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.