Methods for monitoring endoplasmic reticulum stress and the unfolded protein response

Abstract

The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

Figure 1

Detection of transcript levels of UPR target genes by RT-PCR. (a) Upper panel, cartoon of XBP1 splicing during ER stress. Lower panel, schematic representation of various mutant constructs of IRE1. (b) Modulation of XBP1 splicing by mutant IRE1. Total RNA was isolated from HEK 293 cells that were transfected with IRE1 mutants, either untreated or treated with thapsigargin (0.5 μM) 6 hours, and RT-PCR analysis of total RNA was performed to simultaneously detect both spliced and unspliced XBP1 mRNA and GADPH. (c) Induction of UPR target genes upon exposure to thapsigargin. Total RNA was isolated from indicated cells after treatment thapsigargin (Tg), and the expression levels of the indicated genes were determined by real-time RT-PCR, normalizing against GAPDH expression.

Figure 2

Detection of protein levels of UPR target genes. (a) Immunohistochemical detection of CHOP, GRP78, and XBP1 in Multiple Sclerosis patient postmortem brain tissue. Representative images showing upregulation of CHOP (ii) at the edge (LE) of a chronic active lesion, in comparison to (i) NAWM. GRP78 expression was downregulated in the center of a chronic active lesion (iii) when compared to the edge (iv) of actively demyelinating lesions. Sample images illustrate the variety of morphologically distinct cell types that express CHOP or GRP78 including macrophages (Mø), astrocytes (a), and oligodendrocytes (o). Increased expression of XBP1 was found at the edge of a chronic active lesion (vi), when compared to normal-appearing white matter (v). XBP1 immunostaining is also apparent in a large number of oligodendrocytes (o). All immunoperoxidase-stained cells were detected using the chromogen, DAB (brown), and counterstained with hematoxylin for nuclei (blue). Scale bars = 250 μm. Letter codes are as follows: NAWM = normal-appearing white matter; LC = lesion center; LE = lesion edge. Red astrices indicate location of lesion within brain sections analyzed. (b) PC12 cells were treated with 0.25 μM of Tg for 0, 2, 4, 6, and 8 hours. Whole cell lysates were analyzed by Western Blot for GRP78, CHOP, spliced XBP1, phospho-eIF-2α, and total- eIF-2α. β-Actin was used to determine equal loading of samples.

Figure 3

Detection of IRE1 activity using “ER stress-activated indicator” (ERAI) constructs. (a) Schematic presentation of ERAI plasmid obtained by fusing XBP1 and venus, a variant of the green fluorescent protein (adapted from [36] by Iwawaki et al. (2004)). (b) Twenty-four hours after transfection F-XBP1-venus and F-XBP1ΔDBD-venus, 293T cells were left untreated or treated with (1 μg/mL) tunicamycin for 24 hours and then analyzed by fluorescence microscopy.

Figure 4

ER stress-induced processing and nuclear translocation of GFP-ATF6. (a) Twenty-four hours after transfection with pCMVshort-EGFP-ATF6 (WT), 293T cells were left untreated or treated with 1 μg/mL tunicamycin for the indicated periods. Cells were fixed in 4% paraformaldehyde, stained with DAPI, and then analyzed by fluorescence microscopy. (b) Twenty-four hours after transfection with pCMVshort-EGFP-ATF6α(WT), pCMVshort-EGFP-ATF6α(S1P⁻), or pCMVshort-EGFP-ATF6α(S2P⁻), 293T cells were left untreated or treated with 1 μg/mL tunicamycin for the indicated periods and then analyzed by fluorescence microscopy.