Fig 2: Responses of AnxA6-deficient LHCN myotubes to laser ablation. (A-B) Sequences of images showing the response of a wild-type (A) and three different AnxA6-deficient (B) LHCN myotubes to 110-mW laser irradiation in the presence of 2-mM Ca2+ and FM1-43 (green). In these panels, the area of membrane irradiation is marked with a red arrow before irradiation and a white arrow after irradiation. Time at which the image was recorded before (-) or after (+) irradiation is indicated. Scale bars = 20 µm; (C,D) Data represent the FM1-43 fluorescence intensity integrated over whole cell section, which is plotted versus time. For LHCN myotubes (mean ± SEM of 20 cells within 2 independent experiments), the fluorescence intensity reached a plateau after ~80 s (C). For AnxA6-deficient myotubes, very different kinetics were observed from one myotube to another. As an example, one typical experiment with 20 cells is presented in (D). When the presence of a plateau and a maximal fluorescence intensity lower than 20 a.u. were observed, the cell was classified as “repaired cell” (lower part of the graph, as indicated). When the kinetics of fluorescence changes showed a continuous increase with values higher than 90 a.u., the cell was classified as “unrepaired cell” (upper part). Otherwise, cells were classified as “undetermined cells”. Data analysis is presented in Table S1. Red dashed lines divide the three types of response.

Fig 3: Distribution of AnxA5 and AnxA6 within the cap subdomain. LHCN myotubes were transfected with the plasmids pA6-GFP and pA5-mCherry to express AnxA6-GFP (green) and AnxA5-mCherry (magenta) and membrane damage experiments were performed by laser ablation at the edge of the myotube. (A) Follow-up of the recruitment of AnxA6 and AnxA5 within 120 s after membrane damage. Red arrow, area before irradiation; white arrow, area after irradiation; (B) Enlarged images of the damaged area 240 s after membrane damage. Scale bars: 10 µm.

Fig 4: Mechanical force enhances the enrichment of endocytic related proteins in exosomes(A) Exosomes derived from control or force-primed PDLSCs were collected and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis. The identified differential proteins were used to perform heatmap analyses. The colors represent the protein concentration of each sample calculated by peak area normalization.(B) Gene ontology (GO) analysis of the upregulated proteins. BP, biological process; CC, cellular function; MF, molecular process.(C) Western blot showing the expression of ANXA3 and ANXA6 in exosomes derived from control and force-primed PDLSCs.(D) Western blot showing the expression of ANXA3 and ANXA6 in control and force-primed PDLSCs. The same amount of exosomes from each group was used for the western blot. The results are representative of data generated from three independent experiments.Data are presented as the mean ± SD (**p < 0.01). See also Figure S2 and Table S1.

Fig 5: Recruitment of AnxA6 to the damaged membrane site and formation of the cap subdomain in LHCN myotube. (A) LHCN myotubes were transfected with the plasmid pA6-GFP and membrane damage experiments were performed by laser ablation at the lateral edge of the myotube. Red arrow, area before irradiation; white arrow, area after irradiation. Scale bar: 10 µm; (B) Magnified images of the disruption site before (left) and more than 65 s (right) after laser ablation. The dashed white line corresponds to the position of the plasma membrane before irradiation. Scale bar: 5 µm; (C) Distribution of endogenous AnxA6 in a damaged LHCN myotube. The plasma membrane of LHCN myotubes was damaged by laser irradiation. Myotubes were immunostained for AnxA6 and observed in bright-field (left) and fluorescence microscopy (right). White arrow, irradiated area. Scale bar: 5 µm; (D) Distribution of AnxA6 and dysferlin between cap and shoulder subdomains. A LHCN myotube expressing AnxA6-GFP (green) was damaged at the lateral edge and then fixed and immunostained for dysferlin (magenta). Red arrow, irradiated area (E) LHCN myotubes were transfected with the plasmid pA6-GFP and membrane damage experiments were performed by laser ablation on the surface of the myotube. Red arrow, area before irradiation; white arrow, area after irradiation. Scale bar: 20 µm.