(A and B) Rigid lipid nanotubes assembled by ezrin: representative cryo-electron micrographs of ezrinTD inducing randomly-oriented nanotube assembly, named disordered (A) and ezrinWT inducing parallel stacks, named stacked (B). Arrows indicate some of the nanotubes in both cases. Scale bars: 50 nm. The black dots are gold particles. Protein concentrations: 1.2 μM for both ezrinTD and ezrinWT. Nanotube concentration: 0.1 g.L−1. (C) Percentages of membrane nanotubes being disordered, forming stacks or being isolated in the presence of ezrinTD or ezrinWT, deduced from cryo-EM. Numbers of tubes measured are N = 60 and N = 62 from two distinct sample preparations, for ezrinTD and ezrinWT, respectively. Statistic test (chi-square test): for stacked tubes (****) and for isolated tubes (*). (D) Absence of curvature-induced sorting of PIP2 measured using two types of fluorescently-labeled PIP2 (TopFluor PIP2 (N = 14 GUVs, n = 1 sample preparation) and TMR PIP2 (N = 23 GUVs, n = 2 sample preparations)). S: sorting ratio. R: membrane tube radius. (E) and (F) Nanotube pulling assay to probe ezrin positive membrane curvature sensing. (Left) Representative confocal images of ezrinWT (E) and ezrinTD (F) binding on a membrane nanotube pulled from a GUV aspirated in a micropipette. The nanotube is held by a bead (not fluorescent) that is trapped by optical tweezers. Scale bars, 5 μm. (Right) Sorting ratio, S, as a function of tube radius, R, for ezrinWT (E) and ezrinTD (F). φV: ezrin membrane surface fraction. Blue lines indicate S = 1. In (E), measurements were collected from N = 13 GUVs, n > 3 independent sample preparations. In (F), measurements were collected from N = 63 GUVs, n > 3 independent sample preparations. Error bars indicate standard deviations, circles are mean and X symbols are maximum and minimum for each condition, excluding S = 0 data. (G) Sorting of the FERM domain at different membrane tube radii. φV: membrane surface fraction of the FERM domain on the GUVs. S: sorting ratio. R: membrane tube radius. Measurements were collected from N = 5 GUVs, n = 2 sample preparations.