Annexins orchestrate plasma membrane repair in skeletal muscle

ABSTRACT Background/Methods: Skeletal muscle is routinely injured in every day activity. In response, skeletal muscle has an active capacity to resealing membrane disruptions. We previously identified annexin A6 as a modifier of muscle disease. Annexins are membrane-associated Ca2+ binding proteins, and there are 12 different annexins. To define the molecular machinery that directs muscle repair, we developed a new method to visualize membrane resealing and repair in real time. This method uses electroporation to express fluorescently tagged proteins combined with high-resolution confocal microscopy. In this system, muscle fibers are wounded with a laser and then imaged to observe the resealing process. Results: Within seconds of laser-wounding, the annexins A1, A2, and A6 formed a repair cap immediately at the site of injury. Within the myofiber cytoplasm and immediately under, we visualized annexin-free zone. Co-expression of fluorescently tagged annexins A1/A6 and A2/A6 demonstrated that annexin proteins colocalize within the repair cap at the lesion. Expression of a truncated form of annexin A6, which harbors the first 4 of 8 calcium-binding annexin repeat domains, termed ANXA6N32, interfered with normal annexin A6 translocation to the site of membrane disruption. Notably, ANXA6N32 mediated disruption of annexin A6-rich repair cap and was associated with visible leak of intracellular contents into the extracellular space. Additionally, we found actin was recruited to the site of damage localizing within the annexin-free clearance zone beneath the site of sarcolemmal disruption. Annexin A6 cap formation was inhibited in the presence of latrunculin, an actin inhibitor, and at 0mM Ca2+ illustrating annexin A6 cap formation is both actin and Ca2+-dependent. Mutation of Ca2+ binding residues within annexins A1, A2, and A6 reduced repair cap and clearance zone formation and disrupted membrane resealing. Expression of these Ca2+ mutants acted in a dominant-negative fashion, causing delayed wildtype annexin repair cap formation and decreased repair cap size after laser injury. Conclusions: These data define the repair cap complex as an annexin-containing structure formed during sarcolemmal repair that is both actin-dependent and Ca2+-sensitive. Additionally, generation of a proper repair cap after membrane damage requires cooperativity between annexin proteins. We acknowledge the outstanding support of Dr. Constadina Arvanitis at the Center for Advanced Microscopy at Northwestern University.