(703a) Single-Cell Analysis of 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 disturbances including nutrient
deprivation, pathogenic infection, and chemical treatment, collectively termed
'ER stress', induce quality control mechanisms to facilitate the recovery of
cell homeostasis. ER-associated degradation (ERAD), unfolded protein response
(UPR), and autophagy are quality control pathways that occur on 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 and hereditary diseases. 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 recombination strategies 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 [1] have been created to test the effects of
codon-optimized fluorescent protein (FP) variants, small epitope tags (reviewed
in [2]), polylinker length for N- and C-terminal tags, and the inclusion of
essential retrieval sequences for ER luminal chaperones and foldases [3]. Consequently,
our approach enables one to monitor the trafficking effects of ER chaperones
and foldases by incorporating H/KDEL retrieval sequences fused to FP variants,
hence evaluate the spatiotemporal effects of chaperone/co-chaperone
interactions [4]. To investigate
discrete subpopulations of tagged proteins using live-cell imaging methods and super-resolution
techniques (e.g. Fluorescence-Photoactivation
Localization Microscopy, F-PALM and Structured Illumination Microscopy, SIM),
photoconvertible GFP variants and FlAsH-based technology were implemented.

Utilizing FP variants as probes, proteins
were recombinantly expressed from their native promoter in order to monitor
protein trafficking, analyze localization effects of proteins involved in ERAD,
and examine organelle dynamics and morphology as a consequence of local
perturbations to the system [5]. Furthermore, we have confirmed the existence
of cellular variability following UPR activation at the level of single-cell
analysis and evaluated cytoskeleton modifications. It is now evident that
endogenous proteins redistribute in the cell, specifically the ER, in order to
perform essential functions that maintain cell homeostasis. Using novel imagining
techniques such as Focused Ion Beam (FIB) microscopy [6], entire
three-dimensional organelle reconstructions of yeast cells were developed at
electron microscope resolution. Our results confirm organelle connectivity
during inheritance (e.g. lipid droplets derived from perinuclear ER) and following
periods of prolonged stress (e.g. peripheral ER and mitochondria junctions).
High-resolution techniques (e.g. SIM and F-PALM) have led to extraordinary
biological insights involving organelle biogenesis and intracellular communication
[7].

Due
to the intrinsic complexity of biological systems, the integration of
experimental and computational approaches is crucial when investigating the UPR
of S. cerevisiae. To establish that
the UPR is a global response to localized perturbations, we evaluated the transcriptional
effects of UPR activation [8]. Interestingly, microarray analysis and q-PCR
validation have confirmed the novel repression of 206 genes – highly
enriched in protein synthesis and metabolic biological functions –
following ER stress. We have expanded the characterization of the UPR and
reaffirmed diverse, global
consequences for the cell. UPR activation was assessed at the molecular level by
a systematic analysis of yeast deletion strains. Collectively, our experimental
approaches have led to a better understanding of ER homeostasis and cell
regulation.

References

1.    
C. L. Young, D. Raden, J. Caplan, K. Czymmek, A. S. Robinson Optimized Cassettes for Live-Cell Imaging of Proteins and High
Resolution Techniques in Yeast, Yeast, 2012 doi:10.1002/yea.2895.
[Epub 2012 Apr 4]

2.    
C. L. Young,
Z. T. Britton, A. S. Robinson Recombinant
Protein Expression and Purification: A Comprehensive Review of Affinity Tags
and Microbial Applications, Biotechnology Journal, 7(4), Jan 10 2012
doi:10.1002/biot.201100155. [Epub ahead of print]

3.    
C. L. Young, D. L. Raden, A. S. Robinson, Analysis
of Endoplasmic Reticulum Resident Proteins in S. cerevisiae: Implementation of
H/KDEL Retrieval Sequences, 2012 (submitted).

4.    
M. Griesemer, C. Young, A. Robinson, L. Petzold Spatial Localization of Chaperone
Distribution in the Endoplasmic Reticulum of Yeast. IET Systems Biology, 2012
doi:10.1049/iet-syb.2011.0006.

5.    
C. L. Young, D. L. Raden, J. Caplan, B. Chung, K. Czymmek, A. S. Robinson Dynamics of Endoplasmic Reticulum Resident
Proteins and Organelle Morphology in S. cerevisiae, 2012 (in preparation).

6.    
D. Wei, S. Jacobs, S. Modla, S. Zhang, C.
L. Young, R. Cirino, J. Caplan, K. Czymmek High-resolution three-dimensional reconstruction of a whole yeast cell
using focused-ion beam scanning electron microscopy, BioTechniques, 2012 (in press).

7.    
C. L. Young, D. Raden, J. Caplan, K. Czymmek, A. S. Robinson Spatiotemporal Resolution of Protein
Distribution at the Sub-Organelle Level in S. cerevisiae during Cell Division,
2012 (in preparation).

8.    
T. Yuraszeck , C. Young , P. Xu, C. A.
Gelmi, F. J. Doyle III, A. S. Robinson Novel
down-regulation pathways in the Unfolded Protein Response from S. cerevisiae
provide evidence of a complex regulatory response to ER stress, 2012 (under
revision)  co-authorship.