1. Testing the Stability of secNLuc in Culture
Developing a luciferase reporter to monitor ER stress response
Molly Lutrey
Mentored by Dr. Mark Henderson and Dr. Brandon Harvey
Introduction
Methods (cont.)
The secNLuc tool had an increased expression of 4 fold over the course of 24 hours when treated with varying concentrations of thapsigargin as shown in Graph 1. This is indicative of a functioning and responsive reporter. The stability of secNLuc was tested in SY5Y media. SecNLuc remained stable outside the cell for up to 120 hours as shown in Graph 2. A destabilizing domain was attached to the ATF6 reporter in order to prevent its expression during ER stress. When destabilized, there was no secNLuc expression in response to ER stress, but when TMP was added, the secNLuc expression increased during ER stress as displayed in Graph 3.
The 5xATF6 secNLuc tool has been successfully constructed and is ready to be tested in vivo for monitoring disease pathogenesis. The 5xATF6 construct is responsive to thapsigargin treatment, an inducer of ER stress. The UPR is activated in the experiment as the reporter is expressed during treatment. The data from the destabilizing ATF6 experiment supports the theory that ATF6 is what directly drives the expression of the tool. The tool had a higher expression when ATF6 was stabilized with the TMP drug. The future direction for this project is to fuse the tool into a virus so that it may be injected into an SY5Y cell line as a part of the genome. Utilizing the tool in cell line will allow the possibility of running a dual assay with a Gaussia luciferase reporter, Secreted Endoplasmic Reticulum Calcium Monitoring Pathway (SERCaMP), to monitor ER stress and calcium levels.
Results (cont.)
A cell’s endoplasmic reticulum (ER) plays a crucial role in the folding and assembly of membrane and secretory proteins. In order for the ER to maintain its proper function, calcium must be at an ideal concentration. Thapsigargin can disrupt ER calcium levels, inducing an unfolded protein response (UPR) and causing ER stress. As illustrated in Figure 1, the UPR is comprised of multiple, intertwined signaling pathways within the ER that work simultaneously to return the ER to homeostasis. One arm of the UPR consists of a transcription factor, ATF6, which moves to the nucleus to regulate gene expression. (Ron & Walter, 2011). To monitor ATF6 activity a previously characterized reporter was adapted, in which firefly luciferase (F.Luc) enzyme expression is regulated by ATF6 transcription factor binding sites (Arenzana, Kaufman, Prywes, Shen, Tirasophon, & Wang, 2000). The ATF6 binding sites were utilized to control expression of secreted NanoLuc (secNLuc), to develop a more robust sensor of the ER stress response. A plasmid lacking the ATF6 binding sites was created as a control. The sensitivity of secNLuc plasmid provides a potential opportunity to monitor ATF6 activity in vivo during disease pathogenesis.
The 5xATF6-F.Luc plasmid was obtained from Addgene (#11976). The DNA was transformed into Stbl3 cells. The ATF6 promoter fragment, minimal promoter fragment, secNLuc fragment, and 5xATF6-F.Luc plasmid backbone fragment were amplified by PCR. The DNA fragments were separated on an agarose gel by running gel electrophoresis. The desired fragments were purified from the agarose gel, followed by fusion. The first construct contained a minimal promoter controlling secNLuc. The second construct contained a minimal promoter and 5 ATF6 binding sites controlling secNLuc. SY5Y cells were transfected with the plasmid containing ATF6 sites driving expression of the secNLuc enzyme using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, SY5Y cells were treated with varying concentrations of thapsigargin (0 nM-300 nM) over a course of 24 hours. An assay was performed to measure secNLuc expression within the ER stressed cells using coelenterazine.
SY5Y cells were transfected with 5xATF6 secNLuc plasmid using Lipofectamine 2000 (Invitrogen). Seventy-two hours after transfection, 1 mL of media was removed from transfected SY5Y cells and added to a conical tube. A 100 µL sample was taken from the cell media in the conical tube, and stored at 4 °C. The cells were plated at 1.75 × 106 SY5Y cells per mL into a T25 dish. After 24 hours, the cells were transfected with 2 µg of ATF6 plasmid and 8 µg of Destabilized Domain ATF6. Transient transfection was completed using Lipofectamine Two days post transfection, cells were re-plated into 96 well plates and treated with TMP. After 24 hours of thapsigargin, 5 µL of media sample was collected. The secNLuc sample was read utilizing the coelenterazine substrate.
Graph 3: There was more 5xATF6 secNLuc activity as TMP increased. After performing a two-way ANOVA test, the ATF6 concentrations were significantly different while the minimal promoter concentrations were not significantly different.
5xUPRE
Min promoter
Firefly luciferase
LTR
polypurine tract
Cla1 site destroyed
GFP Stuffer
Cla1 site rebuilt
5xUPRE
Min promoter
secNLuc
Ascii site
WPRE
Figure 2: The previously characterized F.Luc plasmid (top) was cut to insert secNLuc, then the secNLuc plasmid was inserted into a Plenti6.3 backbone (bottom).
GRP78 ER
Splicing
mRNA XBP1s
XBP1s
Nucleus
eIf2a P
Reduced translation
Cytosol
Golgi
IRE1
PERK
ATF6
ATF4
Conclusions
XBP1s
Figure 1: Schematic of the UPR pathways as the ER undergoes stress. The pathway produces transcription factors that induce expression of proteins to assist in the proper folding of the misfolded proteins.
IRE1
ATF4
Results P
ATF6
Graph 1: Thapsigargin treatment increases expression of 5xATF6 secNLuc. After performing a two-way ANOVA test, the 5xATF6 activity was significantly different while the minimal promoter activity was not significantly different.
ATF6
Methods
References
Ron, D., & Walter, P. (2011). The Unfolded protein response: from stress
pathway to homeostatic regulation. Science, 334,
Arenzana, N., Kaufman, R. J., Prywes, R., Shen, J., Tirasophon, W., & Wang, Y. (2000).
Activation of ATF6 and an ATF6 DNA binding site by the ER stress response.
The Journal of Biological Chemistry, 275, 1-33.
Testing the Stability of secNLuc in Culture
Graph 2: secNLuc concentration decreased by 3.5% everyday incubating at 37 ºC. After performing a two-way ANOVA test,
37 °C incubation activity was significantly different after 72 hours while 37 °C incubation on cells was not significantly different.
Acknowledgments
A special thank you to my mentors, Dr. Brandon Harvey and Dr. Mark Henderson, and my colleague Steve Yan, as well as to my faculty advisor, Mr. Gareth Davis. This work was supported by the Intramural Research Program at NIDA-NIH.