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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, chemical treatment and heterologous protein expression, 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 mechanisms that occur at various timescales, encompass variations in the spatial organization of multiple organelles, and alter selective protein concentrations and intracellular localization1-7. 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.)8-10. In all cases, as a result of ER stress, there is evidence of atypical, intracellular protein distribution during disease manifestation11-14. Yet, how the accumulation of disease-specific proteins is involved in compromising quality control remains elusive. Beyond therapeutic applications, a better understanding of the cell’s control of ER homeostasis contributes to our fundamental understanding of cellular regulation, both during normal growth, and during expression of proteins of interest.
By implementing DNA recombinant systems combined with high resolution imaging techniques, we have been able to examine protein redistribution, resultant spatial effects, and organelle modifications as a consequence of the cell’s response to ER stress. A system of yeast expression cassettes has 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 retrieval sequences for ER luminal chaperones and foldases. In addition, FlAsH and photoswitchable GFP variants have been incorporated for 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 confirmed the existence of cellular variability during UPR activation. Preliminary data suggests 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 three-dimensional reconstructions of the organelles within a yeast cell at electron microscope resolution. Additionally, we have investigated the effects of cellular disturbances inducing ER stress on a global level by examining protein redistribution and organelle morphology. 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.
The further development and implementation of single cell analysis at high resolution has permitted an additional aim of our research: determination of specific trafficking effects of heterologous proteins in yeast, their localization to sub-compartments of organelles, and elucidation of specific quality control mechanisms. By studying the pathways involved in ERQC, posttranslational modifications, ER export, and vesicle trafficking through the secretory pathway, we have correlated UPR induction and identified specific domains and motifs that affect functional production of heterologous protein in S. cerevisiae.
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