High-throughput functional screening for autophagy-related genes and identification of TM9SF1 as an autophagosome-inducing gene.
Autophagy, a tightly regulated process responsible for the bulk degradation of most long-lived proteins and some organelles, is associated with several forms of human diseases including cancer, neurodegenerative disease and cardiomyopathies. However, the molecular machinery involved in autophagy in mammalian cells remains poorly understood. Here, we describe a high-throughput, cell-based functional screening platform, based on an automated fluorescence microscopy system, which enables acquiring and quantitatively analyzing images of GFP-LC3 dots in cotransfected cells. From a library of 1,050 human cDNA clones, we identified three genes (TM9SF1, TMEM166 and TMEM74) whose overexpression induced high levels of autophagosome formation. In particular, overexpression of TM9SF1, which colocalized with LC3 according to the confocal assay, led to a significant increase in the number of GFP-LC3 dots. The results of transmission electron microscopy and immunoblotting to examine LC3-II levels further confirmed the ability of TM9SF1 to induce autophagy. Furthermore, knockdown of TM9SF1 expression by RNA interference could hamper starvation-induced autophagy. The functional screening platform therefore can be applied to high-throughput genomic screening candidate autophagy-related genes, which would provide new insights into underlying molecular mechanisms that may regulate autophagy in mammalian cells.
He
Pengfei
P
Chinese National Human Genome Center, Beijing, China.
Peng
Zhi
Z
Luo
Ye
Y
Wang
Lan
L
Yu
Peng
P
Deng
Weiwei
W
An
Yunqing
Y
Shi
Taiping
T
Ma
Dalong
D
D000730:Androstadienes / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000235:genetics*; D005820:Genetic Testing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D000077191:Wortmannin
D000730:Androstadienes; D049452:Green Fluorescent Proteins; D018942:Macrolides; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D000077191:Wortmannin; C526109:Anxa3 protein, rat; C108718:TM9SF1 protein, human; C057620:bafilomycin A