Figure 4. ROS depletion affects F-actin dynamics.

(A) F-actin dynamics of control growth cone. Neurons were injected with 20 μM Alexa 568-phalloidin, and time lapse movies were acquired using FSM (Video 7). White line demarks lamellipodia region used for kymographs shown in (C). (B) Same growth cone after 20 min in 25 mM PBN. Note the progressive decrease in F-actin content in the P domain (Video 7). Arrowhead indicates forward movement of actin recycling zone. (C) Kymographs of actin speckles taken in the P domain as indicated with white lines in (A) and (B) reveal leading edge protrusion in control and progressive decrease in F-actin flow from initial control rate of 3.6 μm/min to 2.1 μm/min after PBN treatment (white lines in kymograph). (D) Actin FSM image of another control growth cone. (E) Same growth cone after treatment with 1 μM PAO for 30 min, which causes disassembly of the actin cytoskeleton and forward shift of the recycling zone (Video 8; image is shown after 25 min). A temporary lengthening of filopodial bundles occurred simultaneously with flow reduction. (F) Kymographs of actin speckles taken in P domain as indicated with white lines in (D) and (E) show a decrease in flow rates from 4.0 μm/min in control to 1.5 μm/min after PAO treatment. (G) 4 mM apocynin treatment caused a decrease in actin flow rates, filopodial lengthening and finally F-actin disassembly (Video 9). (H) 50 μM NDGA resulted in an aggregation of actin arcs (arrowhead) (Video 10). (I) Quantification of actin retrograde flow rates. T-test: *p<0.005; n= number of growth cones. (J) 75 mM PBN resulted in an aggregation of actin arcs and contractile effects, similar to NDGA treatment (arrowhead) (Video 11). (K) Treatment with 0.2 mM MnTBAP resulted in temporary filopodia lengthening, arc aggregation (arrowhead) and overall network disassembly. Scale bars: 10 μm.