FIG. 5.

Effects of CRMP-2 mutants on neurite formation in N1E-115 cells and DRG neurons. (A) Localization of GFP-tagged CRMP-2 and CRMP-2 mutants T555A and T555D. Vero cells were transfected with pEGFP-CRMP-2 WT, T555A, or T555D and cultured for 36 h. Transfected cells were fixed and stained with anti-α-tubulin antibody. Arrows indicate the colocalization of GFP-fused protein with microtubules. Bar, 10 μm. (B) The effect of overexpression of CRMP-2 WT, T555A, and T555D on process formation in N1E-115 cells. N1E-115 cells were transfected with myc-GST (as a negative control), CRMP-2 WT, T555A, or T555D. Twenty-four hours after transfection, the cells were cultured in serum-containing medium for 24 h. Transfected cells were fixed and doubly stained by anti-myc antibody (top panels) and anti-α-tubulin antibody (bottom panels). Arrows indicate the processes induced by CRMP-2 constructs. Bar, 40 μm. (C) The percentage of cells bearing neurites (length > 20 μm) in transfected cells. Fifty randomly selected transfected neurons were quantified for each construct. The values shown are means ± standard errors of triplicate experiments. **, significantly different from the cells expressing GST as analyzed by Student's t test (P < 0.01). (D) The percentage of cells bearing axons (length > 1,300 μm) in DRG neuron-transfected cells. DRG neurons were transfected with the plasmids indicated in panel B. After transfection, the neurons were cultured in the NGF-deprived medium for 3 days. Transfected neurons were fixed and doubly stained by anti-myc antibody and anti-neurofilament antibody. The percentage of neurons bearing axons with length > 1,300 μm versus the total number of neurons bearing axons was analyzed. Fifty randomly selected transfected neurons were quantified for each construct. The values shown are means ± standard errors of triplicate experiments. **, significantly different from the cells expressing GST as analyzed by Student's t test (P < 0.01); *, P < 0.05.