Efficient repair cap formation requires actin and Ca²⁺. (A) Myofibers were electroporated with actin (LifeAct-mTurq2) to visualize F-actin and FM4-64–marked laser-induced injury (red). Actin (green) slowly accumulated under the FM4-64–positive lesion (red) in response to laser damage (white arrow), beginning at ∼14 s but best visualized between 50 and 110 s (n = 15 myofibers from n = 4 mice). (B) Coelectroporation of actin (LifeAct-mTurq2) and annexin A6 demonstrated that F-actin accumulated in the annexin-free clearance zone (seen in 15/15 myofibers isolated from n = 4 mice). (C) F-actin reorganized at the site of repair as visualized by the change in periodicity in the plot profile (orange arrow). (D) Latrunculin, which disrupts actin filament formation, resulted in delayed annexin A6 cap formation (n ≥ 7 myofibers from n = 3 mice per condition). (E) The time for annexin A6 cap formation was 13 s compared with 67 s, with and without latrunculin (*, P < 0.01), indicating that rapid annexin A6 repair cap formation requires filamentous actin (n ≥ 7 myofibers from n = 3 mice per condition). (F) Annexin A6 caps failed to form in the absence of Ca²⁺. Incubation of fibers in 0 mM Ca²⁺ + EGTA inhibited annexin A6 cap formation (yellow arrow) up to 110 s of imaging (0 of 13 fibers formed caps). A few fibers retained the ability to form the annexin-free zone despite not forming a repair cap (n = 13 myofibers from n = 3 mice per condition). Bars, 4 µm. Error bars represent SEM.