Characterization of Endoplasmic Reticulum Stress in Single Cells

Abstract

Correct secretory protein folding is essential for cell survival. Misfolding leads to loss-of-function and even toxic-gain-of-function variants in cells. Therefore, cells evolved endoplasmic reticulum (ER) chaperones to facilitate nascent secretory protein folding and resolve misfolded protein burdens in the ER. ER chaperone functions have been extensively characterized in vitro. However, how the chaperones behave in vivo is less well understood. The generalist chaperone BiP binds unfolded clients to facilitate their folding. How BiP is organized in the ER lumen and encounters substrate in vivo has been unclear. My work employed fluorescent photobleaching techniques to investigate chaperone behavior in single live cells. We determined that green fluorescent protein-tagged BiP (BiP-GFP) is mobile throughout the ER and its availability decreases with increasing unfolded protein accumulation in the ER. BiP-GFP is the first tool for directly measuring changes in the unfolded protein burden in live cells. Interaction of BiP with clients is modulated by the co-chaperone ERdj4. A previous study reported ERdj4 as an integral membrane protein. Using FRAP, my work demonstrated that ERDj4 is actually a soluble signal cleaved protein and it is mobile throughout the ER. Additionally, we found that ERdj4 interacts with Derlin-1, a component of ER-associated degradation machinery. This finding provides mechanistic insight into the role of ERdj4 selectively facilitating secretory protein turnover. Finally, my work employed the BiP-GFP reporter to investigate the link between oxidative stress, the unfolded secretory protein burden and activation of an adaptive signaling pathway termed the Unfolded Protein Response (UPR). I found paraquat (Pq)-induced oxidative stress activated the UPR in the absence of detectable increases in unfolded protein burdens. When challenged with an ER stress inducer tunicamycin, Pq-treated cells showed attenuated UPR activation compared to Pq-naive cells---an observation consistent with adaptation. However, Pq-induced UPR does not protect cells against future ER stresses. My work suggests UPR impairment may contribute to the phenotypes associated with aging and other oxidative stress-related pathological processes. My thesis work has showcased how single-cell studies can complement classical biochemistry methods in dissecting ER chaperone function and organization in cells.