Ilp2 gal4

All flies were raised at 25°C in a 12:12 h light:dark cycle. Flies were fed standard cornmeal-dextrose agar media. CASK proteins (full-length wild type β isoform or mutants containing a deletion of either the L27 or CaMK domains) tagged with a C-terminal YFP were expressed in a cell-specific manner using the GAL4/UAS system (Brand and Perrimon, 1993 (link)). All experiments were done on a CASK-β null genetic background [CASK^P18 allele (Slawson et al., 2011 (link))] to ensure that the only CASK-β present was the product of the tagged transgene. Briefly, animals carrying a UAS-CASK-YFP transgene (made from a cDNA encoding isoform B) were crossed to animals carrying GAL4's which expressed in specific cell types. The GAL4's used in this study were: C155-GAL4 (Genotype:P{GawB}elavC155, Bloomington stock 43351; chromosome X), all neurons (Lin et al., 1994 (link)); C164-GAL4 (Genotype:P{GawB}C164, Bloomington stock 33807), a subset of adult neurons including some dopaminergic neurons (Slawson et al., 2011 (link); Slawson et al., in preparation); TH-GAL4, (Genotype:P{ple-GAL4.F}, Bloomington stock 8848; Chromosome 3), dopaminergic neurons (Friggi-Grelin et al., 2003 (link)); and DIlp2-GAL4 (Genotype:P{Ilp2-GAL4.R}, Bloomington stock 37516; Chromosome 2), neurons of the pars intercerebralis which express Drosophila insulin-like peptide 2 (Corl et al., 2004 (link)).

Fly stocks were maintained on standard cornmeal-yeast-agar medium at 25°C, and on a 12/12 hr light/dark cycle. The MiMIC and CRIMIC flies were created in the Bellen lab (see Flypush or Supplementary file 2). UAS-2xEGFP, hs-Cre,vas-dϕC31, Trojan T2A-GAL4 triplet flies were from Dr. Ben White (Diao et al., 2015 (link)). The RMCE conversion of MiMICs with GFSTF and T2A-GAL4 cassettes was described in previous studies (Diao et al., 2015 (link); Nagarkar-Jaiswal et al., 2015a (link); Nagarkar-Jaiswal et al., 2015b (link)). The crossing schemes for CRIMICs are shown in Supplemental Information 1. btl-GAL4, Ilp2-GAL4, repo-GAL4, gcm-GAL4, UAS-mCD8::GFP, UAS-mCD8::RFP, P[acman] flies, and UAS-FLP flies were obtained from the Bloomington Drosophila Stock Center (BDSC, USA). UAS-if was from Dr. Celeste Berg (Beumer et al., 1999 (link)). NP1243-GAL4, NP2222-GAL4, and NP6250-GAL4 are from Kyoto Stock Center (Kyoto DGGR, Japan). Dfs flies were from BDSC or Kyoto DGGR. UAS-cDNA flies were from BDSC or FlyORF (Switzerland). y,w;attP40(y+){nos-Cas9(v+)}/CyO (Kondo and Ueda, 2013 (link)) and y,w;+/+; attP2(y+){nos-Cas9(v+)} (Ren et al., 2013 (link)) were isogenized in this work. See Supplementary file 2 for the genotypes and stock numbers of fly stocks. All references to FlyBase are based on FlyBase 2.0/FB2017_06 (Gramates et al., 2017 (link)).

Flies were raised on standard agar-yeast-based food at 25°C, 50%–60% humidity with 12-hour light: 12-hour dark cycle. Starvation experiments were performed on 1% agar food for 24 hours. upd2^Δ3–62 was kindly provided by J. Hombria [33 (link)]. The following stocks were from Bloomington Drosophila Stock Center (BDSC), yw (#1495), yolk-GAL4 (#58814), UAS-upd2RNAi (#33988, HMS00901) [110 (link)], UAS-domeRNAi (#32860), Ilp2-Gal4 (#37516), 24B-GAL4 (#1757), R57C10-GAL4 (#39171), R38H02-GAL4 (#47352) [111 (link)], UAS-CaLexa (#66542) [65 (link)]. Flies were crossed into a w+ (Canton-S) background (lab stock, originally obtained from BDSC) for all knockdown experiments.