(535g) Single-Cell Analysis of Cellular Quality Control In S. Cerevisiae: How ‘Low' Can You Go? | AIChE

(535g) Single-Cell Analysis of Cellular Quality Control In S. Cerevisiae: How ‘Low' Can You Go?


Czymmek, K. J. - Presenter, Delaware Biotechnology Institute
Robinson, A. S. - Presenter, University of Delaware
Yuraszeck, T. - Presenter, University of California

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.

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.

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.