Superresolution imaging reveals nanoscale reorganization of WASP by membrane geometry. (A) Live-cell 3D STED microscopy of the membrane (labeled with CellMask Deep Red) coupled with confocal imaging of WASP on 500-nm beads show that WASP enriches to the site of highest inward curvature, either across the whole bud (top) or at an invagination neck when present (bottom). Right-hand side depicts the center of intensity of WASP for these two cases, with n_{500nm, no neck} = 17 and n_{500nm, neck} = 11 invaginations collected across eight and four cells, respectively, from the same experiment. Scale bar is 1 μm. (B) WASP enriches to the entire bud at smaller, 200-nm bead-induced invaginations, as reflected in the center of intensity of WASP to the right. n_200nm = 29 invaginations collected across nine cells from one experiment. Scale bar is 1 μm. (C) Time-lapse 3D STED imaging of the membrane reveals evolution from an open- to closed-neck structure at sites of bead-induced invagination. As this occurs, WASP redistributes and moves from the bead surface to the invagination neck. Scale bar is 500 nm. (D) As the membrane closes down around a bead, total WASP levels significantly increase. Mean normalized WASP intensity is 0.42 ± 0.08 a.u. at the first frame of the movie compared with 0.82 ± 0.04 a.u. at the time of neck closing on the single-invagination level. A paired two-tailed t test was used to compare the normalized intensity of WASP at these time points from nine invaginations collected across three experiments (P = 0.001). (E) 2D STED imaging of the cell membrane reveals both open and closed invaginations. At open invaginations, WASP coats the base (white box). As invaginations close down, WASP is reorganized into a focal accumulation at the neck (gray box). Scale bars are 2 μm and 500 nm in the inset. See Video 5 for the dynamics of this process using 2D STED. (F) Model for WASP evolution as the membrane of a cell constricts around a bead.