Fig. 3.

Identification of nGAPs by RNAi. (A) Genes affecting axonal growth. Roles for the identified proteins in growth cone activity were assayed by looking for RNAi-induced reduction of axonal growth. RNAi was performed as described (45). The eight proteins to the left of the hatched line have FI ratios superior to GAP-43 as judged statistically by using the Kruskal-Willis test (red; also shown in red in Fig. 2), and the nine proteins to the right of the hatched line have FI ratios similar to that of GAP-43 as judged by the same test. The axonal lengths of EGFP-positive neurons (no siRNA) were used as the control (blue; also shown in blue in Fig. 2). All siRNAs (except GFP) have P < 0.002 in Wilcoxon rank-sum test (vs. control) (46). The data are shown as mean ± SEM. The number of neurons measured is shown in at the bottom of each bar. (B) Classification of candidate growth cone marker proteins by immunolocalization. We defined the C and P domains of a growth cone by using quantitative analysis of immunostaining images (diagram at top; see SI Text and Fig. S2). We classified the proteins into four groups: group I (ex. Pacs1), predominantly localized in the P region (C [dlt ]P); group II (ex. Syx7), detected in both the C and P regions (C ≃ P); group III (ex. Gnai2), predominantly localized to the C region (C ≫ P); and group IV (ex. Rtn1), specifically localized in the C region (C). In each case, the left diagram is summary of a typical protein distribution for each group. Immunofluorescence micrographs of anticandidate protein antibodies detection in cultured rat cortical neurons are shown in A. Magenta and green show antigen protein and α-tubulin views, respectively. The far views show the merged image. Three groups (groups I-III) are also shown in the legend for Fig. S4. Note that α-tubulin is primarily detected in the axon, although it is also detectable in the central region of the growth cone. See Table 1 for a detailed characterization of each protein and abbreviated names. (Scale bars: 10 μm.)