Figure S3.
The WAVE complex enriches at flattening membrane regions. (A) WAVE complex signal enrichment versus membrane edge velocity in a migrating dHL60 cell. From left to right: Image of the membrane (CellMask DeepRed) at a single time point, image of the WAVE complex (Hem1-EGFP) at a single time point, successive cell outlines of the leading edge over time, WAVE complex localization (solid colors) over time, and graph of normalized WAVE complex signal enrichment versus membrane edge velocity of the entire membrane edge across all time points of the displayed cell (Spearman’s correlation ρ = 0.613). TIRF-SIM images; scale bar: 5 µm. (B) WAVE complex signal enrichment versus membrane edge velocity in a closing TEM. Images (top row): HUVEC cell expressing WAVE complex-EGFP (Nap1-EGFP) treated with Rho-associated protein kinase inhibitor Y27632 (50 µM) to generate TEMs. Left: membrane (CellMask DeepRed). Middle: Inset of membrane channel. Right: Inset of WAVE complex signal. Spinning-disk confocal imaging; scale bars: 40 µm and 5 µm (insets). Left graph: Area of TEM (orange) and the average WAVE complex fluorescence signal (units above background; green) over time (seconds). Right graph: Normalized WAVE complex signal enrichment versus membrane edge velocity at all time points across closure of the displayed transendothelial macroaperture (Spearman’s correlation ρ = 0.556). (C) The WAVE complex fold enrichment at flattening over lagging regions at different image rotations. “Flattening” is defined as the membrane becoming less positively curved (toward flat or negatively curved), “lagging” is defined as the membrane becoming more positively curved, and “rotation” refers to the degree to which the WAVE complex signal is rotated around the TEM. At each rotation, the fold enrichment of WAVE complex (blue) or membrane (orange) signal in flattening over lagging regions is calculated. The WAVE complex peak at zero rotation indicates that the highest enrichment was due to the colocalization of the curvature and fluorescent features of interest in the unrotated configuration, indicating a true correlation. The average ± SD of the enrichment scores for n = 3 TEMs were compared with the null value of 1 (no preference between flattening and lagging) using a two-tailed, nonpaired t test at each rotation value. A particular rotation was marked significant with a star if P < 0.05. avg, average; fluor., fluorescence.

The WAVE complex enriches at flattening membrane regions. (A) WAVE complex signal enrichment versus membrane edge velocity in a migrating dHL60 cell. From left to right: Image of the membrane (CellMask DeepRed) at a single time point, image of the WAVE complex (Hem1-EGFP) at a single time point, successive cell outlines of the leading edge over time, WAVE complex localization (solid colors) over time, and graph of normalized WAVE complex signal enrichment versus membrane edge velocity of the entire membrane edge across all time points of the displayed cell (Spearman’s correlation ρ = 0.613). TIRF-SIM images; scale bar: 5 µm. (B) WAVE complex signal enrichment versus membrane edge velocity in a closing TEM. Images (top row): HUVEC cell expressing WAVE complex-EGFP (Nap1-EGFP) treated with Rho-associated protein kinase inhibitor Y27632 (50 µM) to generate TEMs. Left: membrane (CellMask DeepRed). Middle: Inset of membrane channel. Right: Inset of WAVE complex signal. Spinning-disk confocal imaging; scale bars: 40 µm and 5 µm (insets). Left graph: Area of TEM (orange) and the average WAVE complex fluorescence signal (units above background; green) over time (seconds). Right graph: Normalized WAVE complex signal enrichment versus membrane edge velocity at all time points across closure of the displayed transendothelial macroaperture (Spearman’s correlation ρ = 0.556). (C) The WAVE complex fold enrichment at flattening over lagging regions at different image rotations. “Flattening” is defined as the membrane becoming less positively curved (toward flat or negatively curved), “lagging” is defined as the membrane becoming more positively curved, and “rotation” refers to the degree to which the WAVE complex signal is rotated around the TEM. At each rotation, the fold enrichment of WAVE complex (blue) or membrane (orange) signal in flattening over lagging regions is calculated. The WAVE complex peak at zero rotation indicates that the highest enrichment was due to the colocalization of the curvature and fluorescent features of interest in the unrotated configuration, indicating a true correlation. The average ± SD of the enrichment scores for n = 3 TEMs were compared with the null value of 1 (no preference between flattening and lagging) using a two-tailed, nonpaired t test at each rotation value. A particular rotation was marked significant with a star if P < 0.05. avg, average; fluor., fluorescence.

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