Image-based genome-wide siRNA screen identifies selective autophagy factors

Abstract

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction1–4. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy, and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation5. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. To identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide siRNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to mRNA processing, interferon signaling, vesicle trafficking, cytoskeletal motor function, and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, Smurf1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, Smurf1-deficient mice display an accumulation of damaged mitochondria in heart, brain, and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines Smurf1 as a newly recognized mediator of both viral autophagy and mitophagy.

Contribution of Mitophagy to Cell‐Mediated Mineralization: Revisiting a 50‐Year‐Old Conundrum

“…The ubiquitin ligase Smurf1 has been shown to participate in mitophagy mediation 37. Murine embryonic fibroblasts lacking Smurf1 are incapable of autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria.…”

Peroxisomal protein PEX 13 functions in selective autophagy

“…In MEFs, endogenous Parkin expression is negligible 20 and Parkin overexpression in primary MEFs does not promote the complete clearance of TOMM20 during CCCP or OA‐induced mitophagy as it does in HeLa cells. Instead, damaged mitochondria in primary MEFs undergo Parkin‐independent partial clearance and compaction around the perinuclear region 16. After CCCP treatment, TOMM20 staining showed that damaged mitochondria were compacted around the perinuclear region and partially degraded in Pex13 + / + MEFs, whereas damaged mitochondrial fragments accumulated diffusely throughout the cytoplasm in Pex13 − / − MEFs (Figs 2E and F, and EV2D).…”

“…Since the peroxisome biogenesis function of PEX13 depends on other peroxin family members, we evaluated whether other peroxins are required for mitophagy and general autophagy. Of note, PEX5 and PEX19 are involved in ROS‐induced general autophagy 3, and we previously identified PEX3 as a candidate selective autophagy factor 16. Using pools of four siRNAs targeting each gene, we knocked down PEX3 , PEX5 , PEX13 , PEX14 , or PEX19 in HeLa cells (Fig 5A).…”

“…SIN is a single‐stranded RNA virus in the alphavirus family, and numerous previous studies have shown that SIN viral nucleocapsids are degraded by selective autophagy 16, 17, 18. In HeLa cells stably expressing GFP‐LC3 (HeLa/GFP‐LC3 cells) and infected with SIN, four siRNA oligos that target PEX13 (and decrease PEX13 expression [Fig 1A]) resulted in a decrease in colocalization between mCherry‐capsid and GFP‐LC3 puncta (Fig 1B and C).…”

“…Recombinant Sindbis virus strains SIN‐mCherry.capsid (strain AO30) and SIN‐mCherry.capsid/GFP‐LC3 (strain AO28) were generated, titered, and used for infections as previously described 16, 37, 38. SIN‐mCherry.capsid infections of HeLa/GFP‐LC3 cells were performed at a multiplicity of infection (MOI) of five plaque‐forming units (PFUs) per cell for 10 h. SIN‐mCherry.capsid/GFP‐LC3 infections of primary MEFs were performed at an MOI of 2.5 PFUs per cell for 16 h. Fluorescent microscopy images were analyzed by an observer blinded to experimental condition and the number of mCherry‐capsid puncta, GFP‐LC3 puncta, and colocalized mCherry‐capsid/GFP‐LC3 puncta was counted per cell.…”