Figure 2. WAVE complex forms 230-nm... | Rockefeller University Press

Figure 2.

$WAVE complex forms 230-nm rings in the absence of the actin cytoskeleton. (A) Models of how the WAVE complex could achieve its linear organization. Left: The WAVE complex could achieve its linear organization at the lamellipodial edge in a manner that is dependent on its interactions with the actin cytoskeleton (left) or that is established independently of the actin cytoskeleton (right). Right: Addition of F-actin inhibitor can be used to distinguish these models. (B) Super-resolution TIRF-SIM imaging reveals that the WAVE complex forms rings in the absence of actin polymer. dHL60 cells expressing Hem1-EGFP were acutely stimulated with DMSO (top row) or F-actin inhibitor (+ F-actin inh.; 5 µM latrunculin A final, middle and bottom rows) at 0 s. Bottom row shows the white boxed insets. Scale bars: 5 µm (top) and 1 µm (insets). See Video 2. (C) Conventional TIRF resolution comparison highlights the need for super-resolution microscopy to resolve diffraction-limited WAVE complex puncta as rings. Conventional resolution TIRF images were created by sum-projecting the nine images (three phases × three angles) that construct the TIRF-SIM images of B. Scale bar: 1 µm. See Video 2. (D) The WAVE complex forms stereotypical 230 nanometer sized rings. Left, example measurement of fitting the ring’s perimeter to a perfect circle and calculating the circle’s diameter, d. Scale bar: 500 nm. Right, histogram of diameters of rings across a range of F-actin inhibiting drugs and concentrations, n = 2,418 rings from 103 cells, mode = 230 nm, median = 270 nm (see Fig. S2, A and B).$

WAVE complex forms 230-nm rings in the absence of the actin cytoskeleton. (A) Models of how the WAVE complex could achieve its linear organization. Left: The WAVE complex could achieve its linear organization at the lamellipodial edge in a manner that is dependent on its interactions with the actin cytoskeleton (left) or that is established independently of the actin cytoskeleton (right). Right: Addition of F-actin inhibitor can be used to distinguish these models. (B) Super-resolution TIRF-SIM imaging reveals that the WAVE complex forms rings in the absence of actin polymer. dHL60 cells expressing Hem1-EGFP were acutely stimulated with DMSO (top row) or F-actin inhibitor (+ F-actin inh.; 5 µM latrunculin A final, middle and bottom rows) at 0 s. Bottom row shows the white boxed insets. Scale bars: 5 µm (top) and 1 µm (insets). See Video 2. (C) Conventional TIRF resolution comparison highlights the need for super-resolution microscopy to resolve diffraction-limited WAVE complex puncta as rings. Conventional resolution TIRF images were created by sum-projecting the nine images (three phases × three angles) that construct the TIRF-SIM images of B. Scale bar: 1 µm. See Video 2. (D) The WAVE complex forms stereotypical 230 nanometer sized rings. Left, example measurement of fitting the ring’s perimeter to a perfect circle and calculating the circle’s diameter, d. Scale bar: 500 nm. Right, histogram of diameters of rings across a range of F-actin inhibiting drugs and concentrations, n = 2,418 rings from 103 cells, mode = 230 nm, median = 270 nm (see Fig. S2, A and B).