Measuring ER stress and the unfolded protein response using mammalian tissue culture system

Abstract

The endoplasmic reticulum (ER) functions to properly fold and process secreted and transmembrane proteins. Environmental and genetic factors that disrupt ER function cause an accumulation of misfolded and unfolded proteins in the ER lumen, a condition termed ER stress. ER stress activates a signaling network called the Unfolded Protein Response (UPR) to alleviate this stress and restore ER homeostasis, promoting cell survival and adaptation. However, under unresolvable ER stress conditions, the UPR promotes apoptosis. Here, we discuss the current methods to measure ER stress levels, UPR activation, and subsequent pathways in mammalian cells. These methods will assist us in understanding the UPR and its contribution to ER stress-related disorders such as diabetes and neurodegeneration.

Figure 1

Response categories of the Unfolded Protein Response. Three ER transmembrane proteins, IRE1, PERK, and ATF6, sense ER stress in the ER lumen and become activated regulating a cascade of signaling pathways collectively termed the Unfolded Protein Response (UPR). The UPR has three functions: adaptive response, feedback control, and cell fate. Under the adaptive response, the UPR aims to reduce ER stress and restore ER homeostasis. If the UPR is successful, the UPR signaling pathways are turned off by feedback mechanisms. The UPR also regulates both survival and death factors that govern whether the cell will live or not depending on the severity of the ER stress condition.

Figure 2. Studying ER stress and the Unfolded Protein Response in Mammalian cells

ER stress can be induced by several chemical and physiological inducers. Actual ER stress has been difficult to measure directly. Currently observing ER distension by electron microscopy (EM) and measuring oxidative protein folding (eroGFP) are available. The activation of the UPR master regulators has also been challenging but could be attempted by detecting IRE1α and PERK phosphorylation, and ATF6α cleavage by specific antibodies using western blot (WB). ATF6α translocation can be monitored by fluorescence microscopy of GFP-ATF6α. The downstream outputs of the UPR master regulars are readily measurable. IRE1α splices XBP-1 mRNA which can be detected by quantitative real time PCR (qRT-PCR), XBP-1 venus, and western blot (WB). IRE1α also mediates degradation of ER localized mRNAs, which can be measured by pulse chase assays. PERK phosphorylates eIF2α, which can be detected by specific antibodies. Phosphorylated eIF2α triggers global mRNA translation attenuation, which can be measured by standard methods such as pulse labeling and polyribsome profiling. Activated ATF6α is a transcription factor and regulation of its downstream target genes can be measured by qRT-PCR. The UPR in general regulates several transcription factors and in turn their transcriptional targets can be measured by qRT-PCR and luciferase assays. The UPR also induces expression of ERAD components as well as survival and death components. ERAD and apoptosis can be measured by standard methods.