Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair.
Bottom Line: Compared with wild-type mouse perivascular cells, AnxA5- cells exhibit a severe membrane repair defect.In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair.We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.
Affiliation: Molecular Imaging and NanoBioTechnology, IECB, UMR-5248 CBMN CNRS-University Bordeaux1-ENITAB, Talence F-33402, France.Show MeSH
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Mentions: Membrane damage was applied to both mouse PV cells3637, which express AnxA5 constitutively (wildtype (wt) PV), and to AnxA5-deficient PV cells in which the Anxa5 gene has been inactivated (AnxA5- PV)38 (Supplementary Fig. S1). To induce membrane damage and assess membrane repair, cells were submitted to infrared laser irradiation in the presence of FM1-43 dye, following established procedures11. FM1-43 is a water-soluble dye, which becomes fluorescent on inserting into lipid membranes, yet is unable to cross membranes; therefore, cells with damaged plasma membrane exhibit an increase in fluorescence intensity due to the passive entrance of FM1-43 molecules in the cytosol and their incorporation into intracellular membranes. Irradiation conditions were first adjusted to the minimal power (160 mW) causing conspicuous membrane injury to most wt-PV cells, as indicated by an increase in cytoplasmic fluorescence intensity and the presence of a large, μm-size, disruption at the irradiation site (Fig. 2a). The kinetics of fluorescence intensity changes show that fluorescence intensity increases for ∼90 s, and then reaches a plateau (Fig. 2b, filled circles). This stop in fluorescence increase indicates that cell membranes resealed, stopping further entrance of FM1-43. Membrane resealing correlates with the accumulation of vesicular lipid material near the disruption site (Fig. 2a, frames 3–4). As Ca2+, at mM concentration, is required for membrane repair4, we performed control experiments in the presence of the Ca2+-chelating agent EGTA. With EGTA, wt-PV cells showed a continuous and large increase in fluorescence intensity (Fig. 2c; Supplementary Fig. S2a), followed ultimately by cell death. This response is characteristic of the absence of membrane resealing. This preliminary set of results demonstrates that wt-PV cells possess a machinery that repairs membrane disruptions by means of a Ca2+-dependent mechanism involving the recruitment of intracellular vesicles to the site of membrane injury, in line with current knowledge on cell membrane repair134.
Affiliation: Molecular Imaging and NanoBioTechnology, IECB, UMR-5248 CBMN CNRS-University Bordeaux1-ENITAB, Talence F-33402, France.