, 2005). Mammals contain two Ark family genes, cyclin G-associated kinase (GAK) and adaptor-associated kinase 1 (AAK1), and both have been implicated in vesicular transport (Conner and Schmid, 2002 and Lee et al., 2005). GAK, best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles,

has multiple functions during clathrin cycle (Eisenberg and Greene, 2007). AAK1 has been shown to bind the α subunit of AP2, phosphorylate the cargo-binding μ2 subunit, and promote receptor-mediated transferrin uptake (Conner and Schmid, 2002, Conner et al., 2003 and Ricotta et al., 2002). AAK1 also Epigenetics inhibitor participates in transferrin receptor recycling from the early/sorting endosome in a kinase activity-dependent manner (Conner et al., 2003 and Ricotta et al., 2002). Numb-associated kinase (Nak), the Drosophila Ark family member, contains the conserved Ark kinase domain and several motifs (DPF, DLL, and NPF) mediating interactions with endocytic proteins ( Conner and Schmid, 2002 and Peng et al., 2009). Here, to study the function of Nak in development, we generated nak deletion mutants and RNAi lines and showed that depletion of nak activity

in da neurons disrupts higher-order dendrite development. This function of Nak in dendritic morphogenesis is likely mediated through CME, as Nak exhibits specific genetic interactions with components of CME, colocalizes with clathrin in dendritic puncta, and is required for the presence of clathrin puncta in distal higher-order dendrites. More importantly, live-imaging

HIF activation analysis shows that the presence of these clathrin/Nak puncta at basal branching sites correlates with extension of terminal branches. In addition, we present evidence that the localization of Neuroglian (Nrg) in higher-order dendrites requires Nak, implying that regional internalization of a cell adhesion molecule is crucial for dendrite morphogenesis. To study nak function in development, we generated two nak deletion mutants using nakDG17205, which carries a p[wHy] transposable element ( Huet et al., 2002) in the first next intron of nak ( Figure 1A). The deletion in nak1 extends 1.1 kb toward the 5′ end from the insertion site, removing most of exon 1 of nak. The nak2 allele deletes a 6.2 kb fragment downstream of the insertion site, removing exons 2–7 including the kinase domain. Western analysis of larval extracts with anti-Nak antibodies showed that Nak expression was reduced in nak1 and undetectable in nak2 mutant larvae ( Figure 1B), suggesting that nak1 and nak2 are partial loss-of-function and null alleles, respectively. Adults homozygous for nak1 and nak2 were viable and fertile, indicating that nak is not an essential gene. To understand its role in development, we examined Nak distribution during embryogenesis using immunohistochemistry.