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. 2021 Dec 29;221(2):e202104046. doi: 10.1083/jcb.202104046

Figure 2.

Figure 2.

WASP is recruited to membrane invaginations in a curvature-dependent manner. (A) Experimental strategy of plating cells on beads to induce sites of substrate-controlled plasma membrane curvature. (B) Time-lapse imaging reveals rapid recruitment of endogenous WASP to 500-nm bead-induced invaginations. Scale bar is 1 μm. (C) WASP consistently localizes to 200-nm bead-induced invaginations across the front half of the cell. Scale bar is 5 μm. See also Video 4. (D) WASP KI cells were plated on beads of varying diameter to assess the curvature sensitivity of WASP recruitment. Images are scaled to the same intensity for WASP. Below is a zoomed inset of the boxed regions. Scale bars are 5 μm and 1 μm for the inset. (E) Quantification of WASP signal at 100- and 200-nm beads reveals significantly more WASP at smaller invaginations (sites of higher curvature) when normalized to bead surface area. Mean WASP signal per unit area is 1.01 ± 0.19 a.u./nm2 for 100-nm beads and 0.34 ± 0.07 a.u./nm2 for 200-nm beads on the replicate level. n100 nm = 142 beads and n200 nm = 120 beads each collected across four experiments. Large markers denote the mean of each replicate, which were compared using an unpaired two-tailed t test (P = 0.016). (F) WASP enrichment to 500-nm beads is much stronger than that of other known curvature-sensitive proteins FBP17, clathrin LCa, and WAVE complex. Scale bar is 2 μm. (G) Pearson correlation coefficients for 1.5 × 1.5-μm regions of interest of a bead and the protein of interest. WASP has a significantly higher correlation with bead signal (r = 0.75 ± 0.02) compared with FBP17 (0.14 ± 0.05), clathrin LCa (−0.09 ± 0.04), and WAVE complex (0.19 ± 0.06). The correlation of each protein with beads was compared with the correlation of WASP with beads using an unpaired two-tailed t test. Asterisks mark significance. PWASP/FBP17 = 6.56E-18, PWASP/clathrin = 4.60E-24, and PWASP/WAVE = 5.58E-13. nWASP = 31, nFBP17 = 24, nclathrin = 21, and nWAVE = 31 beads each from one experiment. AU, arbitrary units.