The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies

Ethanol exposure increases mutation rate through error-prone polymerases

International audienceEthanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes

Plasma membrane electron transport in Saccharomyces cerevisiae depends on the presence of mitochondrial respiratory subunits

Most investigations into plasma membrane electron transport (PMET) in Saccharomyces cerevisiae have focused on the inducible ferric reductase responsible for iron uptake under iron/copper-limiting conditions. In this paper, we describe a PMET system, distinct from ferric reductase, which reduces the cell-impermeable water-soluble tetrazolium dye, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-1), under normal iron/copper conditions. WST-1/1-methoxy-phenazine methosulphate reduction was unaffected by anoxia and relatively insensitive to diphenyleneiodonium. Dye reduction was increased when intracellular NADH levels were high, which, in S. cerevisiae, required deletion of numerous genes associated with NADH recycling. Genome-wide screening of all viable nuclear gene-deletion mutants of S. cerevisiae revealed that, although mitochondrial electron transport per se was not required, the presence of several nuclear and mitochondrially encoded subunits of respiratory complexes III and IV was mandatory for PMET. This suggests some form of interaction between components of mitochondrial and plasma membrane electron transport. In support of this, mitochondrial tubular networks in S. cerevisiae were shown to be located in close proximity to the plasma membrane using confocal microscopy

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast

Abstract Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories

Ethanol exposure increases mutation rate through error-prone polymerases

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.Voordeckers K, Colding C, Grasso L, Pardo B, Hoes L, Kominek J, Gielens K, Dekoster K, Gordon J, Van der Zande E, Bircham P, Swings T, Michiels J, Van Loo P, Nuyts S, Pasero P, Lisby M, Verstrepen KJ.Voordeckers K, et al. Among authors: nuyts s. Nat Commun. 2020 Jul 21;11(1):3664. doi: 10.1038/s41467-020-17447-3.Nat Commun. 2020. PMID: 32694532status: publishe