Truncated annexin A6 (ANXA6N32) disrupts dysferlin localization during membrane repair. Muscle fibers were coelectroporated with dysferlin (DYSF)-Venus and full-length annexin A6 (A6)–mCherry or truncated ANXA6N32-mCherry for live-cell imaging after laser damage. (A) Annexin A6 (red) formed a distinct repair cap (yellow arrow) at the site of membrane disruption, whereas dysferlin (green) localized to the shoulder (orange arrow), and a high-magnification image is shown to the right. (B) Fibers expressing ANXA6N32 either did not form a repair cap at all (example, top row) or formed a much smaller repair cap (example, bottom row). In both cases, the annexin-free zone was poorly demarcated compared with that seen with full-length annexin A6. ANXA6N32 disrupted the translocation of dysferlin to the shoulder, consistent with a dominant-negative effect. ANXA6N32 did not disrupt dysferlin’s formation of a clear zone under the annexin A6 cap. High-magnification images are shown on the right. Bars, 4 µm. n = 7 mice per condition. (C) The presence of ANXA6N32 was associated with decreased cap formation compared with full-length annexin A6 (n ≥ 24 myofibers from n = 7 mice per condition). (D) Coelectroporation of dysferlin and ANXA6N32 resulted in smaller annexin repair caps upon damage compared with full-length annexin A6 caps (*, P < 0.0001; n ≥ 13 myofibers from n = 4 mice per condition). (E) Coelectroporation of dysferlin and ANXA6N32 resulted in decreased DYSF-Venus fluorescence at the shoulder 150 s after damage (*, P < 0.05; n ≥ 7 myofibers from n = 3 mice per condition). Error bars represent SEM.