Fig. 6.

Gαo-mediated neurite outgrowth and neuronal morphology in N2a cells require AnkB and AnkG. (A) Overexpression of Gαo stimulates the formation of neurites in parental mouse N2a cells and in cells stably transfected with control shRNA (shluc). Permanent shRNA-induced downregulation of AnkB (shankB) or AnkG (shankG) results in the formation of elongated fibroblast-like cells, increases lamellopodia formation and slightly reduces the percentage of cells growing neurites and the number of neurites per cell. Transient ankyrin double knockdowns achieved by transfection of the shankB and shankG stable cell lines with the shankG and shankB plasmids, respectively, strongly increase the effects observed in single knockdowns. Treatment of Gαo-overexpressing N2a cells with Nocodazole (Noco) mimics the ankyrin double-knockdown phenotypes. Co-expression of EGFP (green) marks transfected cells and staining with phalloidin-Rhodamine (red) and DAPI (blue) is used to visualize F-actin and nuclei, respectively. (B) Quantification of the effects of Gαo overexpression on neurite outgrowth as compared with control transfected (pcDNA3) N2a cells, in shRNA stably transfected cell lines and in the presence of 10 nM Nocodazole. Data represent mean±s.e.m.; horizontal black lines indicate groups of statistical analysis and P-values are given above each bar (ns, not significant). (C) RT-PCR analysis shows the reduction in AnkB and AnkG expression in shRNA stably transfected N2a cells. Expression of the ribosomal protein S12 gene (Rps12) served as control. (D-F) Quantification of effects on the number of neurites per cell (D), cell morphology (E) and lamellopodia formation (F) of overexpression of Gαo in parental and shRNA-treated N2a cells. Data representation and statistical analysis are as in B. (G) Representative images of control transfected (pcDNA3) N2a cells and Gαo overexpression in parental as well as in single and double AnkB and AnkG knockdowns. Nocodazole treatment mimics the effects of Gαo overexpression in ankyrin double knockdowns. (H) Representative images of N2a cells overexpressing EGFP-tagged AnkB or AnkG show a substantial increase in the length of neurites upon co-expression with Gαo, but not alone. Fluorescence as in A. (I) Quantification of total neurite length in H. Data representation and statistical analysis are as in B. (J) Overexpression of Gαo induced the local accumulation of AnkB-GFP at neurite tips (arrowheads), which is not observed in control cells transfected with AnkB-GFP alone. Red fluorescence indicates Gαo immunostaining. Scale bars: 20 µm in A; 10 µm in G,H,J.