Figure 5.

Suppression of GAPDH expression simplifies growth cones, and local ablation of GAPDH in distal axons impairs growth cone dynamics. A, Example of shRNA-mediated knockdown of GAPDH. GFP is expressed by the shRNA expression vector and used to track morphology in transfected neurons. B, Knockdown of GAPDH decreases filopodia number and is rescued by shRNA-resistant GAPDH expression. Mann–Whitney tests. Median shown. C, Knockdown of GAPDH simplifies growth cone morphology and is rescued by shRNA-resistant GAPDH expression. χ² tests for independence. We note that, without live imaging, growth cones scored as having only filopodia may represent growth cones undergoing retraction as the filopodia cannot be strictly differentiated from the retraction fibers in static images. D, Examples of growth cones expressing GAPDH shRNA and expressing RFP (control) or rescue RFP-GAPDH. E, GAPDH overexpression increased the number of growth cone filopodia. Mann–Whitney test. Medians shown. F, GAPDH overexpression increases growth cones with filopodia and decreases the proportion of collapsed growth cone. χ² tests for independence. G, CALI of KR-GAPDH in a RATK experimental design does not adversely affect the morphology of the axon shaft as evidenced by the absence of thinning and/or beading. H, Two examples of RATK in KR-GAPDH-expressing distal axons. Time in seconds is shown. Following illumination/CALI, the dynamic remodeling of the growth cones greatly decreases. Yellow arrow at 600 s qualitatively indicates growth cone retraction in the top sequence. The base of the yellow arrow at 600 s indicates the initial distalmost position of the leading edge of the growth cone (arrowhead at 0 s after illumination) and the tip the current position. Scale bars, 5 μm. I, Example of RATK in RFP-GAPDH-expressing distal axons. After illumination, the growth cone remains dynamic. Scale bar, 5 μm. Percentage of dynamic filopodia (J) and new filopodia (K) formed before and after illumination in RATK using RFP-GAPDH (control) and KR-GAPDH. Repeated-measures ANOVA for control. Dunn's multiple comparison tests between each time point and Pre for KR-GAPDH. Each symbol represents a growth cone. Medians are shown.