Consistent with the notion that Nak functions with AP2, Nrg puncta were also diminished specifically in higher-order dendrites of the α-Adaptin-RNAi da neurons ( Figures 7G and S6). These results suggest that the localization of Nrg to higher-order dendrites requires Nak-mediated endocytosis. We then examined the effect on dendrite development by Nrg overexpression. When the neuronal-specific Nrg long form (Nrg180) was overexpressed in da neurons, the higher-order dendrites were shortened and reduced (compare Figures 7H and 7I, and see quantification

in Figure 8A, column 17), similar to the defects in nrg loss-of-function mutants. These dominant-negative selleck screening library effects by Nrg180 overexpression might be due to excess adhesion property that inhibits cellular extension. We then tested whether the dendritic defects induced by excess Nrg could be overcome by enhancing the

activity of Nak-mediated endocytosis. Consistent with this idea, coexpression of Nrg and Nak in da neurons not only restored but also further enhanced dendrite arborization (Figures 7J and HTS assay 8A, column 18), similar to the effect by Nak overexpression ( Figure 8B, column 6). The dephosphorylation on mammalian L1 Tyr-1176 (Y1176) is known to be crucial for L1 endocytosis ( Schaefer et al., 2002). The conserved Y1185 in the Drosophila Nrg was mutated to Asp (NrgY1185D) to mimic constitutive phosphorylation, which would disrupt Nrg endocytosis. Overexpression of NrgY1185D in da neurons also caused shortening and reduction of dendrites (Figures 7K and 8A, column 19). However, these dendritic defects could not be restored by the overexpression of Nak (Figures 7L and 8A, column 20). Thus, Nrg endocytosis is essential for Nak to overcome dendritic defects induced by Nrg overexpression.

Taken together, these results strongly suggest that Nak regulates the proper distribution of Nrg no through endocytosis in dendrite development. During larval development, arborization of higher-order dendrites fills in the gaps between pioneer dendrites to cover the entire receptive field, and is important for coping with the increasing epidermal surface (Parrish et al., 2009). Here, we show that disruption of nak during dendrite arborization of da neurons significantly reduces both number and length of dendritic branches. Multiple classes of da neurons were analyzed for the lack of Nak activity, which suggests that its general role in higher-order dendrite morphogenesis. The function of Nak in dendrite arborization is required cell autonomously, as dendritic defects in nak mutants could be rescued by neuron-specific expression of wild-type Nak. Several lines of evidence suggest a functional link between Nak and AP2, the endocytosis-specific clathrin adaptor, in dendrite morphogenesis. First, coimmunoprecipitation results show that Nak predominantly associates with AP2. Second, Nak colocalized well with GFP-Clc and α-adaptin but not with AP1 and AP3 in S2 cells (Figures S7A–S7D).