A genome-wide imaging-based screening to identify genes involved in synphilin-1 inclusion formation in Saccharomyces cerevisiae

Abstract

Synphilin-1 is a major component of Parkinson’s disease (PD) inclusion bodies implicated in PD pathogenesis. However, the machinery controlling synphilin-1 inclusion formation remains unclear. Here, we investigated synphilin-1 inclusion formation using a systematic genome-wide, high-content imaging based screening approach (HCI) in the yeast Saccharomyces cerevisiae. By combining with a secondary screening for mutants showing significant changes on fluorescence signal intensity, we filtered out hits that significantly decreased the expression level of synphilin-1. We found 133 yeast genes that didn’t affect synphilin-1 expression but that were required for the formation of synphilin-1 inclusions. Functional enrichment and physical interaction network analysis revealed these genes to encode for functions involved in cytoskeleton organization, histone modification, sister chromatid segregation, glycolipid biosynthetic process, DNA repair and replication. All hits were confirmed by conventional microscopy. Complementation assays were performed with a selected group of mutants, results indicated that the observed phenotypic changes in synphilin-1 inclusion formation were directly caused by the loss of corresponding genes of the deletion mutants. Further growth assays of these mutants showed a significant synthetic sick effect upon synphilin-1 expression, which supports the hypothesis that matured inclusions represent an end stage of several events meant to protect cells against the synphilin-1 cytotoxicity.

Perturbing proteomes at single residue resolution using base editing

“…Competent cells of target genes were transformed with the appropriate mutant plasmids as well a the original plasmid bearing the wild-type gene and the empty vector 63 , and transformants were selected by plating on SC-U (MSG). Multiple independent colonies per transformation were then put on sporulation media until sporulation could be confirmed by microscopy.…”

FMN reduces Amyloid-β toxicity in yeast by regulating redox status and cellular metabolism

“…The synthetic genetic array (SGA) approach has been applied to yeast neurodegeneration models, such as AD, Huntington's disease (HD) 24 and Parkinson's disease (PD) 25 , demonstrating its robust capability to help identify genes and mechanisms underlying phenotypes of interest, such as modeling toxicity of human disease proteins. In previous studies, the nonessential genomewide deletion library has been applied to Aβ42 expression strain to identify proteins and cellular processes affecting intracellular Aβ42 aggregation and toxicity 26,27 .…”

Perturbing proteomes at single residue resolution using base editing

“…Competent cells of target genes were transformed with the appropriate mutant plasmids as well 582 a the original plasmid bearing the wild-type gene and the empty vector (Zhao et al 2016), and 583 transformants were selected by plating on SC-U (MSG). Multiple independent colonies per 584 transformation were then put on sporulation media until sporulation could be confirmed by 585 microscopy.…”