Genetic screens in worm identify autophagy genes specific to metazoan

I never under-estimate the power of genetic screen.

Here is one example performed in C.elegans.

Volume 141, Issue 6, 11 June 2010, Pages 1042-1055
DOI: 10.1016/j.cell.2010.04.034

C. elegans Screen Identifies Autophagy Genes Specific to Multicellular Organisms

Ye Tian1, 2, 5, Zhipeng Li1, 5, Wanqiu Hu3, 5, Haiyan Ren1, 5, E. Tian1, Yu Zhao1, Qun Lu1, Xinxin Huang1, Peiguo Yang1, Xin Li1, Xiaochen Wang1, Attila L. Kovács4, Li Yu3, The Corresponding Author and Hong Zhang1, The Corresponding Author

1 National Institute of Biological Sciences, Beijing 102206, P.R. China
2 College of Life Sciences, Beijing Normal University, Beijing 100875, P.R. China
3 State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China
4 Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest 1117, Hungary

The molecular understanding of autophagy has originated almost exclusively from yeast genetic studies. Little is known about essential autophagy components specific to higher eukaryotes. Here we perform genetic screens in C. elegans and identify four metazoan-specific autophagy genes, named epg-2, -3, -4, and -5. Genetic analysis reveals that epg-2, -3, -4, and -5 define discrete genetic steps of the autophagy pathway. epg-2 encodes a coiled-coil protein that functions in specific autophagic cargo recognition. Mammalian homologs of EPG-3/VMP1, EPG-4/EI24, and EPG-5/mEPG5 are essential for starvation-induced autophagy. VMP1 regulates autophagosome formation by controlling the duration of omegasomes. EI24 and mEPG5 are required for formation of degradative autolysosomes. This study establishes C. elegans as a multicellular genetic model to delineate the autophagy pathway and provides mechanistic insights into the metazoan-specific autophagic process.

Identification of epg-2, -3, -4, and -5

gfp::pgl-1, sepa-1::gfp, or t12 g3.1::gfp animals were used for EMS mutagenesis and F3 progeny were screened for accumulation of PGL-1, SEPA-1, or T12G3.1 aggregates in late-stage embryos. From 30,000 genomes screened (10,000 genomes for each reporter), 158 mutant alleles were isolated (28 from the gfp::pgl-1 screen, 68 from the sepa-1::gfp screen, and 62 from the t12 g3.1::gfp screen). Details of the mapping and cloning of the mutants are in the Extended Experimental Procedures.

Mapping and Cloning of epg-2, -3, -4, and -5

Three-factor mapping was performed to determine the genetic position of epg-2, -3, -4, and -5. From the cross between epg-3 and dpy-18 unc-25 (carrying the gfp::pgl-1 reporter) animals, 142 out of 146 Dpy non-Unc and 1 out of 72 Unc non-Dpy animals carried the epg-3 mutation. From the cross between epg-4 and sma-3 unc-32 (carrying the gfp::pgl-1 reporter) animals, 10 out of 10 Unc non-Sma and 1 out of 42 Sma non-Unc animals carried the epg-4 mutation. Fosmids or PCR fragments in the region where epg-3 and epg-4 were mapped were used to perform transformation rescue. Formation of somatic PGL granules was examined in epg-3 and epg-4 animals carrying various transgenes.

epg-5 was placed on chromosome II. From the cross between epg-5 (bp450) and bli-2 dpy-10 (carrying the sepa-1::gfp reporter), 13 out of 15 Bli non-Dpy recombinants carried the epg-5 mutation. Fosmids in this region were used to perform transformation rescue. Three out of seven lines carrying fosmid WRM0637aA03 and 5 out of 8 lines carrying the PCR fragment C56C10.12 rescued the bp450 phenotype.

epg-2 was mapped on chromosome I. Single-nucleotide polymorphism (SNP) mapping placed epg-2 between pas47511 (10.59) and pas45008 (9.34). The fosmid WRM0614 dC06 in this region rescued the epg-2 mutant phenotype.

The molecular lesions in epg-2, -3, -4, and -5 mutants were determined by sequencing PCR products from the corresponding genomic sequence. Transcripts derived from epg-3(bp405) and epg-4(bp425) mutants were also sequenced.

Btw, this is my first ever blog post from the sky, through SSH tunnel. I am writing now sitting in the dark of Delta flight 1080 heading to Raleigh/Durham. Thanks to FEDEX for this free WiFi access!