Actin filament clearance in the P-domain precedes C-domain organelle engorgement. (A) Actin filament FSM dynamics (top panel) and corresponding DIC (bottom panel) time-lapse sequence of a targeted growth response. Dashed green line denotes actin filament flow and yellow dashed lines mark the extending C-domain organelle boundary. Red arrow heads denote clearance of actin bundles from growth corridor. Green arrow denotes C-domain associated actin structures. (B) Actin filament FSM time-lapse sequence and corresponding (C) flow vector maps of a targeted growth response from white rectangle region of interest shown in the inset of Figure 1B. Red hatched line marks filopodial turnover in the T-zone. White dashed region (C) shows an area with actin structure and strong flow attenuation during the growth phase. (D) Graphs plot the average speed and direction of flow over time in the target interaction corridor in the P-domain (red box, inset) and C-domain (green box, inset), respectively. Blue line and diamonds indicate the net direction of movement where 0° represents retrograde flow and 180°, flow towards bead site. Purple line and filled squares denote flow speed in the retrograde direction, whereas open purple squares designate anterograde flow speed towards adhesion site. Arrowheads in (D) denote the time interval shown in (B and C). Turquoise circles in (A, B, and C) mark bead site. Scale bar, 10 μm. The regions of interest in (A) and (B) are 27×5.4 and 22×3 μm respectively.

(A) DIC (top panel) and corresponding MT FSM (bottom panel) time-lapse images of a growth cone 420 seconds after placement and restraint of an apCAM coated target bead. (B) DIC (top panel) and MT FSM (bottom panel) images of the same growth cone later in time during the growth phase. Dashed white lines outline the C-domain organelle boundary and turquoise circles denote the bead site. (C) Kymograph analysis of the region of interest marked by yellow line in (A); DIC channel (top panel) and MT channel (bottom panel). In (C): yellow dashed lines mark the organelle boundary; green arrow heads highlight dynamic MTs interrogating the target interaction site during the latency period; and red dashed line denotes the initial needle position. The needle is pulled away from the growth cone at the 720 second time point. (D) DIC (top) and MT (bottom) time-lapse montages of the target interaction corridor. White dashed line marks organelle boundary. The initial position of the bead is marked by red line with arrow showing displacement of bead and needle towards the C-domain during the targeting response. (E) and (F) show time-lapse montages of single MTs from the interaction corridor. Green arrows identify the terminal speckles of the growing MTs while the solid yellow lines mark internal reference speckles. Red arrow in (F) identifies a MT shortening event. Yellow arrow head in (E) marks un-looping MT. Scale bar, 10 μm.

(A) Actin (left panel), MTs (middle panel), and actin/MT overlay (right panel) images showing region of interest from inset. Yellow arrow shows co-alignment of arcs and MTs. (B) Time-lapse sequence of a region of interest from inset showing actin channel/ flow vector map (left column) and MT/ flow vector map (right column). (C) Graph shows average direction over time for actin filaments (red diamonds) and MTs (green circles) from the white hatched region of interest in the inset. 90° represents retrograde flow and 180°, flow towards bead site. Double black arrows show time interval from (B). (D) Plots show the DCS (blue circles) and VMCS (purple squares) between the actin and MT speckle features from the same region of interest as (C). A score of one for the coherency and coupling measurements defines vector populations with identical directionality or velocity. Scale bar, 10 μm.

Cytoskeletal response underlying substrate evoked growth. A) The “latency” phase begins after bead restraint and is characterized by localized actin assembly near the bead binding site which acts as a scaffold for docking signaling proteins. During this time, filopodial actin bundles (red) and the less polarized actin network (pink) move by fast retrograde flow in the P domain (black arrow). Dynamic MTs (green) enter the P domain by polymerizing parallel to filopodia. MTs are simultaneously carried rearward by tight coupling to retrograde flow and do not accumulate at the nascent adhesion site. MTs in the C-domain (yellow) are restricted by interactions with actomyosin arcs (blue). B) The start of the “growth phase” is marked by strengthening of the adhesion site and engagement of the molecular clutch. Flow slows in the interaction corridor (blue zone) and is accompanied by actin filament restructuring and continued actin recycling. This process removes a barrier to C-domain (outlined gray zone) advance towards the adhesion site. C) Later, tension develops (red arrows) between the restrained bead substrate and contractile arcs, causing arcs to straighten and compress MTs into the C-domain (blue arrows). We hypothesize this action facilitates MT bundling and subsequent cross-linking necessary for consolidation of a new segment of the axon shaft.

