Figure 4.
WAVE complex rings associate with plasma membrane saddle points. (A) TIRF-SIM imaging of the WAVE complex (Hem1-EGFP; cyan) reveals rings localized at the boundary where the plasma membrane (DiD-labeled; magenta) exits the TIRF-SIM plane. Right: Insets and a graph of a linescan across a ring. Scale bars: 5 µm and 500 nm (insets). See Video 3. (B) Schematic of the proposed organization of the WAVE complex ring structure (green) around the neck of a plasma membrane invagination (magenta) as seen from the top, x-y plane (left) or the side, x-z plane (right). (C) Transmission electron micrographs of a cross-section of dHL60s treated with latrunculin B (500 nM). Red arrows point to membrane invaginations. Insets (i and ii) shown below. Scale bars: 500 nm (top) and 200 nm (insets). Graph comparing the number of membrane invaginations per serial section of cells treated with or without F-actin inhibitor (500 nM latrunculin B). Graph shows mean ± SEM of the number of invaginations per cell; (+) F-actin inhibitor n = 37 cells, (−) F-actin inhibitor n = 18 cells; Mann–Whitney test with two-tailed; ****, P < 0.0001. (D) Graph comparing the length across the neck of invaginations by electron microscopy (EM) to the inner diameter of the fluorescent Hem1 rings of dHL60s treated with latrunculin B (500 nM). Schematic depicts where measurements were made. Graph shows mean ± SEM; EM of neck invaginations n = 190 from 38 cells, fluorescent ring inner diameter n = 222 from 4 cells; Mann–Whitney test with two-tailed; ns, not significant; P = ∼0.39 > 0.05. This finding supports the possibility that the WAVE complex enriches around the necks of membrane invaginations. (E) Schematic of a plasma membrane invagination’s saddle curvature. The necks of invaginations display saddle geometry of positive curvature in one axis (orange; the curve around the invagination neck) and negative curvature in the other axis (blue; the curve perpendicular to the invagination neck). (F) Curvature comparison of the necks of invaginations and tips of protrusions. Electron micrographs of dHL60s treated with F-actin inhibitor (top; 500 nM latrunculin B) and dHL60s treated with chemoattractant (bottom; 100 nM fMLP) show that the curvature at the necks of invaginations and the tips of protrusions are similar. Graph shows mean ± SEM; invaginations n = 190 from 28 cells, protrusions n = 59 from 19 cells; unpaired t test, two-tailed; ns, not significant; P = 0.074 > 0.05. Curvature, κ, defined as 1/r where r is the radius. Scale bar: 100 nm.

WAVE complex rings associate with plasma membrane saddle points. (A) TIRF-SIM imaging of the WAVE complex (Hem1-EGFP; cyan) reveals rings localized at the boundary where the plasma membrane (DiD-labeled; magenta) exits the TIRF-SIM plane. Right: Insets and a graph of a linescan across a ring. Scale bars: 5 µm and 500 nm (insets). See Video 3. (B) Schematic of the proposed organization of the WAVE complex ring structure (green) around the neck of a plasma membrane invagination (magenta) as seen from the top, x-y plane (left) or the side, x-z plane (right). (C) Transmission electron micrographs of a cross-section of dHL60s treated with latrunculin B (500 nM). Red arrows point to membrane invaginations. Insets (i and ii) shown below. Scale bars: 500 nm (top) and 200 nm (insets). Graph comparing the number of membrane invaginations per serial section of cells treated with or without F-actin inhibitor (500 nM latrunculin B). Graph shows mean ± SEM of the number of invaginations per cell; (+) F-actin inhibitor n = 37 cells, (−) F-actin inhibitor n = 18 cells; Mann–Whitney test with two-tailed; ****, P < 0.0001. (D) Graph comparing the length across the neck of invaginations by electron microscopy (EM) to the inner diameter of the fluorescent Hem1 rings of dHL60s treated with latrunculin B (500 nM). Schematic depicts where measurements were made. Graph shows mean ± SEM; EM of neck invaginations n = 190 from 38 cells, fluorescent ring inner diameter n = 222 from 4 cells; Mann–Whitney test with two-tailed; ns, not significant; P = ∼0.39 > 0.05. This finding supports the possibility that the WAVE complex enriches around the necks of membrane invaginations. (E) Schematic of a plasma membrane invagination’s saddle curvature. The necks of invaginations display saddle geometry of positive curvature in one axis (orange; the curve around the invagination neck) and negative curvature in the other axis (blue; the curve perpendicular to the invagination neck). (F) Curvature comparison of the necks of invaginations and tips of protrusions. Electron micrographs of dHL60s treated with F-actin inhibitor (top; 500 nM latrunculin B) and dHL60s treated with chemoattractant (bottom; 100 nM fMLP) show that the curvature at the necks of invaginations and the tips of protrusions are similar. Graph shows mean ± SEM; invaginations n = 190 from 28 cells, protrusions n = 59 from 19 cells; unpaired t test, two-tailed; ns, not significant; P = 0.074 > 0.05. Curvature, κ, defined as 1/r where r is the radius. Scale bar: 100 nm.

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