Figure 5.
WAVE complex enriches to TEM tunnel saddle points. (A) Schematic of a TEM closure and saddle geometry. Right: ChimeraX renderings of spinning-disk confocal imaging of a HUVEC cell that was treated with Y27632 (50 µM) to induce TEMs, labeled with a membrane dye (CellMask DeepRed), and then fixed with paraformaldehyde. Top: Tilted view. Bottom: View from inside the cell facing a TEM and its saddle geometry. Scale bars: 5 µm (top) and 2 µm (bottom). (B) The WAVE complex localizes to TEMs. A HUVEC cell expressing EGFP-tagged Nap1, a WAVE complex subunit (cyan), and stained with membrane dye CellMask DeepRed (magenta) was treated with Y27632 (50 µM). Spinning-disk confocal imaging, scale bar: 2 µm. Left graph: Linescan across the TEM; WAVE complex (cyan) and membrane dye (magenta). Right graph: Violin plot of enrichment, which was measured as the ratio between the signal intensity per unit area at the TEMs compared with the background intensity per unit area. A value of 1 indicates no enrichment at the TEM (dotted line), and a value >1 indicates enrichment. Each time point throughout TEM closure was considered as a single data point; WAVE complex and membrane both had n = 164 from eight TEMs from at least three independent experiments per condition. (C) Schematic of different potential molecular behaviors during TEM closure. As TEMs close, the local concentration (signal per unit length) could increase, remain constant, or decrease. A saddle geometry sensor would increase its local concentration until the TEM closure reaches the sensor’s preferred positive radius of curvature. (D) Time-lapse images of the WAVE complex (top) and the membrane (bottom) as a TEM closes. Images in each set have the same intensity scale in a lookup table that eases the visualization of signal enrichment. The dotted line outlines the TEM membrane mask. Spinning-disk confocal imaging; time in seconds; scale bar: 2 µm. See Video 4. (E) WAVE complex enriches to closing TEMs. Left: Graph of the WAVE complex (circles) and membrane (triangles) signal per unit perimeter as a TEM closes. Each color represents a single TEM over time. The WAVE complex shows higher enrichment at smaller (more positively curved) TEMs, suggesting a preference for membrane saddles with high positive curvature. Right: Graph of mean ± SEM of the Spearman’s ρ correlation coefficient of each TEM shown on the left graph; the WAVE complex and membrane each had n = 8 TEMs from at least three independent experiments; unpaired t test, two-tailed; **, P = 0.0012 < 0.0021.

WAVE complex enriches to TEM tunnel saddle points. (A) Schematic of a TEM closure and saddle geometry. Right: ChimeraX renderings of spinning-disk confocal imaging of a HUVEC cell that was treated with Y27632 (50 µM) to induce TEMs, labeled with a membrane dye (CellMask DeepRed), and then fixed with paraformaldehyde. Top: Tilted view. Bottom: View from inside the cell facing a TEM and its saddle geometry. Scale bars: 5 µm (top) and 2 µm (bottom). (B) The WAVE complex localizes to TEMs. A HUVEC cell expressing EGFP-tagged Nap1, a WAVE complex subunit (cyan), and stained with membrane dye CellMask DeepRed (magenta) was treated with Y27632 (50 µM). Spinning-disk confocal imaging, scale bar: 2 µm. Left graph: Linescan across the TEM; WAVE complex (cyan) and membrane dye (magenta). Right graph: Violin plot of enrichment, which was measured as the ratio between the signal intensity per unit area at the TEMs compared with the background intensity per unit area. A value of 1 indicates no enrichment at the TEM (dotted line), and a value >1 indicates enrichment. Each time point throughout TEM closure was considered as a single data point; WAVE complex and membrane both had n = 164 from eight TEMs from at least three independent experiments per condition. (C) Schematic of different potential molecular behaviors during TEM closure. As TEMs close, the local concentration (signal per unit length) could increase, remain constant, or decrease. A saddle geometry sensor would increase its local concentration until the TEM closure reaches the sensor’s preferred positive radius of curvature. (D) Time-lapse images of the WAVE complex (top) and the membrane (bottom) as a TEM closes. Images in each set have the same intensity scale in a lookup table that eases the visualization of signal enrichment. The dotted line outlines the TEM membrane mask. Spinning-disk confocal imaging; time in seconds; scale bar: 2 µm. See Video 4. (E) WAVE complex enriches to closing TEMs. Left: Graph of the WAVE complex (circles) and membrane (triangles) signal per unit perimeter as a TEM closes. Each color represents a single TEM over time. The WAVE complex shows higher enrichment at smaller (more positively curved) TEMs, suggesting a preference for membrane saddles with high positive curvature. Right: Graph of mean ± SEM of the Spearman’s ρ correlation coefficient of each TEM shown on the left graph; the WAVE complex and membrane each had n = 8 TEMs from at least three independent experiments; unpaired t test, two-tailed; **, P = 0.0012 < 0.0021.

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