Muscle growth by sarcomere divisions

Figures

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  Fig. 1.. Muscle growth and sarcomere addition. (A) Schematic of a mature Drosophila flight muscle sarcomere bordered by two Z-discs containing α-actinin (green), which cross-links polar actin filaments (red). Central bipolar myosin filaments (blue) are cross-linked by obscurin at the M-band and form the A-band region of the sarcomere. The myosin-free zone is the I-band region. The titin homolog Sallimus (Sls) links the Z-disc to the myosin filaments, and the second titin homolog Projectin is located at both ends of the myosin filaments. Scheme was adapted from (2) including data from (21). (B) Schematic of a Drosophila hemithorax at pupal stages with the six dorsal longitudinal flight muscles (DLMs) in magenta and the tendon cell epithelium in green. DLM4 is highlighted and was used for quantifications in (E). The stable connection of the flight muscles to the tendon cell extensions (green lines) during the growth phase from 32 to 40 hours after puparium formation (APF). h, hours. (C and D) Confocal images of pupae of the indicated stages displaying the six flight muscles stained for actin (phalloidin in red) and Shot (anti-Shortstop in gray, also labeling the tendon extensions) in (C).…

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  Fig. 2.. Flight muscle end growth live. (A) Schematic of a Drosophila hemithorax at 34 to 36 hours APF with the six DLMs in magenta and the tendon cell epithelium in green. DLM1 is highlighted, and the estimated area used for live imaging of myofibril terminal ends is marked by an orange box. (B and C) About 34 hours APF, a pupa expressing Sls-GFP with a focus on the anterior flight muscle end. Stills from movie S1 are shown (B), and a kymograph of a selected region is shown (C). The myofibril ends, marked with yellow arrowheads, move to the left, while muscle fiber length increases and thus moves away from the reference point marked with the orange arrowhead. No obvious sarcomere addition is seen at the ends. (D and E) About 36 hours APF, a pupa expressing Mhc-GFP with a focus on the anterior flight muscle end. Stills from movie S2 are shown (D), and a kymograph of a selected region is shown (E). The myofibril ends, marked with yellow arrowheads, move to the left, while muscle fiber length increases and thus moves away from the reference point marked with the orange arrowhead. No obvious sarcomere additions at the ends are seen. Scale bars, 10 μm. At least three pupae were imaged for…

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  Fig. 3.. An automated detection tool for flight muscle sarcomeres. (A) Summary of automated myofibril tracing from a confocal z-stack of flight muscles stained for sarcomere proteins as input to traced sarcomeres as output; Z-discs and M-bands of a selected myofibril are labeled (see Materials and Methods, fig. S2, and movies S3 and S4). (B) Normalized mean intensity profiles computed from detected sarcomeres of actin (phalloidin in red), Sallimus (Sls-Nano2 in orange), Obscurin (anti-Obs in dark blue), and myosin (Mhc-GFP in light blue) shown as mean (black) ± SD (shaded regions) at 36 and 40 hours APF. 36 hours APF: Five samples from two animals, n = 5063 sarcomeres; 40 hours APF: Eight samples from three animals, n = 12,794 sarcomeres. Histograms of sarcomere length bins are shown to the right. h, hours.

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  Fig. 4.. Sarcomere detection reveals sarcomere division by myosin segregation. (A) Top: Merged mean intensity profiles for the same proteins as in Fig. 3B sorted by sarcomere length bins using the 40-h APF flight muscles (same eight samples as in Fig. 3; n = 120 to 9580 sarcomeres per bin, mean profiles from oriented individual profiles with the larger of the two obscurin signals oriented computationally to the left). Below are representative confocal images with highlighted sarcomeres matching the length categories. Color code used in the profiles above is indicated on the left. Two distinct myosin and Obscurin signals are visible in sarcomeres longer than 2.4 μm (green arrow and arrowheads); concomitantly, Sls is moving away from the Z-discs (marked by yellow arrows) to form a new Z-disc (yellow arrowheads). Scale bar, 2 μm. (B and C) Peak-to-peak distances of Z1, Z2, S1, S2 (Sallimus), and M1, M2 (Obscurin) peaks computed from the mean intensity profiles shown in (A). Distances Z1-M1 and Z2-M2 (in gray) do not change when sarcomere length increases, while the distances M1-S2 and M2-S1 increase with slope ≈1 (C), suggesting that daughter sarcomeres slide relative to each other as rigid…

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  Fig. 5.. Live imaging of sarcomere division. (A) Stills from a time-lapse movie of Mhc-GFP–expressing flight muscles of a 36-hour APF pupa acquired with two-photon microscopy (see movie S7). Arrows indicate the myosin filament stacks of one selected myofibril. Sarcomere length is indicated with the yellow brackets. The dividing myosin filament stack is labeled with magenta arrows. The increase in sarcomere length correlates with the segregation of the two new myosin peaks. Scale bar, 2 μm. (B) Mhc-GFP intensity profiles along the highlighted myofibril in (A) for different color-coded time points. Distances between Mhc-GFP peaks are highlighted for two frames (t: −10 min in blue and t: 10 min in red). Time 0 min indicates the putative start of the division event. a.u., arbitrary units; d, distance. (C) Interpeak distances of intensity profiles from (B) as a function of time. Color code corresponds to the inset in (B). (D) Normalized total Mhc-GFP intensity of the tracked dividing sarcomere. Note the increase in MhcGFP amount over time.

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  Fig. 6.. Dividing sarcomeres in larval muscles. (A) SEM segmentation of L3 larval Z-discs in a 3D volume starting from the muscle surface using the slices of fig. S9. Each color indicates voxels that were associated with one complete Z-disc at the lateral myofibril edge. See also movie S9. Scale bar, 7 μm. (B) Living Drosophila L3 larval muscles labeled with Mhc-GFP (green) and Sls-Cherry (magenta) display myosin rings sorted by increasing diameters (yellow), which show increasing amounts of Sls in their center (red arrowheads). The different images were collected from different animals. Scale bar, 5 μm. (C) Mhc-GFP in living late L2 larval muscle (dorsal oblique 2), before fluorescence recovery after photobleaching (FRAP; left), after FRAP (middle; FRAP region marked by red rectangle), and after a 7-hour recovery with feeding, resulting in molding to L3 larva (right; approximate region shown as high magnification in (D) is marked with yellow rectangle). The yellow line marks 31 sarcomeres before FRAP, which increases to 36 sarcomeres after a 7-hour recovery (which are more contracted compared to the L2 state). Scale bars, 50 μm. (D) High magnification of yellow region in (C) with Mhc-GFP…

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  Fig. 7.. Sarcomere division model. Working model of the tension-induced sarcomere division in Drosophila flight muscle sarcomeres. The growth of the skeleton, marked by integrin-based attachments, induces high tension, which triggers the division of the highlighted middle sarcomere. Thus, the sarcomere number increases from three to four. For details, see Discussion.