Thick filament‐associated myosin undergoes frequent replacement at the tip of the thick filament

Figures

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  Fig. 1. Photoconversion analysis of KikGR‐Myh3 in the myofibrils. (A‐B) Localization of photoconvertible KikGR‐Myh3 in the myofibrils. (A) A green fluorescence signal of KikGR‐Myh3 was detected in the thick filament of myofibrils at preconversion. The fluorescence color of KIkGR‐Myh3 shifted from green to red with exposure to UV light (yellow rectangles). Bar, 5 µm. (B) Alternation of KikGR‐Myh3 fluorescence signals was monitored at 10 min intervals up to 10 h after photoconversion. Merged images with green and red fluorescence signals are indicated in the dashed rectangular area in A. Bar, 5 µm. (C‐D) Symmetrical and biphasic changes of KikGR‐Myh3 fluorescence intensities in the myofibrils. (C) Green and red fluorescence signals of KikGR‐Myh3 were measured in the myofibrils at the indicated time points. The circle symbol of each color shows measurement value, and white curves show curve‐fitting results. (D) Mobile fractions (%) were 55.24 ± 7.86% for green fluorescence and 66.98 ± 3.90% for red fluorescence. T1/2 (h) were 2.55 ± 0.59 for green fluorescence and 1.38 ± 0.38 for red fluorescence. Values represent the mean ± SE. p values were calculated by Student’s t‐test. n = 7 for each…

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  Fig. 2. Changes across time in the green and red fluorescence distribution of KikGR‐Myh3 in the sarcomeres. (A) A scheme of image‐processing to compare the distributions of red and green fluorescence of KikGR‐Myh3 in each sarcomere. First, the fluorescence signal was line‐scanned along myofibrils to obtain mean fluorescence intensities in a sarcomere with 10 min intervals up to 200 min (dashed rectangular area in Fig. 1A). Second, a single peak between turning points of green fluorescence intensity was extracted as a fluorescence waveform. Finally, the data was curve fitted with a polynomial formula, and a fluorescence max value at the y‐axis and a max distance from the center at the x‐axis were normalized to 1.0. The normalized distance of −1.0~1.0 (between Z‐bands) indicates the width of a sarcomere, and 0.0 indicates the center of the sarcomere (M line). The two ends of the thick filament correspond to approximately −0.8 and 0.8. (B‐C) Overlaid graphs of fluorescence waveforms. Twenty line‐scanned images taken from 0 to 200 min at 10 min intervals following photoconversion were overlaid. The unprocessed fluorescence intensity and the normalized fluorescence intensity are shown in B…

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  Fig. 3. Visualization of the incorporation of newly synthesized myosin into the thick filaments. (A) Experimental procedure of different fluorescence pulse‐chase labeling using Halo‐Myh3. First, myotubes expressing Halo‐Myh3 were incubated with the Oregon‐Green ligand (green fluorescence) for 16 h. Next, myotubes were reacted with the TMR ligand (red fluorescence) to label newly synthesized myosin for 7, 15, or 30 min after washout of Oregon‐Green ligand. Finally, myotubes were fixed and observed by confocal microscopy. (B) Representative images of myotubes expressing Halo‐Myh3 with fluorescence pulse‐chase labeling. Bar, 10 µm.

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  Fig. 4. A variety of incorporation patterns of newly synthesized Halo‐Myh3 into the thick filaments. (A) A scheme to categorize newly synthesized myosin insertion sites in the thick filaments. First, each sarcomere was line‐scanned. Second, line‐scanned data were curve‐fitted with a polynomial formula. Third, a green waveform and its corresponding red waveform were extracted. Fourth, the green and red fluorescence intensities were normalized as a max value of 1. Finally, waveforms were classified into eight patterns based on the red peak position. (B) Eight patterns of newly synthesized Halo‐Myh3 incorporation into the thick filaments. Line‐scanned images were categorized into eight groups based on the waveforms of red fluorescence signals compared to green fluorescence signals: both sides, predominantly right side, predominantly left side, right side, left side, center, random fashion, and no incorporation. Solid lines show the average value ± SE (pale‐colored width) in the upper graphs. The lower panels are drawn by enumeration of individual subtraction of the green fluorescence signal from the red fluorescence signal. In the no incorporation group, red fluorescence intensities are…

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  Fig. 5. Comparison of distance between the thick filament center and the peak signal of Halo‐Myh3 with red fluorescence. (A) A scheme to evaluate the insertion site of newly synthesized myosin with red fluorescence on the thick filaments. First, the insertion area of newly synthesized myosin was compared using waveforms categorized to the side group. Second, the distance between the red fluorescence peak and center was measured as the myosin insertion area from the tips of the thick filament to red fluorescence peak point(s). Finally, the measured distance was plotted in the histogram and the peak shift was evaluated during pulse‐chasing labeling. (B) A histogram of newly synthesized myosin incorporation sites on the thick filaments. The frequency of the myosin incorporation site, defined as the distance between the red fluorescence peak and the center of the thick filament, is shown (upper panel). The frequency was further divided into the three categories: Both sides, Predominantly R + L, and One side (R + L) (lower panel). The x‐axis indicates the normalized distance from the center of the thick filament. The center of the thick filament is located at 0, and the tip of the thick…