Titin-dependent biomechanical feedback tailors sarcomeres to specialized muscle functions in insects

Figures

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  Fig. 1.. Evolution of titin homologs in animals. (A) Left: Evolutionary tree representing animals with striated or non-striated muscles. Right: Representative titin homologs (longest isoforms) are drawn to scale, with highlighted Z-repeats (gray), Ig domains (brown), PEVK (dashed magenta), Fn3 domains (green), and kinase domain (blue). All chordates have a long titin protein with I-band and A-band features, whereas Ecdysozoa species have two titin homologs separating I-band and A-band features into two proteins. The sizes of the mouse titin, the two Drosophila titins (Sallimus and Projectin), and the two C. elegans titins (Titin and Twitchin) are indicated. The latter four are highlighted by orange boxes. For organisms with recent genome duplications only one titin is shown (Danio rerio, ttn2, XP_021334745.1; Xenopus laevis titin-like isoform X10, XP_018091448.1; see table S1). (B) Domain scheme of Drosophila and C. elegans titin isoforms: Sls (short and long), Projectin, C. elegans Titin, and Twitchin. PEVK-rich regions are highlighted in magenta. (C) Flight, leg, and larval muscles of sls[32-V5] stained for actin (magenta) and V5 (green). Scale bars, 5 μm. (D) Electron microscopy image…

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  Fig. 2.. Reducing long titin PEVK length causes shorter larval sarcomeres. (A and B) Wild-type (w[1118], N = 39), sls[31-V5] (N = 21), sls[32-V5] (N = 22), and sls[∆31-32] (N = 19) third instar larval VL3 muscles stained for actin (phalloidin). Scale bar, 100 μm. (B) Quantification of sarcomere length from muscle images as shown in (A). Note the shorter sarcomeres in sls[32-V5] and sls[∆31-32]. Tukey’s multiple comparisons test, ****P < 0.0001. Plots are Tukey’s box and whisker plots with median as lines, first quartile and second quartile as box, and whisker size is 1.5 times the interquartile range (IQR). All animals measured are represented as dots and average values are noted as numbers. This applies for all figures. (C and D) Wild-type (w[1118], N = 22), sls[31-V5] (N = 12), sls[32-V5] (N = 23), and sls[∆31-32] (N = 18) third instar larval VL3 muscles stained with N- and C-terminal anti-Sls nanobodies (Sls-Nano2 in green and Sls-Nano42 in magenta). Scale bar, 5 μm. (D) Quantification of Sallimus length, sarcomere length, and ratio Sls/sarcomere length from muscle images as shown in (C). Tukey’s multiple comparisons test, ****P < 0.0001. (E and F) Wild-type (w[1118], N = 39),…

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  Fig. 3.. Extra titin PEVK elongates thin and thick filaments in a muscle type-specific way. (A) sls[23-YPet] and sls[23-extraPEVK-YPet] third instar larval VL1 muscles stained for actin. Scale bar, 100 μm. (B) Quantification of sarcomere length of wild type (w[1118], N = 17), sls[∆31–32] (N = 13), sls[23-YPet] (N = 18), and sls[23-extraPEVK-YPet] (N = 14). (C and D) sls[23-YPet] (N = 23 for Sls, 13 for actin and 22 for myosin length) and sls[23-extraPEVK-YPet] (N = 18, 11, and 18) VL1 muscles stained for myosin, actin (green), and Sls-C-terminal nanobody (Sls-Nano48, magenta). Scale bar, 5 μm. Mann-Whitney test, *P < 0.05, **P < 0.01, and ***P < 0.001. (D) Quantification of Sls, actin, and myosin filament length from images as shown in (C). (E and F) Flight muscles of sls[23-YPet] (N = 14) and sls[23-extraPEVK-YPet] (N = 15) stained with C-terminal anti-Projectin nanobody (Proj-Nano37) and actin (phalloidin). Scale bar, 3 μm. (F) Quantification of sarcomere length and distance between Proj-C-termini [shown by white bracket in (E)]. Mann-Whitney test, *P < 0.05. (G and H) DNA PAINT imaging of sls[23-YPet] (N = 14) and sls[23-extraPEVK-YPet] (N = 12) stained with either anti-Projectin…

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  Fig. 4.. Sls molecular forces in different muscle types in vivo. (A) In vivo expression of sls[23-YPet] and sls[32-YPet] in third instar larval, adult abdominal, and adult flight muscles. Scale bar, 10 μm. Note that sls[32-YPet] is not expressed in flight muscles. (B) Quantification of sarcomere length in heat fixed third instar larval VL3 muscles of sls[23-YPet] (N = 22) and sls[32-YPet] (N = 22). Mann-Whitney test, ****P < 0.0001. Plots are Tukey’s box and whisker plots with median as lines, and first quartile and second quartile as box and whisker size are 1.5 times the IQR. All animals measured are represented as dots. (C and D) FLIM-based FRET quantification of sls[23-TS] and sls[23-stTS] (C) and sls[32-TS] and sls[32-stTS] (D) in larval, abdominal, or flight muscles (n ranges from 10 to 40 per muscle and genotype, see source data S4). (E) Sarcomere length and I-band length (calculated by measuring the A-band length; see fig. S6E) in alive MhcGFP expressing embryos (N = 20) and larvae at different larval instars (L1, N = 24; L2, N = 17; L3, N = 18). Tukey’s multiple comparisons test, ****P < 0.0001. (F) FLIM-based FRET quantification of sls[23-TS] in alive embryos (N = 16) and…

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  Fig. 5.. A titin-instructed biomechanical feedback controls filament length. (A) Mathematical model of a Sls instructed biomechanical feedback regulating I-band and A-band length: Actin and myosin filaments grow until a constant actin/myosin overlap (αC) is reached in the relaxed state (stretched titin). If in the relaxed state α < αC, actin and myosin filaments can grow at their ends (highlighted by darker colors). If α = αC is reached in the relaxed state, then growth stops as frequent contractions are initiated and then recruitment in the contracted state is blocked (red STOP signal). Such a stable A-band length is reached that scales with the I-band length. See section “A biomechanical feedback model” and the Materials and Methods for details. (B and C) Wild-type control (N = 9) and hypercontractile Mhc[S1]/+ (N = 12) and troponin I (wupA[hdp2], N = 11) mutant flight muscle sarcomeres were stained with Sls-N-terminal to label the Z-discs (magenta) and anti-myosin light chain nanobody (NB1-Mlc1) to label myosin (green). Scale bar, 5 μm. (C) Sarcomere length from (B). Tukey’s multiple comparisons test, ****P < 0.0001. (D and E) Larval sarcomeres of wild type (Prm-GFP, Mef2-GAL4; N =…

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  Fig. 6.. A sarcomere type phase diagram. A phase diagram that illustrates how two variables, developmental muscle contractions (resulting in changes of actin-myosin overlap αC) and I-band titin length, can generate sarcomeres with vastly different dimensions, which are specialized for maximum frequency at high force (flying muscles, red), large motion amplitude (crawling muscles, magenta), or maximum force generation (cutting muscles, blue).