FIGURE 1:
Site-directed actin assembly drives inductopodial motility and requires Arp2/3 complex activity. (A) Inductopodia are induced by unrestrained beads derivatized with anti-apCAM (4E8) antibody landing on the growth cone. Growth cone domains: P, P domain; T, T zone; C, C domain. (B) DIC image (left, cf. Supplemental Movie 1) taken immediately before fixation and confocal phalloidin-stained fluorescent image (right) showing the actin tail of an inductopodium (bracket and green arrow; bead positions in B and C: green circles). Direction of inductopodial movement is indicated by white arrow. (C) Multiple inductopodia with F-actin visualized with phalloidin (left) and Arp2/3 complex with p34Arc antibody (right). (D) 4E8 beads were applied to a growth cone in control conditions (cf. Supplemental Movie 2). Arbitrarily colored bead tracks are shown superimposed over DIC image of the growth cone. Scale bar: 5 μm. Inset, Retrograde flow shown as a color map of flow speed |vflow| calculated from flow-coupled beads only, with arrows indicating flow direction. (E) Bead tracks for the same growth cone in the presence of 50 μM CK666 (cf. Supplemental Movie 2). Inset, Map of |vflow| in 50 μM CK666. (F) Bead-velocity classification scheme. Beads are classified as being flow coupled when the flow-subtracted bead/inductopodial speed |vind| is below a threshold of 2 μm/min (blue), or when |vind| is above threshold, as retrograde, lateral, or anterograde (purple, yellow, or green, respectively) according to flow-subtracted bead-velocity angles relative to flow, θrel (light blue arrow). The latter three classifications can be grouped into a nonflow or “propulsive” velocity classification category (red). (G) Track corresponding to the dotted white track indicated by arrow in D with color coding for classified motility states. Local flow direction is indicated by black arrows. Scale bar: 1 μm. (H) Plot of observed positions, bead velocities (white arrows), and flow-corrected velocities (red arrows) of the initial portion of the track (orange box in G) shown at 5 s intervals with color-coded motility classification (circles). Velocities are also color coded: flow velocity vflow in black, uncorrected bead velocity vtot in white, and flow-corrected bead/inductopodial velocity vind in red. Scale bar: 1 μm. (I) Kinematic analysis of bead track shown in G and H. Kymograph of bead position relative to leading edge (dotted line) vs. time (top panel) showing color-coded motility states, with retrograde flow (solid black lines); velocity vs. time graph (bottom panel) with uncorrected (white) and flow-corrected (red) speed traces. (J) The P domain of each growth cone was subdivided into four annular zones for analysis of motility (cf. Supplemental Figure S1). Probabilities of the four velocity states in the front one-fourth (zone 1) or rear three-fourths (zones 2/3/4) of the P domain of the growth cone in D and E are shown as stacked histograms in control vs. 50 μM CK666 conditions. (K) Changes in flow-coupled (blue) vs. propulsive (red) state probabilities resulting from Arp2/3 complex inhibition are shown for multiple growth cones. Each line represents paired probabilities for control vs. CK666; solid lines, experiments in which [CK666] = 50 μM; dashed lines, [CK666] = 20 μM. ngc = 9 paired growth cones; total number of beads nb = 592 (control), 247 (20 mM CK-666), and 342 (50 mM CK 666).