Team:KOREA/Demonstrate
Demonstrate
We decided to measure the membrane potential to check that our protein works properly. We don’t know how to design a linker sequence between the transmembrane domain of Rhodopsin and the intracellular domain of DRD2. In other fusion proteins, We searched for previous papers on how to design, but did not find the exact method, and made three candidates. Since our design lacked evidence, We had to make sure that our fusion protein worked. If our fusion protein works, it will initiate the dopamine signaling pathway. Thus, we selected the U-87 MG cell that dopamine signaling pathways downstream is expressed. There were several opinions about how we check that the dopamine signaling pathway has initiated. Measuring cAMP concentration, Measuring Membrane potential, and Measuring activation of Gi/o. Among them, we choose to measure the membrane potential with membrane potential dye. We do experiments with DiOC2. DiOC2 emit red fluorescence in resting potential and emit green fluorescence in action potential. When we first design the fusion protein, we didn't know how to design the linker sequences between the transmembrane domain of rhodopsin and the cytoplasmic domain of the DRD2. n other fusion proteins, we searched the preceding paper for how to design but did not find the exact method, so we made three candidates. Since our design lacked enough evidence, we had to make sure our fusion proteins were working properly. If our fusion protein works, it will initiate the dopamine signaling pathway. So we used a U-87 MG cell that expressed the dopamine downstream signaling pathway. There were several opinions on how to identify dopamine downstream. We learned how to measure the concentration of cAMP, how to measure the difference in membrane potential, and how to measure the activity of Gi / o. Among them, the membrane potential dye was used to measure the difference in membrane potential. It was difficult to measure Gi / o activity and the concentration of cAMP was likely to change due to other factors. We experimented with DiOC2. DiOC2 originally emits red wavelengths of light and then turns to green when the membrane potential depolarized. If the fusion protein we designed is working properly, in the presence of light the rhodopsin on the membrane is stimulated by the light and Gi /o and Gbr are separated to promote the release of potassium from the GIRK channel. As potassium escapes, the color dyed with DiOC2 becomes green.
This is GGGGS 2.5D image cultured in dark condition. there is many green background noise and red signal in cell. Red signal says that cells are in resting potential.
This is GGGGS 2.5D image in cultured in light condition. there is a significant difference between dark conditions. There are many green signals is in cell and clear background. The green signal says that the K+ ion channel is open. Just like we expected before the experiments, membrane potential has changed. This means our protein works properly in the human neuronal cell.
Without blue light, the GIRK channel isn't activated and DiOC2 stays red. When light stimulation is given to the WT(wild type) cells, DiOC2 keeps red state unrelated to the light stimulation. The image above shows the cells dyed with red DiOC2. The red peak distinctively implies the inactivation of cells.
When blue light stimulation is given to the cell transfected with the chimera protein, it's potassium channel GIRK is opened to induce hyperpolarization which causes DiOC2 to turn to the green from red. There are strikingly remarkable green peaks on the image above which means the activation membrane potential.
Light exposure time is 1 hours.
a and d are GS. In a, the light source is too strong and the background is too bright. It's hard to identify the cell shape, but you still see a red wavelength signal. At d the red signal turned green overall. However, the fluorescence of the cells does not differ significantly from the background, so the background looks blurry. b and e are GGGGS. In b we see a neat red signal. In e, the cell shows a green signal that is distinct from the background. c and f are GSGGGSGGGSGGGS. In c, only the background shows green fluorescence and there are no signals in cells. In f, it's green, except for the lower right red signal. When we compared the controls, we chose GGGGS because GGGGS changed the cell membrane potential neatly.
