Fig. 3: M305L actin causes IFM hypercontraction and enhances Ca²⁺ sensitivity.

a Fluorescent micrographs of dorsal longitudinal IFMs (DLMs) of two-day-old Act88F^WT and Act88F^M305L Drosophila. Act88F^WT/+, Act88F^M305L/+, Act88F^WT/Act88F^WT;+, Act88F^M305L/Act88F^M305L;+ heterozygotes, and Act88F^WT/Act88F^WT homozygotes displayed normal DLM morphology with the six fibers spanning the length of the thorax. Act88F^M305L/Act88F^M305L homozygotes, however, demonstrated hypercontracted and torn DLMs. Scale bar = 250 µm. b Confocal images of consecutive sarcomeres along a single IFM myofibril from transgenic flies. Red, TRITC-phalloidin-labeled actin; Cyan, immunolabeled α-actinin. WT/+ = Act88F^WT/+, 305/+ = Act88F^M305L/+, WT/WT;+ = Act88F^WT/Act88F^WT;+, 305/305;+ = Act88F^M305L/Act88F^M305L;+, WT/WT = Act88F^WT/Act88F^WT, and 305/305 = Act88F^M305L/Act88F^M305L. IFM thin filament lengths did not significantly differ among the genotypes (Supplementary Fig. 5B) as determined by a Kruskal-Wallis one-way ANOVA with Dunn’s post hoc test (n = 200–211). Scale bar = 2.5 µm. c The power-pCa relationship of Act88F^M305L/+ IFM fibers revealed a significant leftward shift in Ca²⁺ sensitivity (see Table 2), indicating less Ca²⁺ is required for activation. Significance was assessed via an unpaired two-tailed t-test (n = 10). Data points are mean ± SEM and were fit by the Hill equation. Source data are provided as a Source Data file.