A genetic screen in Drosophila reveals novel cytoprotective functions of the autophagy-lysosome pathway.

Fly Strains and Collections The P-element insertion stocks were obtained from the Drosophila Genetic Resource Center of the Kyoto Institute of Technology ( http://kyotofly.kit.jp/cgi-bin/stocks/data_search.cgi ), or the Szeged Stock Center, Hungary ( http://expbio.bio.u-szeged.hu/fly/index.php ). The Kyoto stocks were generated and characterized by the BruinFly project [41] ( http://www.bruinfly.ucla.edu/ ). UAS-RNAi stocks [42] were obtained from the Vienna Drosophila RNAi Center ( http://stockcenter.vdrc.at/control/main ) or the National Institute of Genetics of Japan in Shizuoka ( http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp ). Other fly strains used: w; uas-dSec61α k155 /CyO (kind gift of Masayaki Miura, University of Tokyo), y w hsflp; Actin>CD2>GAL4, UAS-GFP (“flip out”), y w hsflp; Actin>CD2>GAL4, UAS-GFP-ATG8a , y w hsflp; r4-mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/+, y w hsflp ; UAS-2xeGFP FRT40A Fb-Gal4 (“40AGFP”), y w hsflp; UAS-dsRed FRT40A fb-GAL4; UAS-GFP-Atg8a , y w hsflp; Cg-GAL4 UAS-GFP-Atg8a FRT42D UAS-myrRFP , y w; P{lacW}Xbp1 k13803 /CyO (Bloomington Stock Center, #11104); UAS-xbp1-RB (kind gift of H.D. Ryoo [43] ).

EMS Mutagenesis and Screening Procedures

Mature isogenized males of the genotype y w; FRT40A iso1 were fed overnight with 25 mM EMS in a 1.5% sucrose solution before a brief incubation in a clean vial and normal feeding. Males were crossed to virgin female y w hsflp; sp/SM6b-TM6B , and males of the genotype y w hsflp; FRT40A*/SM6-TM6B were retrieved. Individual males of the genotype FRT40A*/SM6-TM6B or multiple males of the genotype FRT40A P/CyO were crossed with 10–15 virgin females of the genotype y w hsflp; UAS-2xeGFP FRT40A Fb-GAL4 or y w hsflp; UAS-dsRed FRT40A fb-GAL4 UAS-GFP-Atg8a . Eggs were collected in a normal food vial for 6–8 hours before a 1 hour heat shock at 37°C. Fat bodies were isolated from early third instar larvae and stained simultaneously with DAPI and 100 µM LysoTracker Red (L-7528, Molecular Probes) diluted in PBS. Fat bodies were then mounted in PBS on standard glass slides and coverslips and imaged on a Zeiss Axioskop 2 outfitted for epifluorescence. Mutant clones were identified by the lack of GFP and the close proximity of twin spot clones with two copies of the GFP expression cassette. All lines with a noticeable difference in size or LysoTracker staining in mutant clones vs. neighboring cells were noted. For phenotypic categorization, 3 clones per animal were visually inspected in a minimum of 3 animals, and multiple clones were imaged. For most crosses, 5 or more larvae were analyzed. For LT3 classification in Table S1 , at least one clone per animal in multiple animals, but not necessarily all animals, was observed.

Imaging Fixed Tissues and Antibody Staining

For confocal imaging of fluorescent ATG8 fusion proteins and antibody staining, larvae were everted and fixed in 4% formaldehyde in PBS at RT for 90 minutes before washing with PBS/0.01 triton X-100 (PBS-TX), staining with DAPI, washing in PBS, and dissection and mounting in FluoroGuard (Bio-Rad). Anti-Hsc70-3 guinea pig polyclonal antibody (a kind gift of H.D. Ryoo) was diluted 1∶200 in PBS-TX. Everted larvae were incubated in primary antibody for 2 hours at room temperature or overnight at 4°C, washed 3 times in PBS-TX, incubated in Texas Red-conjugated anti-GP secondary antibody for 1–2 hours at RT, washed and stained in DAPI and mounted as above, and imaged on a Zeiss Axioskop2 microscope (Plan-Apochromat 63×1.40 NA objective) equipped with a CARV spinning disc confocal system and a Hamamatsu ORCA-ER digital camera.