Left column shows (A) DIC, as well as corresponding (B) actin filament FSM and (C) actin filament flow vector map images of a growth cone for a time-point 280 seconds after placement and restraint of an apCAM coated target bead. White arrowheads in (A) demarks intrapodia. Double headed arrow in (A) indicates target interaction axis. Right column shows (D) DIC, (E) actin filament FSM, and (F) flow vector map images of the same growth cone 610 seconds after bead restraint following the C-domain growth response. Dashed white lines in (A and D) outline the C-domain organelle boundary. Star marks filopodial actin rib in P-domain and green arrow head filopodial root in T-zone (B and E). Yellow arrowheads in (A, B, D, and E) denote the position of actin arcs surrounding the C-domain organelles. Turquoise circles in (B) and (C) mark bead site and turquoise arrows in (E) and (F) show actin filaments accumulated at the new adhesion site. In (C) and (F) Colors encode flow speed in μm min^-1; and vector arrows, flow direction. Scale bar, 10 μm.

Columns (A-D) show a multimode time-lapse sequence of a targeted growth response; DIC (top panel), actin filament FSM (middle panel), and MT FSM (bottom panel). White hatched lines denote boundary of leading edge. Yellow hatched lines mark C-domain organelle border. Columns (A) and (B) show the latency period. Note the presence of filopodial actin bundles before growth corridor formation and dynamic P-domain MTs (green arrow heads). Columns (C) and (D) show the engorgement response. Note the reduction in actin bundles in the corridor (denoted by hatched red line in C) followed by C-domain MT invasion and advance of the leading edge. Turquoise circles mark bead site. Scale bar, 10 μm.

(A) DIC and corresponding (B) actin filament FSM and (C) flow vector map time-lapse montage taken from the left side lateral aspects of the growth cone in Figure 1. Dashed lines outline C-domain in (A). Yellow arrows in first and last frame of (B) show straightening of normally curved arcs and red arrow heads show arcs embedded in actin meshwork in the target interaction corridor. Turquoise circles in (A, B, and C) show bead site. (D) Graph of the average flow speed and direction for the arcs in the white hatched region of interest in (C). Blue line and diamonds indicate direction where 90° represents retrograde flow and 180°, flow towards bead site. Purple lines and filled squares denote arc speed towards the C-domain whereas open purple squares designate speed towards adhesion site. (E) Time-lapse sequence of region outlined by white box in (A) showing the actin channel (left) and flow vector map (right). Yellow hatched line marks T-zone boundary and star denotes time-point showing arc vector angles perpendicular to peripheral flow. Scale bar, 10 μm.

(A) DIC image of a control substrate evoked growth response 984 sec after apCAM bead placement and restraint. (B) DIC image of the targeting response of the same growth cone following a 20 minute pretreatment with 20 μm of the Rho Kinase inhibitor Y27632, 444 sec after bead placement. Hatched white lines in (A and B) mark the C-domain organelle boundary. Note the spread appearance of the C-domain during the targeting response following drug treatment (black calipers). C) Schematic of growth response stages (top). Graph (bottom) shows Rho Kinase inhibition reversibly decreased (69.4% ± 6.0% of controls) the latency phase. Data are expressed as mean ± SEM. Statistical analysis was performed with two-tailed paired t-test. * indicates p < 0.01; n=7. (D and E) Actin filament FSM images of the corresponding control and drug treatment at time points shown in (A) and (B). (F) Flow vector maps from turquoise box regions of interest in (D, control) and (E, treatment) showing latency and growth time points. (G) Graph shows the reduced coherency of the F-actin vectors in drug treatment versus control over time. A coherency score of one means all vectors are parallel and close to 0 if their directions are random. (H) F-actin/MT immunofluorescence images showing control (left panel) and Rho Kinase inhibited (right panel) targeted growth responses. (I) F-actin immunofluorescence Images showing the growth corridors from (H). Yellow arrows mark arcs surrounding the growth corridor. Turquoise circles mark bead site in (D and E). Scale bar is 10 μm.