Part:BBa_K5182022
For testing the UVR8-COP1 light control system mechanism
SJTU-BioX-Shanghai 2024
Background
To verify the feasibility of the UVR8 photosensing system in mammalian cells, we designed a set of fluorescent labeling experiments to monitor the dynamic behaviors of UVR8 and COP1 under UV-B treatment in real time by fluorescence signal changes. In this experiment, the UVR8 protein was fused with green fluorescent protein (GFP), and the COP1 protein was fused with nuclear localization signal (NLS) as well as red fluorescent protein (mCherry). The RUP2 gene in plants, on the other hand, was linked to the tetracycline-inducible promoter (TRE promoter), and RUP2 (REPRESSOR OF UV-B PHOTOMORPHOGENESIS 2) is a core protein that regulates the response to ultraviolet B (UV-B) in plants, and RUP2, as a negative regulator of the UV-B signaling pathway, can transfer UVR8 from the UVR8-COP1-SPA4 complex, which promotes the re-dimerization and thus inactivation of UVR8 [1]. This design allowed us to monitor the dynamic interaction of UVR8 and COP1 by observing changes in the localization and intensity of fluorescence and to test the existence of a negative feedback regulatory mechanism in our optogenetic system by tetracycline-induced RUP2 production.
BBa_K5182022——COP1-NLS-mcherry
This Composite part will express red fluorescence in the nucleus in the absence of UVB signal; when UVB signal is received, UVR8 enters the nucleus and forms a polymer with COP1, and GFP co-localizes with mcherry to produce yellow fluorescence.
Usage and Biology
In this composite part, we connect COP1 with mcherry and NLS to form a composite part that can be used to observe the nuclear localization of COP1 in real time. When there is no UVB signal in the environment, COP1 will exists in the nucleus. When UVB signal is present in the environment, UVR8 conformation changes, dimers depolymerize, and monomeric UVR8 will carry GFP into the nucleus. then, UVR8 will interact with COP1 protein after receiving UVB signal[2]. When UVR8 interacts with COP1 and polymerizes together, we can observe yellow fluorescence in the nucleus due to the co-localization of GFP and mcherry. Through these composite parts, we have built a set of molecular modules to finely analyze the mechanism of optogenetic system.
Fig1. UVB signaling response mechanism of UVR8 and COP1.(a) In the absence of UVB signal, UVR8 and COP1 exist outside and inside the nucleus, respectively. (b) After receiving UVB signal, UVR8 carries GFP into the nucleus and interacts with COP1, which co-localizes with mcherry to produce yellow fluorescence.
Characterization
We observed the cell slides with different treatments under confocal microscope, and a field of view was found using a 20x magnification. Subsequently, find a field with more representative cells in different channels, and a multi-channel film was used to prepare for cell counting.
Fig2. 20x confocal microscope multichannel combination plots of UVR8-GFP and COP1-NLS-mcherry plasmid-transfected 293t cells.（NO UVB）
Fig3. 20x confocal microscope multichannel combination plots of UVR8-GFP and COP1-NLS-mcherry plasmid-transfected 293t cells.（UVB）
Table1. Statistics of the number and proportion of cells with dark nuclear fluorescence
Fig4. statistical chat of nuclear fluorescence status
Transfection of pIRESN2-GFP-UVR8, pSNV2-CHER-NLS-COP1, without UV irradiation, found that the proportion of cells with dark green nuclei was 27.78% on average, after UV irradiation, the proportion decreased to about 6.91%.
In summary, we transfected plasmids pIRESN2-GFP-UVR8 and pSNV2-CHER-NLS-COP1 into cell lines. After ultraviolet irradiation, we found that a large part of the green fluorescence originally in the cytoplasm entered the nucleus. we have verified that our system can be induced into the nucleus by ultraviolet light, which lays the foundation for further experiments. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
- ↑ Tilbrook, K., Arongaus, A. B., Binkert, M., Heijde, M., Yin, R., & Ulm, R. (2013). The UVR8 UV-B photoreceptor: perception, signaling and response. The Arabidopsis Book/American Society of Plant Biologists, 11.
- ↑ Wang Y, Wang L, Guan Z, et al. Structural insight into UV-B–activated UVR8 bound to COP1[J]. Science Advances, 2022, 8(16): eabn3337.
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