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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

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.
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fig4: 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.

Mentions: A key regulatory step in ATF6 activation is its transport from the ER to the Golgi body, where it is processed by S1P and S2P proteases [38, 39]. The cytoplasmic fragment of ATF6, thereby liberated from the membrane, translocates into the nucleus and activates transcription of its target genes [38, 39]. A GFP-ATF6 fusion protein, which relocates from the ER to the nucleus via the Golgi apparatus in response to ER stress, can be used to monitor activation of ATF6 by fluorescent microscopy [38, 39]. One limitation of this approach, however, is that overexpression can sometimes alter the subcellular localization and kinetics of protein trafficking. This problem has been addressed to some extent by expressing GFP-ATF6 from a shortened CMV promoter which has a deletion of 430 base pairs from the 5′ side. The short promoter possesses considerably lower activity than the full promoter and GFP-ATF6 expressed using the short CMV promoter is localized exclusively to the ER and translocates to the nucleus similarly to endogenous ATF6 [39]. For detection of GFP-ATF6, 293T cells were transfected with pCMVshort-EGFP-ATF6 (WT), pCMVshort-EGFP-ATF6 (S1P−), and pCMVshort-EGFP-ATF6 (S2P−) plasmids. pCMVshort-EGFP-ATF6 (S1P−) and pCMVshort-EGFP-ATF6 (S2P−) have a mutation that abrogates the cleavage by S1P or S2P, respectively. 24 hours post transfection, cells were treated with 1 μg/mL tunicamycin. As shown in Figure 4(a), the wild-type GFP-ATF6 was translocated to the nucleus via the Golgi apparatus. Both EGFP-ATF6 (S1P−) and EGFP-ATF6 (S2P−) were localized in 293T cells similarly to the wild-type GFP-ATF6 (Figure 4(b): a–c). In contrast to wild-type GFP-ATF6 (Figure 4(b): a, d, g), GFP-ATF6_(S1P−) (Figure 4(b): b, e, h) and EGFP-ATF6 (S2P−) (Figure 4(b): c, f, i) remained associated with the Golgi apparatus even 4 hours after tunicamycin treatment. These results demonstrate that cleavage by S1P and S2P is critical for the processing of GFP-ATF6 and that only the processed product, GFP-ATF6, can enter the nucleus. The advantage of GFP is that its intrinsic fluorescence allows the translocation of ATF6 to be continuously followed in single living cells and the whole process recorded over time using, for example, time-lapse photography.


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

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

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

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getmorefigures.php?uid=PMC2821749&req=5

fig4: 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.
Mentions: A key regulatory step in ATF6 activation is its transport from the ER to the Golgi body, where it is processed by S1P and S2P proteases [38, 39]. The cytoplasmic fragment of ATF6, thereby liberated from the membrane, translocates into the nucleus and activates transcription of its target genes [38, 39]. A GFP-ATF6 fusion protein, which relocates from the ER to the nucleus via the Golgi apparatus in response to ER stress, can be used to monitor activation of ATF6 by fluorescent microscopy [38, 39]. One limitation of this approach, however, is that overexpression can sometimes alter the subcellular localization and kinetics of protein trafficking. This problem has been addressed to some extent by expressing GFP-ATF6 from a shortened CMV promoter which has a deletion of 430 base pairs from the 5′ side. The short promoter possesses considerably lower activity than the full promoter and GFP-ATF6 expressed using the short CMV promoter is localized exclusively to the ER and translocates to the nucleus similarly to endogenous ATF6 [39]. For detection of GFP-ATF6, 293T cells were transfected with pCMVshort-EGFP-ATF6 (WT), pCMVshort-EGFP-ATF6 (S1P−), and pCMVshort-EGFP-ATF6 (S2P−) plasmids. pCMVshort-EGFP-ATF6 (S1P−) and pCMVshort-EGFP-ATF6 (S2P−) have a mutation that abrogates the cleavage by S1P or S2P, respectively. 24 hours post transfection, cells were treated with 1 μg/mL tunicamycin. As shown in Figure 4(a), the wild-type GFP-ATF6 was translocated to the nucleus via the Golgi apparatus. Both EGFP-ATF6 (S1P−) and EGFP-ATF6 (S2P−) were localized in 293T cells similarly to the wild-type GFP-ATF6 (Figure 4(b): a–c). In contrast to wild-type GFP-ATF6 (Figure 4(b): a, d, g), GFP-ATF6_(S1P−) (Figure 4(b): b, e, h) and EGFP-ATF6 (S2P−) (Figure 4(b): c, f, i) remained associated with the Golgi apparatus even 4 hours after tunicamycin treatment. These results demonstrate that cleavage by S1P and S2P is critical for the processing of GFP-ATF6 and that only the processed product, GFP-ATF6, can enter the nucleus. The advantage of GFP is that its intrinsic fluorescence allows the translocation of ATF6 to be continuously followed in single living cells and the whole process recorded over time using, for example, time-lapse photography.

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