Fig 2: Expression and subcellular distribution of ANX in human myoblasts and myotubes. (A) Cellular content in endogenous ANXA1 to A4 in LHCN myoblasts (Mb) and myotubes (MT) was quantified through western blot analysis. Black arrows indicate bands corresponding to ANX. The four western blot experiments were performed simultaneously with the same protein extracts (10 µg). The experimental series was repeated at least three times with independent samples. GAPDH, used as a loading control, was immunodetected together with ANXA3 and ANXA4, but not ANXA1 and ANXA2 that exhibit a similar molecular weight (37 kDa). The whole membranes, as well as quantitative analysis, are presented in Figure S1. (B) Subcellular localization of endogenous ANXA1 to A4 (green) in LHCN myotubes by immunocytofluorescence. White asterisks indicate nuclei in one myotube per image. Results in LHCN myoblasts are presented in Figure S2. Scale bars: 50 µm.

Fig 3: Model of cell membrane repair in human skeletal muscle cells. (A,A’) Entry of Ca2+ induces the recruitment of ANX to injured sarcolemma, notably ANXA5 [15], ANXA6 [17] and ANXA4. It induces also the recruitment of ANXA1/A2-bearing intracellular vesicles. (B,B’) Membrane tension is reduced by depolymerization of actin and exocytosis of lysosomes [37]. Increase in sarcolemma surface leads to excess membrane at the disruption site, on which ANXA6 is associated [17]. ANXA5 forms 2D arrays that strengthen the sarcolemma and limit the expansion of the tear [15]. Intracellular vesicles are recruited to the disruption site. (C,C’) Aggregation of intracellular vesicles forms the lipid patch that plugs the rupture. ANXA4 and ANXA6 induce folding of the extensions of sarcolemma in order to form a tight structure. (D,D’) Accumulation of ANX leads to folding and curvature of membranes [38] and the formation of the cap subdomain [16,17]. ANXA2 may promote repolymerization of cortical actin [7]. At this time, some vestiges of the lipid patch (rich in ANXA1 and ANXA2) and sarcolemma fragments (rich in ANXA4 and ANXA6) are present in the extracellular milieu. (E,E’) Integrity of sarcolemma is restored by membrane reconstitution and elimination of the cap subdomain by macrophages (not represented) [39]. Adapted from [17].

Fig 4: Model of cell membrane translocation and recruitment to the site of membrane injury of ANX according to their Ca2+ sensitivity. (A) As observed in Skrahina et al. [32], treatment with ionomycin, a Ca2+ ionophore, induces a gradual influx of Ca2+ in the whole cell (green arrows). At t1, first ANX to be translocated to the plasma membrane are therefore the most Ca2+ sensitive, such as ANXA2 and ANXA4 (red circles). At tn, high increase in the intracellular Ca2+ concentration causes the sequential translocation of ANX less sensitive to Ca2+ (blue circles). The order of translocation is as follows: ANXA2, ANXA4, ANXA6, ANXA1, and ANXA5. Data from [32]. (B) According to the current study, we propose that a local rupture in the plasma membrane causes a massive entry of Ca2+, creating a large gradient. At t1, the high Ca2+ concentration close to the disruption site induces the binding to plasma membrane of the lesser Ca2+-sensitive ANX, such as ANXA1, ANXA6 and ANXA5 (blue circles). Instead, high Ca2+-sensitive ANX, such as ANXA2 and ANXA4 (red circles), interact with membranes distant from the site of rupture. In human skeletal muscle cells, the order of recruitment to the site of membrane injury is the following: ANXA1, ANXA6/A5 [17], ANXA4, and ANXA2. Once membrane has been resealed (tn), ANX present in the cap subdomain, which is localized outside the cell where the Ca2+ concentration is high, remain in interaction with membranes. Inside the cell, most ANX, except eventually ANXA2, which is the most Ca2+-sensitive, are released from membranes when Ca2+ homeostasis is restored.

Fig 5: CLEM imaging of ANX in damaged LHCN myotubes. ANXA1-GFP (A), ANXA2-GFP (B), or ANXA4-GFP (C) expressing LHCN myotubes were damaged by laser ablation (red arrow) and immunostained for ANX using a secondary antibody coupled to gold nanoparticles (black particles). Fluorescence images obtained about 90 s after laser ablation are presented (left-hand image) together with TEM images (middle and right-hand images). Respective middle and right-hand images have been collected from different sections. Right-hand images show ANX (black particles) interacting with circular lipid structures (black arrow). Scale bar for fluorescence images: 10 µm; for TEM: 1 µm.