations, respectively, demonstrating the actin and myosin filament lattice within the myofibrils. Scale bars, 1 µm (A) and 500 nm (B). (C and D) Reconstructed images of Alexa Fluor 488-phalloidin (actin) filaments (C) and Alexa Fluor 647 anti-myc (MyBP-C’s C0 domain; D) within 400-nm-thick transverse cryo-sections of mouse papillary muscle imaged by STORM. Scale bars, 5 µm.

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  Figure 2.. Visualization of actin and MyBP-C within individual sarcomeres by STORM and detection of the actin hexagonal lattice. (A and B) Reconstructed images (see Materials and methods) of Alexa Fluor 488-phalloidin (actin filaments; A) and Alexa Fluor 647 anti-myc (MyBP-C’s C0 domain; B) within 400-nm transverse cryo-sections of mouse papillary muscle imaged by STORM. Images show part of one cell. Fluorescently labeled polygonal structures within the cell are myofibrils, and dark spaces are mostly mitochondria (Fig. S1). Scale bar, 1 µm. (C) Merged reconstructed images in A and B. Scale bar, 1 µm. (D) Individual actin filaments (green) within a single sarcomere. Scale bar, 50 nm. (E) The sum of greatest cross-correlation values resulting from cross correlation of fluorescence images with templates containing two identical conjoined hexagons (inset) of various sizes (range, 17.5–37.5 nm, n = 45 myofibrils). (F) Only templates with 25–30-nm interactin distances (white points in dashed rectangles) overlapped with one another within each sarcomere, suggesting that these were regions of particularly good preservation of the actin filament lattice. Scale bar, 50 nm.

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  Figure S3.. Identification of myosin and actin lattice and quantification of interactin spacing in actin STORM and EM images. (A) Templates containing two identical adjoining hexagons with different interactin spacing (17.5–37.5 nm) used in template matching by cross-correlation analysis. (B) The tail of the distribution of cross-correlation values indicative of cross correlations with the highest values. Such values were summed from analysis of 45 sarcomeres and plotted to determine the best fits (Fig. 2 F). (C) The highest cross-correlation values from the cross correlation of a negatively stained EM image of transverse papillary muscle with 17.5-, 22.5-, and 27.5-nm hexagonal templates. The intensities from the thick filaments were removed from the image using the brush tool in ImageJ (Schneider et al., 2012) to mimic the STORM images. The template matches with 22.5-nm interactin distances (blue points) overlapped with one another, highlighting the region of the sarcomere with particularly good preservation of the myosin and actin filament lattice. (D) The sum of greatest cross-correlation values resulting from cross correlation of the two-hexagonal templates of various sizes (range,…

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  Figure 3.. Relative positions of actin and MyBP-C in sarcomeres from active muscle preparations determined by combining STORM with particle averaging. (A) Examples of partial hexagons cropped from active muscle sarcomeres in the actin STORM images. (B) Average reconstructed actin fluorescence image generated from 120 actin ROIs originating from different sarcomeres. (C) 3-D plot of the average actin image shown in B, with the z axis (intensity) representing localization certainty. The distance between two neighboring actin filaments was 29 ± 1 nm. (D) ROIs of the MyBP-C reconstructed images cropped from areas identical to those shown for actin in A. (E) Average reconstructed MyBP-C image, as in B for the actin. (F) 3-D plot of the average MyBP-C image shown in E. (G) Merged images shown in B and E. Actin (green) and MyBP-C (magenta). (H) Merged 3-D images shown in C and F. Actin (green) and MyBP-C (magenta). Scale bars, 25 nm. a.u., arbitrary unit.

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  Figure S4.. Geometry and reconstructed images of DNA origami scaffolds used to evaluate the resolution of the STORM analyses. (A) Illustrative representation of Alexa Fluor 647–labeled DNA origami scaffolds (i.e., nanorulers) having 3–4 Alexa Fluor 647 molecules spaced at 6-nm intervals, with their centers being separated by 31.1 nm. (B) Orange and blue points (n = 2,671) are the average centroid position of stochastic signals that persisted for two or more consecutive frames when imaging the nanorulers. Red and green symbols are the centroid positions determined for each half of 68 different nanorulers by STORM analysis. (C) The reconstructed image for the orange and blue points shown in B, rendered with Gaussian distributions representing each point’s localization uncertainty. This image demonstrates the average positions of the Alexa Fluor 647 molecules on each side of the 68 nanorulers. Scale bar, 25 nm. (D) 3-D plot of the average reconstructed image shown in C, with the z axis being intensity, representing localization uncertainty. (E) The intensity profile along the x axis of the average reconstructed image (black), fitted with sum (red dashed line) of two single Gaussian (blue and…

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  Figure S5.. Comparison of high-resolution STORM images reconstructed with Alexa Fluor 647 versus Alexa Fluor 488 for actin and MyBP-C. (A–D) Actin (A and B) and MyBP-C (C and D) are shown. A broad, toroid signal was observed for MyBP-C using both color fluorophores.

