Figure 1.
Endogenous WASP forms puncta stabilized by cell-substrate interactions.(A) TIRF microscopy of endogenous WASP reveals localization to the tip of the lamellipod and to ventral puncta throughout the front half of the cell and to a lesser extent the uropod. Color map is inverted. Each frame is 30 s. See also Video 1. (B) Kymograph along the line in A highlights the stationary nature of WASP puncta relative to the substrate. Scale bar is 2 μm. (C) Endogenous WASP and endogenous clathrin LCa fail to colocalize. Color map is inverted for single-channel images. See also Video 2. (D) Spatial distribution of WASP (green) and clathrin (magenta) puncta and their corresponding line scans confirm consistent spatial separation of these proteins in coexpressing cells. n = 11 cells collected across three experiments with >1,000 puncta measured for each marker. Mean relative position of WASP is 0.71 ± 0.01 and mean relative position of clathrin is 0.29 ± 0.02 on the single-cell level. P = 8.82E-10 by a paired two-tailed t test on the mean puncta position of each marker in each cell. (E) Treatment of cells with EDTA, a calcium and magnesium chelator that blocks integrin-based adhesion, causes normally stationary WASP puncta to experience retrograde flow relative to the substrate. Left depicts a single frame of endogenous WASP with a line used to construct the kymograph to the right. See also Video 3. (F) Spatial distribution of the position of disappearance of WASP puncta reveals earlier extinction in the presence of EDTA. n = 12 cells per condition collected across three experiments with ∼300 puncta measured for each condition. Mean relative position of puncta disappearance is 0.55 ± 0.03 in the absence of EDTA and 0.66 ± 0.02 in the presence of EDTA on the single-cell level. P = 0.005 by an unpaired two-tailed t test on the mean position of puncta disappearance for each cell. For relative positions, the cell rear is defined as 0 and the cell front as 1. Scale bars are 5 μm unless otherwise specified. max, maximum; min, minimum.

Endogenous WASP forms puncta stabilized by cell-substrate interactions. (A) TIRF microscopy of endogenous WASP reveals localization to the tip of the lamellipod and to ventral puncta throughout the front half of the cell and to a lesser extent the uropod. Color map is inverted. Each frame is 30 s. See also Video 1. (B) Kymograph along the line in A highlights the stationary nature of WASP puncta relative to the substrate. Scale bar is 2 μm. (C) Endogenous WASP and endogenous clathrin LCa fail to colocalize. Color map is inverted for single-channel images. See also Video 2. (D) Spatial distribution of WASP (green) and clathrin (magenta) puncta and their corresponding line scans confirm consistent spatial separation of these proteins in coexpressing cells. n = 11 cells collected across three experiments with >1,000 puncta measured for each marker. Mean relative position of WASP is 0.71 ± 0.01 and mean relative position of clathrin is 0.29 ± 0.02 on the single-cell level. P = 8.82E-10 by a paired two-tailed t test on the mean puncta position of each marker in each cell. (E) Treatment of cells with EDTA, a calcium and magnesium chelator that blocks integrin-based adhesion, causes normally stationary WASP puncta to experience retrograde flow relative to the substrate. Left depicts a single frame of endogenous WASP with a line used to construct the kymograph to the right. See also Video 3. (F) Spatial distribution of the position of disappearance of WASP puncta reveals earlier extinction in the presence of EDTA. n = 12 cells per condition collected across three experiments with ∼300 puncta measured for each condition. Mean relative position of puncta disappearance is 0.55 ± 0.03 in the absence of EDTA and 0.66 ± 0.02 in the presence of EDTA on the single-cell level. P = 0.005 by an unpaired two-tailed t test on the mean position of puncta disappearance for each cell. For relative positions, the cell rear is defined as 0 and the cell front as 1. Scale bars are 5 μm unless otherwise specified. max, maximum; min, minimum.

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