Arf4 Regulates Dendritic Spine Development Figure 10. ASAP1, an Arf4 GAP, inhibits dendritic spine formation, and both Arf4-WT and Arf4-Q71L partially rescue this inhibition. (A2D9) Representative examples of WT neurons transfected at DIV5 with FUGW2-GFP plasmid alone (A, A9), or together with ASAP1-Flag (B, B9), ASAP1-Flag plus Arf4-WT-HA (C, C9), or ASAP1-Flag plus Arf4-HA Q71L (D, D9) and analyzed at DIV14. Lower (A2D) and higher (A92D9) magnification images are shown. (n = 11-13 neurons per condition). (E2F) Quantification of the effect of ASAP1 alone or together with Arf4-HA-WT or Arf4-HA-Q71L on dendritic spine density (E) and morphology (F). All data are mean6SEM. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0046340.g010 spine density, suggesting that restricting Arf4 to its GDP-bound state prevents the development of mature spines. caused by endogenous mouse Arf4 knockdown, demonstrating the specificity of Arf4-shRNA9s effects. Arf4 is required for normal spine development We next asked whether the absence of endogenous Arf4 perturbs spine development. We transfected neurons at DIV5 with a FUGW2-based vector encoding GFP and one of two mouse Arf4-short hairpin RNA (shRNA) sequences, and then imaged the cells at DIV14. Both Arf4-shRNA1 and Arf4-shRNA2 markedly decreased Arf4 protein levels in Neuro-2a cells 48 hours post- transfection (Figs. 9A and B). Knockdown of Arf4 significantly decreased total spine density (Figs. 9D–E9) as well as the individual densities of all spine subtypes except filopodia (Fig. 9H), compared to controls (Figs. 9C and C9). To verify the target specificity of Arf4-shRNA, we co-expressed human Arf4-HA—whose expres- sion is resistant to mouse shRNA knockdown—with mouse Arf4- shRNA. Expression of human Arf4-HA completely rescued the effects on spine density (Figs. 9F and G) and morphology (Fig. 9H) ASAP1, an Arf4 GAP, negatively regulates spine development Previous studies have shown that ASAP1 functions as an Arf4 GAP and forms a complex with Arf4 [32]. We found that overexpressed ASAP1 is localized to dendrites and dendritic spines in mouse primary neurons, similar to Arf4 (Figs. 10B and B9). Neurons transfected at DIV5 with ASAP1-Flag, together with FUGW2-GFP for visualization of spines, showed a significant decrease in total spine density (Figs. 10B, B9, and E) as well as in stubby and mushroom spine density at DIV14 (Fig. 10F). To determine whether the spine-promoting effect of Arf4 is regulated by ASAP1, we co-transfected primary neurons with ASAP1-Flag and either Arf4-HA-WT or constitutively active Arf4-HA-Q71L, together with FUGW2-GFP. Both WT (Figs. 10C and C9) and constitutively active (Figs. 10D and D9) Arf4 partially blocked ASAP1-induced PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e46340