Fig 2: ANXA4 induces membrane curvature in supported membranes. a Schematic of supported membrane model composed of primary and secondary membranes. Non-vesicular membrane patches with open edges were used in the subsequent experiments (POPC/POPS, 9:1 molar ratio). b Sequential images before and after addition of ANXA4-GFP protein to a membrane patch stained with DiD in the presence of Ca2+ (last image GFP channel) (also, see Supplementary Movie 2). c Similar experiment as in b but without POPS in the supported membrane. d, e Mutants of ANXA4 proteins including d Ca4Mut or e TrimMut. Note: TrimMut exhibits >10 times slower rolling activity. f Fluorescence and matching AFM image of membrane patch after completed rolling induced by ANXA4 and g AFM-topography graph of two colliding membrane rolls. h Schematic of ANXA4-induced membrane rolling including a numeric estimate of the number of turns N and layer spacing d. i Calculation of time constant (t) for membrane rolling and corresponding data for ANXA4. ANXA4-GFP shows similar rolling activity (data not shown). j Rolling time constants for ANXA4 and A4 ?N, respectively. Error bars represent S.D. for three–five independent experiments. P values based on t-test: *P = 0.05, ***P = 0.001

Fig 3: Proposed model for plasma membrane repair initiated by ANXA4 and ANXA6. a Binding of ANXA6 to wound edges induces local in-plane folding, which contracts the hole edges. b In uninjured cells, ANXA6 and ANXA4 are distributed uniformly as monomers in the cytoplasm. Upon local plasma membrane injury, Ca2+ influx results in recruitment of ANXA6 and ANXA4 to the membrane wound edges. ANXA6 initiates constriction of hole edges and may also cross-bridge patch vesicles translocated to the injury site. ANXA4 self-assembles into trimers that induce local out-of-plane curvature. The combined forces of constriction and curvature accelerate wound closure eventually leading to fusion of membrane edges. Vesicles recruited and fused to the wound edges contribute to repair by reducing wound size. The curvature structure triggered by ANXA4 can take several shapes including I round or II funnel-shaped or a more flat structure (not shown). Subsequent repair steps include local actin polymerization to fully restore the membrane after injury

Fig 4: ANXA4 counteracts plasma membrane injuries triggered by detergent, hypo-osmotic stress, and heat shock. a Immunofluorescence images showing endogenous ANXA4 protein localization in HeLa cells (white arrows) exposed to digitonin (20 µg/ml, 15 min), hypo-osmotic stress (25 mOsm/l, 10 min) or 47 °C heat shock (5 min) (scale bar, 20 µm). Plasma membrane integrity assays in cells overexpressing ANXA4-RFP or Ctrl-RFP and b treated with digitonin (20 µg/ml) in HeLa cells, or c MCF7 cells exposed to hypo-osmotic stress (25 mOsm/l) and assayed with impermeable Hoechst-33258. Also, see Supplementary Fig. 1. d Images showing FM1-43 dye uptake in MCF7 cells exposed to hypo-osmotic stress and e corresponding intensity plot. f Plasma membrane integrity upon 55 °C heat shock in MCF7 cells expressing ANXA4 wild type, or g Ca2+-binding-deficient mutants, h mutant lacking N terminus (aa 4-13) (?N). i Residues mutated in the Ca2Mut/Ca4Mut and the TrimMut ANXA4 mutants are indicated with yellow and pink sticks, respectively, on the crystal structure of ANXA4 (PDB entry code: 2zoc) displayed in transparent light blue cartoon with bound calcium ions as blue spheres. j ANXA4 (light blue) is structurally aligned on a subunit of homotrimer observed in the crystal structure of ANXA5 (transparent gray, PDB entry code: 1a8a) and the residues mutated in the ANXA4- TrimMut mutant displayed as pink spheres. The two structures are aligned to a root-mean-square deviation (RMSD) value of 0.942 Å over 302 Ca atoms (PyMOL Molecular Graphics System, Version 1.7.6.4, Schrödinger, LLC). k Membrane integrity upon 55 °C heat shock in MCF7 cells expressing ANXA4-TrimMut as compared to wtANXA4 and control. Means of three independent experiments measuring >40 cells for each condition. Error bars represent S.D. for three independent experiments. P values based on t-test: *P = 0.05, **P = 0.01, ***P = 0.001

Fig 5: ANXA4 is needed for repair in MCF7 cells. a Immunoblot of lysates from MCF7A4--CRISPR cells with disrupted ANXA4 gene expression. Hsc70 served as internal control for equal loading. b Plasma membrane repair kinetics upon laser injury measured by membrane impermeable FM1-43 dye influx in b CRISPR clones and in c MCF7A4--CRISPR (#1) cells expressing wild-type ANXA4-RFP, ANXA4 TrimMut-RFP or Ctrl-RFP. Error bars represent S.D. for at least eight independent cells per condition. P values based on t-test: *P = 0.05, **P = 0.01. d Sequential 3D images of MCF7A4---CRISPR (#1) with reintroduced ANXA4-RFP and exposed to a large injury by shooting through the cell by laser (injury site marked by white arrow). ANXA4-RFP translocation to wound edges and constriction is marked by yellow arrow (also, see Supplementary Movie 1). e Example of ANXA4-RFP-induced curvature and funnel formation (yellow arrow) created from the bottom of the plasma membrane after laser injury. f Trimer-deficient ANXA4-RFP mutant (A4-TrimMut) failed to induce curvature upon laser injury (see also Supplementary Movie 1)