Figure 4

XMAP215 knockdown (KD) leads to changes in microtubule (MT) lattice and F-actin flow rates. (A-C) Micrograph of mKate2-tubulin at low levels in control growth cone (A), overlaid with flow vectors calculated by QFSM software at three different time points (B), and (C) rose plot of MT flow directions within all control growth cones, thresholded to include only vectors above the mean, in control. Zero degrees denotes flowing in the retrograde direction, towards the axon, while 180 degrees corresponds to the anterograde direction, with MTs flowing outwards in the direction of new growth (direction of arrow). (See Additional file 4A) (D-F) The same type of data as in (A-C), but for XMAP215 KD growth cones. (See Additional file 4B) (G-J) Micrograph of F-actin labeled by fluorescent kabC in control growth cone (G) and XMAP215 KD (I), with overlay of F-actin flow vectors from QFSM software at three different time points (H,J). (See Additional file 4C,D) (K) Quantification of F-actin speckles using QFSM software. Note that wild-type flow rates are slower than traditional kymograph-measured F-actin retrograde flow rates because manual analysis tends to selectively measure prominent, fast-flowing speckles, while QFSM measures every F-actin speckle within the entire growth cone. For analysis of F-actin flow quantification, only the top quartile of flow vectors for each movie was utilized. This excludes any false positive speckle detection from analysis, and also enhances the likelihood of identifying differences in maximum rates of F-actin retrograde flows. (L-N) Micrograph overlays of EB1-GFP tracks from a two-minute timelapse image series in control (L) and XMAP215 KD (M). (N) Quantification of the percentages of growth cones with co-linear versus non-co-linear tracks. Box-and-whisker plots indicate the mean (diamond), median, extrema and quartiles for each group. ns not significant. n = number of growth cones examined. Scale bar is 5 μm.