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. 2017 Jan 1;28(1):98–110. doi: 10.1091/mbc.E16-04-0228

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© 2017 Buck et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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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 |v_flow| 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 |v_flow| in 50 μM CK666. (F) Bead-velocity classification scheme. Beads are classified as being flow coupled when the flow-subtracted bead/inductopodial speed |v_ind| is below a threshold of 2 μm/min (blue), or when |v_ind| is above threshold, as retrograde, lateral, or anterograde (purple, yellow, or green, respectively) according to flow-subtracted bead-velocity angles relative to flow, θ_rel (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 v_flow in black, uncorrected bead velocity v_tot in white, and flow-corrected bead/inductopodial velocity v_ind 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. n_gc = 9 paired growth cones; total number of beads n_b = 592 (control), 247 (20 mM CK-666), and 342 (50 mM CK 666).