Study On Tunable Optogenetic System And Regulating The Signaling Pathway In Mammalian Cells

Optogenetics uses expression of genetic encoded proteins and combined with optical controlling and laser-based imaging, which offers a brand-new, damage-free, reversible, non-invasive and super temporal-spatial resolution research tool. Early studies of using optogenetics are mainly focus on neuroscience, which utilized the first generation of optogenetic components to observe and control specific neural activities in the brain, and got unprecedented achievements. In recent years, with the development of different optogenetic components, the application of optogentics has been constantly expanded. Nowadays, using optogenetic technology to study signal transduction in cells becomes a hot area of research. These photoactivatable proteins use protein conformation, such as allosteric, oligomerization and depolymerization to control and regulate intracellular signaling molecules. In addition, the advance of other research tools, such as microscopic imaging, light stimulation and mathematical analysis methods make optogenetics fully accessible for researches.To date, the optogenetic components that were discovered and engineered by genetic engineering and protein engineering are roughly consists of six types. These photoactivatable proteins use conformation allosteric and interaction of two proteins to control protein interactions and position. These tools can help researchers to achieve light controlling on a variety of signaling pathways and cellular activities in cells.In this thesis work, I firstly designed and developed a tunable LED light stimulation system, used CRY2/CIBN optogenetic modules and combined with Ring TIRFM system to achieve optically tunable activation of PI3K/PI(3,4,5)P3/Akt signaling pathway in mammalian cells. Ultimately, this system was applied to study of the epithelial-mesenchymal transition (EMT) regulation in A549 lung cancer cells. The thesis project consists of the following work:(1) Read the literature and analyzed the theoretical basis for the research. Before carrying out the project, many related papers have been read to learn the latest researches in this field. An optically tunable optogenetic research approach based on CRY2/CIBN components were put forward, which used LED array as light source to stimulate transfected cancer cells and studying of EMT process in tumor. At last, through theoretical analysis of the feasibility of this project, I obtained theoretical basis for this research.(2) Designed and established the LED stimulation system. Based on previous literature and theoretical analysis, LED stimulation system was designed and set up under the conditions of low cost and power, convenient and stable performance. Blue light LED lamps (460 nm-465 nm) were used as light resource to set up a 4x4 LED array. STC89C52 chip was selected as the whole system Microprogrammed Control Unit and welded on the PCB. The host control software was programmed on the Visual C++6.0 platform. The connection between the upper and lower machine was achieved by Bluetooth, which permits wireless communication and control even in the cell incubator.(3) Using CRY2/CIBN based optogenetic module to achieve optically tunable PI3K/PI(3,4,5)P3/Akt signaling activation in Hela cells. Different intensity of blue light laser, LED stimulation schemes, duration of imaging and exposure were chosen to design the experiments schemes. The degree of the PI(3,4,5)P3 levels in Hela cells were detected using Ring TIRFM and I found that the phosphorylation of Akt was in a dose-dependent manner with light exposure as revealed by immunofluorescence staining and western blot.(4) Using the established optogenetic system to achieve optical regulation of EMT in A549 lung cancer cells. The LED stimulation parameters were settled down to design the experiment schemes. The levels of Akt phosphorylation in A549 cells were detected by western blot after LED stimulation. The expression of two protein markers for EMT induction, E-cadherin and Vimentin were measured and the role of PI3K signaling in the regulation of EMT process was investigated.