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  Figure S6.. Relative positions of actin and MyBP-C in sarcomeres from relaxed and active muscle preparations determined by combining STORM with particle averaging. (A and B) Average reconstructed actin image (A) and 3-D plot (B) generated from 134 actin hexagons originating from different relaxed muscle sarcomeres in the hearts treated with blebbistatin before fixation. (C and D) Average reconstructed MyBP-C image (C) and 3-D plot (D) from areas of the MyBP-C images corresponding to those shown for actin in A. The z axis is intensity, representing localization uncertainty. (E) Merged images shown in A and C. Actin (green) and MyBP-C (magenta). (F) Merged 3-D images shown in C and D. Actin (green) and MyBP-C (magenta). (G–L) Similar data to that shown in A–F but from active muscle sarcomeres. 120 individual actin reconstructions were used to generate the average images. The interactin distances and uncertainties were similar between the active and relaxed sarcomeres. a.u., arbitrary unit.

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  Figure 4.. Comparison of the localization N terminus of MyBP-C within the actin lattice in active and relaxed muscle sarcomeres with in silico models with known binding geometries. (A) Illustrative representations of MyBP-C’s three binding partner interactions considered using in silico models. (B) In silico image generated with 20% of MyBP-C N termini bound to actin filaments and the remaining molecules randomly distributed in the space between the thick and thin filament surfaces. Dashed line and yellow arrow demonstrate the generation of the radial intensity profile plotted in C. (C) Radial intensity profiles of the fluorescence intensity of MyBP-C’s N terminus between the center of the actin hexagon and vertices of the actin filaments in the in silico image shown in B (dashed line) and the experimental data for the active (red line) and relaxed (black line) muscle preparations. (D) RMSDs between the radial intensity profiles, as shown in C, of the active (red) and relaxed (black) muscle images and the in silico images generated with a fraction of MyBP-C N termini bound to a binding partner (Fig. S6, Fig. S7, and Fig. S8). Error bars represent the SEM of the RMSDs from six in silico…

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  Figure S7.. Comparison of the radial intensity profile of MyBP-C fluorescence from the center of the actin hexagon in active and relaxed sarcomeres to in silico models with MyBP-C bound to actin. (A) Illustrative representations of MyBP-C bound to actin. (B) The high-resolution cryo-EM structure of MyBP-C’s C0 domain (orange) bound to a cardiac thin filament (green; PDB accession no. 6CXI). The binding domain of an antibody (magenta; PDB accession no. 1IGT) was placed in close proximity to the C0 domain, and the fluorophores (represented as magenta stars) were added to the cysteines presumed to be accessible for direct conjugation of the Alexa Fluor dyes. Six fluorophores were expected to be bound to a single antibody. (C) White areas are target zones for random placement of the fluorophores for when MyBP-C is bound to the actin, as shown in A, for in silico models. (D) Red areas are regions of the image in which fluorophores cannot be randomly placed due to steric exclusion from the myosin and actin filaments. (E) Average in silico reconstructed images generated with a fraction of the fluorophores bound to actin and the remaining fluorophores randomly distributed throughout the image except…

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  Figure S8.. Comparison of radial intensity profile of MyBP-C fluorescence from the center of the actin hexagon in active and relaxed sarcomeres to in silico models with MyBP-C bound to myosin, when myosin adopts the IHM and is stabilized on the surface of the myosin thick filament. (A) Illustrative representations of MyBP-C bound to myosin heads, which adopt the IHM. (B) Projection of a cross section from the 3-D EM reconstruction of cardiac muscle thick filament (Zoghbi, et al., 2008; EMD-1465). MyBP-C’s C0 domain (orange; from PDB accession no. 6CXI) with an antibody (magenta; PDB accession no. 1IGT) was placed in close proximity to the outer surface of the RLC of myosin thick filament (4). The red arrow indicates the 4-nm distance between the center of the thick filament and the surface of the C0 domain. The black arrow indicates the 14-nm distance between the center of the thick filament and centroid position of fluorophore with the antibody attached to the C0 domain. The pink dashed circle indicates the potential localization of the fluorophore around each thick filament due to this binding geometry. (C) White areas are target zones for random placement of the fluorophores for when…