Single-Cell Analysis of Cellular Quality Control In S. Cerevisiae: How ‘Low' Can You Go?

Within the secretory pathway of eukaryotic cells, the endoplasmic reticulum (ER) is responsible for maintaining the fidelity of protein synthesis and maturation. A variety of insults including nutrient deprivation, pathogenic infection, and chemical treatment, collectively termed ‘ER stress', induce quality control mechanisms to recover cell homeostasis. ER associated degradation (ERAD), unfolded protein response (UPR), and autophagy are quality control pathways that occur at various timescales, encompass variations in the spatial organization of multiple organelles, and alter select protein concentrations and intracellular localization. Surprisingly, all three pathways are activated in several neurodegenerative (e.g. Alzheimer, Parkinson, Sclerosis, Huntington, etc.) and hereditary diseases (e.g. Marinesco-Sjögren syndrome, Woozy mouse, nonpolyposis colorectal cancer-associated small-bowel cancer, prostate cancer, etc.). In all cases, as a result of ER stress, there is evidence of atypical, intracellular protein distribution during disease manifestation. Yet, how the accumulation of disease-specific proteins is involved in compromising quality control remains elusive.

By implementing DNA recombinant systems combined with high-resolution imaging techniques, we have determined that protein redistribution, resultant spatial effects, and organelle modifications are a consequence of the cell's response to ER stress in yeast, S. cerevisiae. In pursuit of a thorough analysis of protein redistribution at the subcellular level, multiple yeast expression cassettes have been created to test the effects of codon-optimized fluorescent variants, small epitope tags, polylinker length for N and C terminal tags, and the inclusion of essential retrieval sequences for ER luminal chaperones and foldases. A photoconvertible GFP variant (i.e. mEos2) and six-residue tetracysteine motif required for FlAsH (fluorescein arsenical helix binder)-based technology was used to investigate discrete subpopulations of tagged proteins using live-cell imaging methods, correlative microscopy, and super-resolution techniques. Using fluorescent protein (FP) variants as probes, we have expressed endogenous proteins with FP fusions in order to continuously monitor protein trafficking; analyzed localization effects of proteins involved in ERAD; examined organelle dynamics under various environmental conditions to initiate ER quality control (ERQC) and co-localization with cytoskeleton structures, actin and β-tubulin; while confirming the existence of cellular variability during UPR activation and its direct correlation to age at the level of single-cell analysis.

It is now evident that endogenous proteins involved in quality control redistribute within the cell, specifically the endoplasmic reticulum (ER), in order to perform essential functions that maintain cell homeostasis. Using novel techniques to image cells such as correlative microscopy, a combination of confocal light microscopy (CLM) and transmission electron microscopy (TEM), as well as Focused Ion Beam (FIB) microscopy, we have developed entire three-dimensional organelle reconstructions of yeast cells at electron microscope resolution. Interestingly, our microarray analysis and q-PCR validation have established novel down-regulation of selective genes in S. cerevisae as a consequence of ER stress. Subsequently, we investigated the effects of cellular disturbances inducing ER stress on a global level by examining select proteins in various organelle targets and elucidating the dramatic changes in organelle morphology.