Arp2/3 complex and CP distribute homogeneously within the actin network during actin assembly. GST-pWA–coated beads were added to a motility medium containing 4 μM Alexa Fluor 568–actin monomers, 12 μM profilin, and (A) 210 nM Alexa Fluor 488–Arp2/3 complex, 25 nM CP, or (B) 150 nM Arp2/3 complex, 45 nM Alexa Fluor 488-CP. Images were taken at the indicated times during polymerization. Alexa Fluor 488 is colored in green and Alexa Fluor 568 in red. In (A) different motile beads were imaged after 30 min of actin assembly, and in (B) a montage of time-lapse images of a motile bead taken at 20-min intervals shows the comet tail formation. Line scans of the fluorescence intensities taken along the actin gel, as indicated by the dashed curves, are displayed under each corresponding image. For easier comparison in (A), we normalized the fluorescence by the integrated fluorescence of each image. Scale bar: 5 μm. (See also Movie S1.)

Kinetic modeling of ADF/cofilin recruitment within the comet tail. In silico reconstitution of the elongation of the Arp2/3-mediated actin branched network according to the kinetic model (Supplemental Data). (A and B) The graphs represent a montage of the evolution over time of the ratio of ATP- and ADP-loaded actin subunits within the simulated network assembled in the presence of 4 μM of profilin-actin complex, 25 nM CP, and ADF/cofilin as indicated. The ATP and ADP-P_i states of the nucleotide were incorporated into one, and named ATP. Simulations of the elongating comet tail were drawn and positioned with the front bead edge aligned with the origin of x-axis and the rear one with the interface bead/actin network. The color-coding of the comet tail corresponds to that of the curves. (B) Given the acceleration of P_i release on actin subunits by ADF/cofilin and the higher affinity for ADP-loaded subunits, the proportion of the ADP-loaded subunits diminished in favor of both ADP-ADF–loaded subunits and old ADP-ADF–loaded ones. The latter population accounted for severing by ADF/cofilin. (C) Superimposition of predicted ATP/ADP-P_i cap lengths (line) and experimental ADF/cofilin exclusion zones measured in Figure 2, A and D, respectively (dots); experimentally, the cap length, i.e., the zone of exclusion of ADF/cofilin, was represented by the red zone in the comet tail (Figure 2, A and D). (See also Movie S4.)

Evidence for the macroscopic stochastic dynamics of ADF/cofilin severing activity. (A) GST-pWA–coated beads were added to a motility medium containing 4 μM Alexa Fluor 568–actin monomers, 150 nM Arp2/3 complex, 25 nM CP, and 0.6 μM Alexa Fluor 488-ADF/cofilin under the same conditions as in Figure 2A. The dark blue arrowhead points to the motile bead and the light blue arrowhead follows the thick actin shell assembled prior to symmetry breaking and its fragmentation into small meshwork pieces. Alexa Fluor 568-actin is colored in red, and Alexa Fluor 488-ADF/cofilin in green. (B) is a magnification of the area outlined by a dashed box in (A). The signal in (B) corresponds to fluorescence of Alexa Fluor 568-actin only. Scale bar: 5μm. (See also Movie S6.)

ADF/cofilin is progressively recruited to the actin comet tail during reconstituted bead motility. GST-pWA–coated beads were added to a motility medium containing 12 μM profilin, 4 μM Alexa Fluor 568–actin monomers, 150 nM Arp2/3 complex, 25 nM CP, and (A) 0.3 μM Alexa Fluor 488–ADF/cofilin or (D) 0.6 μM Alexa Fluor 568–ADF/cofilin. Images were taken at the indicated times during polymerization and analyzed as in Figure 1. Blue arrowheads indicate the site of abundant binding of ADF/cofilin and its abrupt dissociation due to the subsequent actin network fragmentation. Scale bar: 5 μm. (B and E) A time course of motility rates during actin comet tail formation was measured. (C and F) Line scans of the fluorescence intensities taken along the actin network, as indicated by the dashed lines in D, were analyzed for the different times of acquisition (as in Figure 1). Graphs represent the ratio of the fluorescence intensity for ADF/cofilin to that of actin at different times. A zoom of the vicinity around the bead is presented, whereas the inset shows the entire ratio of the full actin network. (See also Movies S2 and S3.)

ADF/cofilin severs short actin filaments and creates new filament barbed ends during reconstituted bead motility. (A) Schematic representation of the photobleaching experiments of Alexa Fluor 488–labeled CP in a dense actin network nucleated from the pWA-coated beads in the absence of ADF/cofilin (left) and in the presence of ADF/cofilin (right). Once dissociated from the end of filaments according to its kinetic dissociation constant k₋ (limiting step), bleached CP is rapidly replaced by unbleached Alexa Fluor 488-CP diffusing from the surrounding medium (following a kinetic constant equal to its association constant rate k₊ multiplied by the protein concentration in the medium). Filaments are also damaged and fragmented by the laser light during the photobleaching process. These new artificial barbed ends are rapidly capped. In the presence of ADF/cofilin, filaments are further fragmented, leading to the rapid recruitment of CP. (B and C) Time-lapse images of FRAP experiments of Alexa Fluor 488-CP on the propulsive actin network nucleated from GST-pWA–coated beads in the absence (A) or presence (B) of 0.3 μM Alexa Fluor 568-ADF/cofilin under conditions similar to Figure 2A. Images were taken at the indicated times during polymerization. Time-lapse images were acquired by fluorescence microscopy. The photobleached zones, indicated by a dashed rectangle on the images, were obtained by a local intense laser illumination. For a better visualization of CP fluorescence intensity, images are also shown in pseudocolor. Scale bar: 5 μm. (D) Graph representing the time course of fluorescence intensity changes for Alexa Fluor 488-labeled CP within the bleached zones. For an easier comparison, data were normalized by fluorescence intensity prior to bleaching. Blue, data corresponding to the experiment shown in (B) in the absence of ADF/cofilin; red, those corresponding to experiment (C) in the presence of ADF/cofilin. Means and statistics obtained for the kinetics of the fluorescence recovery after photobleaching of Alexa Fluor 488-CP in different experiments are presented. Curve fits were obtained using a double exponential function in KaleidaGraph. The dissociation constant was fixed to the mean experimental value found (k₋ = 0.00155 s⁻¹) in order to solve the other kinetic constant k_obs, therefore permitting the calculation of the half-time t_1/2 and the free concentration of CP in the medium (using k_+th = 3 μM⁻¹·s⁻¹). Data are represented as mean ± SE. (See also Movie S5.)