Limits...
Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

Samali A, Fitzgerald U, Deegan S, Gupta S - Int J Cell Biol (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Ireland, Galway, Galway, Ireland.

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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2821749&req=5

fig3: 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.

Mentions: ER stress-dependent splicing of XBP1 has been used to develop fluorescent reporter constructs by fusing XBP1 sequence to venus, a variant of green fluorescent protein which enables the activation of IRE1 to be monitored [36, 37]. The design of the XBP1-venus reporter is shown in Figure 3(a). In this construct, the gene encoding venus is cloned downstream the 26-nt ER stress-specific intron of human XBP1 [36]. Under normal conditions, the mRNA of the fusion gene is not spliced, and its translation terminates at the stop codon near the joint between the XBP1 and venus genes. However, during ER stress, the 26-nt intron is spliced out, leading to a frame shift of the chimeric XBP1-venus mRNA, similar to that of the endogenous XBP1 mRNA. Translation of the spliced mRNA produces an XBP1-venus fusion protein and cells experiencing ER stress can be detected by monitoring the fluorescence activity of venus. As venus expression can only occur from the spliced form of the XBP1-GFP mRNA, its presence signals the activation of IRE1. Upon transfection of the XBP1-GFP reporter into cells, tunicamycin treatment results in detectable fluorescence in the nucleus, whereas negligible fluorescence is detected in any compartment under normal conditions [36, 37]. Moreover, venus expression during tunicamycin treatment has been shown in splicing assays to correlate with the extent of splicing of the UPR intron from XBP1/GFP mRNA [36, 37]. We have used 293T cells to detect activation of IRE1 using two different XBP1-venus reporter plasmids: F-XBP1-venus and F-XBP1ΔDBD-venus (Figure 3(a)). In F-XBP1ΔDBD-venus construct, DNA-binding domain (DBD) of XBP1 is deleted. F-XBP1ΔDBD-venus construct is recommended for use as overexpression of F-XBP1ΔDBD-venus does not affect induction of UPR target genes and can be used to detect activation of IRE1 similar to F-XBP1-venus construct. F-XBP1ΔDBD-venus construct has been used to generate a transgenic mouse model for monitoring ER stress (discussed later). Twenty-four hours post transfection, cells are induced to undergo ER stress by incubating with appropriate concentrations of tunicamycin, for 24 hours. In the cells transfected with F-XBP1-venus construct, tunicamycin treatment leads to appearance of green fluorescence in the nucleus (Figure 3(b)). However in the cells transfected with F-XBP1ΔDBD-venus construct, tunicamycin treatment leads to appearance of green fluorescence in the cytosol (Figure 3(b)). One important point to note is that overexpression of F-XBP1-venus construct interferes with induction of UPR target genes in a dominant-negative manner [36]. The major drawback, however, is the relatively large amount of GFP that needs to be expressed in the cell for visualization by microscopy. Thus, there will be a time lag between actual IRE1 activation and its detection by the accumulation of GFP.


Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

Samali A, Fitzgerald U, Deegan S, Gupta S - Int J Cell Biol (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821749&req=5

fig3: 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.
Mentions: ER stress-dependent splicing of XBP1 has been used to develop fluorescent reporter constructs by fusing XBP1 sequence to venus, a variant of green fluorescent protein which enables the activation of IRE1 to be monitored [36, 37]. The design of the XBP1-venus reporter is shown in Figure 3(a). In this construct, the gene encoding venus is cloned downstream the 26-nt ER stress-specific intron of human XBP1 [36]. Under normal conditions, the mRNA of the fusion gene is not spliced, and its translation terminates at the stop codon near the joint between the XBP1 and venus genes. However, during ER stress, the 26-nt intron is spliced out, leading to a frame shift of the chimeric XBP1-venus mRNA, similar to that of the endogenous XBP1 mRNA. Translation of the spliced mRNA produces an XBP1-venus fusion protein and cells experiencing ER stress can be detected by monitoring the fluorescence activity of venus. As venus expression can only occur from the spliced form of the XBP1-GFP mRNA, its presence signals the activation of IRE1. Upon transfection of the XBP1-GFP reporter into cells, tunicamycin treatment results in detectable fluorescence in the nucleus, whereas negligible fluorescence is detected in any compartment under normal conditions [36, 37]. Moreover, venus expression during tunicamycin treatment has been shown in splicing assays to correlate with the extent of splicing of the UPR intron from XBP1/GFP mRNA [36, 37]. We have used 293T cells to detect activation of IRE1 using two different XBP1-venus reporter plasmids: F-XBP1-venus and F-XBP1ΔDBD-venus (Figure 3(a)). In F-XBP1ΔDBD-venus construct, DNA-binding domain (DBD) of XBP1 is deleted. F-XBP1ΔDBD-venus construct is recommended for use as overexpression of F-XBP1ΔDBD-venus does not affect induction of UPR target genes and can be used to detect activation of IRE1 similar to F-XBP1-venus construct. F-XBP1ΔDBD-venus construct has been used to generate a transgenic mouse model for monitoring ER stress (discussed later). Twenty-four hours post transfection, cells are induced to undergo ER stress by incubating with appropriate concentrations of tunicamycin, for 24 hours. In the cells transfected with F-XBP1-venus construct, tunicamycin treatment leads to appearance of green fluorescence in the nucleus (Figure 3(b)). However in the cells transfected with F-XBP1ΔDBD-venus construct, tunicamycin treatment leads to appearance of green fluorescence in the cytosol (Figure 3(b)). One important point to note is that overexpression of F-XBP1-venus construct interferes with induction of UPR target genes in a dominant-negative manner [36]. The major drawback, however, is the relatively large amount of GFP that needs to be expressed in the cell for visualization by microscopy. Thus, there will be a time lag between actual IRE1 activation and its detection by the accumulation of GFP.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Ireland, Galway, Galway, Ireland.

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.

No MeSH data available.


Related in: MedlinePlus