Fig. 5

Paralysis leads to actin lengthening in both relaxed mutant and Tricaine treated embryos, subsequent movement restoration in recovered embryos leads to actin length complete rescue. (A,B) H-H measurement. Confocal images of actin filaments stained with phalloidin (1/40) were taken (upper panel 24 hpf control embryo and lower panel 24 hpf Tricaine treated embryo) and measurements between H bands, across Z discs and sarcomeres, were made. (C) 17–24 hpf paralysis caused a significant lengthening of actin filaments H-H at 24 hpf (*p < 0.05) (n = 20 in 5 controls, n = 15 in 3 treated embryos, t-test). At 42 hpf, recovered embryos (17–24 hpf paralysis) showed a full recovery (n = 50 in 10 control embryos, n = 40 in 8 recovered embryos, unpaired t-test), in contrast with treated embryos (17–42 hpf paralysis) which did not recover (n = 50 in 10 control embryos, n = 135 in 27 treated,*p < 0.05). (D) Immotile relaxed mutants, rr, showed a very significant lengthening of actin filaments H-H at 24 hpf (***p < 0.001) (n = 115 in 23 immotile relaxed mutants rr, n = 120 in 24 motile control siblings Rr/RR and at 42hpf (***p < 0.001) (n = 105 in 21 immotile relaxed mutants rr, n = 135 in 27 motile control siblings Rr/RR). Interestingly, in contrast with the Tricaine treated immotile embryos, the immotile relaxed mutant (rr) actin is still very significantly longer than the motile control siblings (Rr/RR) actin at 42 hpf. It is noticeable that in the complete absence of movement up to 42 hpf actin filaments showed a significant shortening between 24 and 42 hpf in between both treated and control embryos and immotile relaxed mutants and motile control siblings, respectively (n = 15 in 3 24 hpf treated, n = 135 in 27 42 hpf treated embryos, *p < 0.05 unpaired t-test; and n = 115 in 23 24 hpf immotile relaxed mutants rr, n = 105 in 21 42 hpf immotile relaxed mutants rr, *p < 0.05 unpaired t-test).