Flipout Expression of UAS-driven Genetic Constructs

UAS expression stocks were crossed to virgins of y w hsflp; Actin>CD2>GAL4, UAS-GFP, y w hsflp; Actin>CD2>GAL4, UAS-GFP-ATG8a or y w hsflp; r4-mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/+, and incubated at 25°C for 3–4 days. Leaky expression at 25°C of the heat-shock inducible FLP leads to stochastic “flip out” activation of GAL4 expression [44] in a minority of fat body cells, activating UAS-driven expression of GFP and the inserted RNAi, allowing assessment of these cells' level of lysosomal and autophagic activity in otherwise normal animals. Fat bodies were either stained unfixed with LysoTracker and DAPI, or were fixed and imaged as noted. Gene Ontology The GO Term Mapper ( http://go.princeton.edu/cgi-bin/GOTermMapper ) was used to bin individual GO categories into more general parent or “slim” terms to facilitate a broader analysis of functional categories. FlyBase IDs for the LysoTracker-positive and -negative groups with duplications eliminated were uploaded as text files to the GO Term Mapper and run against the FlyBase gene association file. Each group of genes was run separately in all three GO databases, and were compared to one another and to the overall fly genome using Microsoft Excel.

EMS Mutagenesis and Screening Procedures

Mature isogenized males of the genotype y w; FRT40A iso1 were fed overnight with 25 mM EMS in a 1.5% sucrose solution before a brief incubation in a clean vial and normal feeding. Males were crossed to virgin female y w hsflp; sp/SM6b-TM6B , and males of the genotype y w hsflp; FRT40A*/SM6-TM6B were retrieved. Individual males of the genotype FRT40A*/SM6-TM6B or multiple males of the genotype FRT40A P/CyO were crossed with 10–15 virgin females of the genotype y w hsflp; UAS-2xeGFP FRT40A Fb-GAL4 or y w hsflp; UAS-dsRed FRT40A fb-GAL4 UAS-GFP-Atg8a . Eggs were collected in a normal food vial for 6–8 hours before a 1 hour heat shock at 37°C. Fat bodies were isolated from early third instar larvae and stained simultaneously with DAPI and 100 µM LysoTracker Red (L-7528, Molecular Probes) diluted in PBS. Fat bodies were then mounted in PBS on standard glass slides and coverslips and imaged on a Zeiss Axioskop 2 outfitted for epifluorescence. Mutant clones were identified by the lack of GFP and the close proximity of twin spot clones with two copies of the GFP expression cassette. All lines with a noticeable difference in size or LysoTracker staining in mutant clones vs. neighboring cells were noted. For phenotypic categorization, 3 clones per animal were visually inspected in a minimum of 3 animals, and multiple clones were imaged. For most crosses, 5 or more larvae were analyzed. For LT3 classification in Table S1 , at least one clone per animal in multiple animals, but not necessarily all animals, was observed.

Supporting Information Table S1 Full listing and phenotype data for all strains tested. Table lists all P-element insertion stocks and RNAi insertion stocks tested, from a total of four stock centers. Phenotype designations are as follows: for RNAi expression experiments LysoTracker positive phenotypes were listed simply as LT+. For FRT40A mitotic clones, LT+ phenotypes were classified as LT1 (intense punctate staining); LT2 (mild to moderate, with some punctae); LT3 (mild or intermittent phenotype, including diffuse as opposed to punctate staining); S (small cell size without LysoTracker phenotype); and L (apparent cell lethal). Type (mitotic or RNAi) and source of constructs are listed, and can be sorted by any field. Gene disruption assignments for P-element insertions were downloaded from previously published descriptions and initial characterizations of the P-element collections: http://www.genetics.org/cgi/content/full/163/1/195/DC1 [45] and http://www.bruinfly.ucla.edu/subsets.php?id=3 . Where two genes were potentially disrupted, both are listed. Additional information was obtained by personal communication from the curators of the BruinFly project and from FlyBase release 5.15. For BruinFly stocks, the primary ID listed is the Kyoto Stock Center ID; BruinFly ID is listed separately. Insertion site refers to the genomic location of the P-element insertion or the chromosome of insertion of the UAS-RNAi insert, as appropriate. (0.47 MB XLS) Click here for additional data file.