Flies were maintained on standard yeast/cornmeal/agar media. In climbing tests, a total of 90 adults per genotype were recorded and scored in cohorts of three to six flies, after tapping them down in a plastic graduated cylinder (Juhász et al., 2007 (link)). In clonal analyses, RNAi cells were generated spontaneously in larvae carrying hs-Flp; upstream activation sequence (UAS)-Dcr2; Actin>CD2>Gal4 UAS-RNAi (and UAS-GFP or UAS-LAMP1-GFP as a knockdown cell marker), and mutant cell clones (marked by lack of GFP expression) were generated by heat shocking 2–4-h embryos of the genotype hs-Flp; ubi-GFP FRT2A/Syx17[LL] FRT2A in a 38°C water bath for 1 h (Juhász et al., 2007 (link), 2008 (link); Pircs et al., 2012 (link)). Expression of mCherry-Atg8a was driven by a fat body–specific r4 promoter in our screen (transgenic flies were provided by T. Neufeld, University of Minnesota, Minneapolis, MN; Pircs et al., 2012 (link)). We used the GFP knockin line dLAMP[CPTI001775] (Drosophila Genetic Resource Center) to label lysosomes, w[1118] as control, UAS-GFP-KDEL and Pdi[G00198] as ER reporters (Bloomington Drosophila Stock Center), Atg2[EP3697] (Berry and Baehrecke, 2007 (link)), Atg7[d77]/Atg7[d14] (Juhász et al., 2007 (link)) mutants, and SNARE loss-of-function strains listed in Table S1. Knockdown of usnp was induced by Actin-Gal4 for Western blots and collagen-Gal4 for EM in L3 stage larvae, and overexpression of UAS-p35 or UAS-DIAP1 in adult neurons was mediated by elav-Gal4 (all obtained from Bloomington Drosophila Stock Center).
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Drosophila Climbing and Clonal Analyses
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Variable analysis
independent variables
- Genotype of flies (e.g., Syx17[LL] mutant)
- Presence or absence of heat shock (for clonal analysis)
- Expression of RNAi, GFP, LAMP1-GFP, mCherry-Atg8a, GFP-KDEL, or Pdi[G00198]
- Climbing ability of adult flies
- Presence or absence of mutant cell clones (marked by lack of GFP expression)
- Standard yeast/cornmeal/agar media for fly maintenance
- Plastic graduated cylinder for climbing tests
- W[1118] as control genotype
- Knockdown or overexpression of various genes (e.g., usnp, p35, DIAP1) in specific tissues or developmental stages
- Positive control: w[1118] as control genotype
- Negative control: Not explicitly mentioned
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