Annexin-A6 in Membrane Repair of Human Skeletal Muscle Cell: A Role in the Cap Subdomain.

Coralie Croissant,Flora Bouvet,Sisareuth Tan,Anthony Bouter,Céline Gounou

doi:10.3390/cells9071742

Abstract

Defects in membrane repair contribute to the development of some muscular dystrophies, highlighting the importance to decipher the membrane repair mechanisms in human skeletal muscle. In murine myofibers, the formation of a cap subdomain composed notably by annexins (Anx) is critical for membrane repair. We applied membrane damage by laser ablation to human skeletal muscle cells and assessed the behavior of annexin-A6 (AnxA6) tagged with GFP by correlative light and electron microscopy (CLEM). We show that AnxA6 was recruited to the site of membrane injury within a few seconds after membrane injury. In addition, we show that the deficiency in AnxA6 compromises human sarcolemma repair, demonstrating the crucial role played by AnxA6 in this process. An AnxA6-containing cap-subdomain was formed in damaged human myotubes in about one minute. Through transmission electron microscopy (TEM), we observed that extension of the sarcolemma occurred during membrane resealing, which participated in forming a dense lipid structure in order to plug the hole. By properties of membrane folding and curvature, AnxA6 helped in the formation of this tight structure. The compaction of intracellular membranes—which are used for membrane resealing and engulfed in extensions of the sarcolemma—may also facilitate elimination of the excess of lipid and protein material once cell membrane has been repaired. These data reinforce the role played by AnxA6 and the cap subdomain in membrane repair of skeletal muscle cells.

Highlights

Plasma membrane of numerous cell types, such as cardiac or skeletal muscle cells during contraction [1,2], epithelial cells during the passage of food bolus [3] or endothelial cells submitted to blood flux [4], endure mechanical stress that can induce membrane damages
In order to investigate the relative expression of AnxA6 in human skeletal muscle cells, western-blot analysis was performed from the healthy human myogenic cell line LHCN, which was established from satellite cells isolated from pectoralis major adult muscle [31]
The observation of a small cluster of AnxA5-mCherry in the cap subdomain could correspond to AnxA5 arrays that interacted with the edges of the torn membrane and were packaged in the cap after membrane resealing. These results indicated that membrane repair in human skeletal muscle cells is based on the formation of a cap subdomain, composed notably by AnxA6

Summary

Introduction

Plasma membrane of numerous cell types, such as cardiac or skeletal muscle cells during contraction [1,2], epithelial cells during the passage of food bolus [3] or endothelial cells submitted to blood flux [4], endure mechanical stress that can induce membrane damages. It has been observed that up to 20% of skeletal muscle cells can be damaged during physical exercise [1]. The absence or failure of membrane resealing leads to cell death and may contribute to the development of degenerative diseases, such as muscular dystrophies. (formerly 2 L) are, respectively characterized by mutations in the dysferlin and anoctamin-5 genes, leading to several dysfunctions including a failure in cell membrane repair process [5,6]. Extracellular mM Ca2+ concentration enters the cell. The increase of intracellular Ca2+ concentration triggers the repair machinery in eukaryotic cells ensuring a rapid resealing of large plasma membrane ruptures [7,8,9]. Several proteins have been identified as belonging to the membrane repair machinery such as AHNAK, acid sphingomyelinase, ESCRT complexes, MG53, S100 proteins, SNAREs, synaptotagmins, calpains, caveolins, dysferlin or

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