Figure 4. Actin network disassembly in the rear of detergent-extracted keratocyte cytoskeletons is ATP-dependent and blebbistatin-sensitive, consistent with a direct role for myosin II in this process.

a–d, ATP triggers an acute loss of actin network in the rear region of the cell, where myosin II is localized (compare Fig. 1f). a, A detergent-extracted and phalloidin-labeled keratocyte cytoskeleton⁶. b, The same cytoskeleton 7 min after addition of 1 mM ATP. c, Overlay of initial frame (a, cyan) and frame at 7 min (b, yellow); regions with increase, decrease, or no change in net intensity appear yellow, cyan, or white, respectively. d, Time evolution of fluorescence intensities (normalized at t = 0) in the indicated regions. Time points for a mock buffer wash (chevron) and ATP addition (black arrowhead) are indicated. e–h, In a cell treated with 50 μM blebbistatin for 30 min prior to extraction, addition of ATP does not induce a loss of actin network. There is a slow loss of fluorescence due to photobleaching or background dissociation. i–l, The F-actin severing protein villin rapidly disassembles the lamellipodial actin network, demonstrating that this part of the cytoskeleton is not protected against a general disassembling activity. 0.1 μM GST-villin was added instead of ATP (arrow in l). m–t, Addition of GST-villin (arrows in p, t) in addition to ATP (arrowheads in p, t), in either order, results in complete, rapid disassembly of the actin network. Compare Supplementary Movie 4.