Figure 5.

Expression of the Nter domain of TIVAMP inhibits neurite outgrowth. (A) Effect of GFP, GFP plus TeNT, GFP plus BoNT E, or GFP-Nter-TIVAMP on neurite outgrowth. PC12 cells transfected with the indicated constructions and treated with staurosporine were fixed and direct fluorescence images were recorded. Representative fields of the distinct phenotypes found are shown. Note the long neurites displayed both by the GFP and the GFP+TeNT–transfected cells compared with the shorter ones displayed by the GFP+BoNT E and the GFP-Nter-TIVAMP-transfected cells (arrowheads). (B) GFP-Nter-TIVAMP and BoNT E inhibit neurite length. Percentage of neurites longer than 20 μm. A minimum of 50 transfected cells of each type was recorded in blind, and the length of all their neurites was measured. The mean values (±SE) of percentage of neurites longer than 20 μm from three independent experiments are shown. *P < 0.03 (Student's t test). Note the lack of effect of TeNT and that BoNT E and GFP-Nter-TIVAMP had a similar inhibitory effect on neurite length. (C) Number of neurites per cell. The same randomly chosen transfected cells were use to quantify the number of neurites per cell. Shown is the number of cells, expressed as the percentage of transfected cells, displaying 1, 2, 3, or > 4 neurites. The mean values (±SE) of three independent experiments are shown. Note the lack of effect of TeNT and that both BoNT E and GFP-Nter-TIVAMP enhanced the percentage of cells without neurites. Bar, 25 μm.