Fig. 8.

Suppression of microtubule plus tip dynamic instability blocks actin filament dependent growth cone elaboration in response to acute NGF treatment but does not alter the ATP/ADP ratio. (A) Examples of EB3 comets (yellow arrows) in growth cones before (pre) and after treatment for 10 min with DMSO followed by 10 min with NGF (top row) or before and after treatment for 10 min with 100 nM nocodazole (Noco) followed by 10 min with NGF (bottom row). Graphs in B and C show the quantification of the number of comets. Note that in the nocodazole+NGF treatment condition there are no visible comets. (B) Graph of the effects of NGF treatment on the number of EB3 comets in growth cones. The pre group represents the untreated no NGF condition, the DMSO group (control for nocodazole treatment) is not different at any time points (2–10 min of treatment) from any time points in the pre-treatment group (one-way ANOVA). For analysis of the effects of NGF the pre and DMSO treatment groups were thus compiled and used for comparison to each time point after NGF treatment using Dunn's multiple comparison tests. NGF increased the number of comets in growth cones at 10 min of treatment. (C) Graph of the effects of treatment with nocodazole on the number of EB3 comets in growth cones. By 4 min of treatment the number of comets in growth cones decreases as assessed using Dunn's time point-matched multiple comparison tests between pre and nocodazole treatment. The time points after NGF treatment were compared to the compiled data from 8 and 10 min of nocodazole treatment, when the effect of nocodazole is greatest prior to treatment with NGF, using Dunn's multiple comparison tests. Subsequent treatment with NGF did not elevate the number of comets relative to 8-10 min of nocodazole treatment at any time point. (D) Treatment with nocodazole blocks the NGF-induced increase in growth cone area. Bonferroni post-hoc time point-matched tests yield P>0.05 at all time points. (E) Treatment with nocodazole does not affect the ATP/ADP ratio after NGF treatment. Bonferroni post-hoc time point-matched tests yield P>0.05 at all time points. (F) Treatment with nocodazole does not affect the ATP/ADP ratio in no NGF treatment condition. Bonferroni post-hoc time point-matched tests yield P>0.05 at all time points. (G) Treatment with nocodazole for 30 min does not alter intracellular pH in growth cones cultured in continuous NGF. Results are shown as mean±s.e.m. DMSO, n=8; 100 nM Noco, n=31. P-value calculated with an unpaired two-tailed Welch t-test.