Figure 10.

Wnt5a gradients induce redistribution and reorganization of dynamic microtubules (MTs) in growth cones. (A) Schematic of measurement of the distribution of EB3 comets on MT plus ends in the presence of a pipette gradient of Wnt5a locally applied at the right side of the growth cone. Individual EB3 comets are detected by fluorescence (green dots) and their center of intensity (cross) is determined. (B) Localization of the center of intensity of EB3 comets evoked by a Wnt5a gradient in 10 growth cones in independent experiments including the growth cone shown in (C,D). Negative numbers indicate EB3 localization to the side of the growth cone farthest from the Wnt5a source. Change of the center of intensity = −0.35 ± 0.14 μm (p < 0.05, Wilcoxon signed rank test). (C,D) Composite images of the same growth cone showing redistribution of EB3 comets over time 30 min before and during 30 min of application of the Wnt5a gradient (n = 601 images before Wnt5a and 601 images during Wnt5a application). The maximum intensity was computed for each pixel from the entire image stack by using algorithms in Metamorph. Using these values stacks of all images were superimposed and pixels assigned values in pseudocolor to indicate fluorescence intensity.