Study On Single Molecule Imaging Of EGFR(Epidermal Growth Factor Receptor)using A Novel Small Molecule Fluorescent Probe

Epidermal growth factor receptor(EGFR)is a receptor tyrosine kinase,and with growth factors such as epidermal growth factor and transforming growth factor as ligands.After binding with the ligands,EGFR spontaneously polymerizes,activates its kinase activity and autophosphorylates,and conducts to multiple signaling pathways in the cell,such as MAPK,PI3K-Akt signaling pathway,etc.Since EGFR was found,many clinical data have shown that EGFR plays an important role in some diseases,especially in tumors.Amplification and multiple site mutations of EGFR are found in some cancer tissues.Recently with the rapid development of precision medicine,some small molecule epidermal growth factor receptor tyrosine kinase inhibitors came into market.For example,the third generation of EGFR small molecule inhibitor osimertinib(also known as AZD9291)that specifically covalently binds to EGFR mutations such as EGFR T790 M,etc.Although osimertinib can effectively prolong the survival of patients for about 8-10 months,the resistance to it still occur due to mutations such as C797 S of EGFR or continuous activation of bypass signal pathway.Thus there is still a great demand for further research on EGFR.Recent development in super-resolution microscopy based on single molecule fluorescence imaging breaks the diffraction limitation and allows to record and reconstruct fluorescent images with resolution far beyond the diffraction limited resolution of optical microscopy.Some fluorescent emitters,like some fluorescent proteins and small fluorescent molecules,can keep most in the dark state in the specific reducing solution under the strong laser illumination,and in each time only a few number of them can be activated to emit fluorescence.In this case,the image of each fluorescent emitter can be analyzed to determine its spatial location,and then the process is repeated until all the fluorescent emitters are activated and located.Finally,the location information of all the fluorescent emitters is statistically generated and reconstructed to a super resolution image.By this method,two neighbouring fluorescent emitters,whose distance is below the diffraction limit and can not be distinguished in one imaging process,can be distinguished at different time points.Thus,a super resolution image withthe best resolution of 10-20 nm can be achieved in this case breaking the diffraction limit.The key point of the super resolution imaging is the development of high-precision single light activated and photoswitched fluorescent molecules for cell structure and protein labeling.At present,some specific fluorescent proteins or fluorescent sequences can be utilized for single molecule imaging.Dipyrromethene boron difluoride(BODIPY),one of the commonly used fluorescent compounds,has the specification.Prof.Hu Youhong's group in Shanghai Institute of Materia Medica,Chinese Academy of Sciences has developed a small molecule fluorescent probe P48 to label EGFR by covalently combining bodipy and osimertinib.In chapter 1-4 of the thesis we focused on the investigation of the properties of P48,the effect of P48 on EGFR and downstream signal pathways,the binding of P48 to EGFR and single molecule imaging of P48.Firstly,we measured the photophysical properties,the fluorescence photostability at different pH,and the "on-off target" effect in the cell suspension of P48 probe.Secondly,we performed native polyacrylamide gel electrophoresis on the protein lysates of cells incubated with P48.By comparing the results of fluorescence imaging and immunoblotting,we found a good overlap between P48 and EGFR in gel.Pretreatment of osimertinib also attenuated the intensity of P48 in gel.These revealed the binding of P48 to EGFR.Thirdly,we employed Western blotting to test the activity of EGFR in three cell lines H1975,PC9 and A549,and the effect of P48 on their EGFR and related downstream pathways.Finally,we carried out single molecule imaging experiments with P48,and measured some single molecule properties of it.We also analyzed the dynamic distribution and aggregation degree of P48 in living cells at single molecule level.The statistical results of all the activated fluorescent molecules showed that the aggregation degree of P48 probe was significantly strengthened after EGF stimulation,which again revealed the binding of P48 to EGFR.In chapter 5 of the thesis we used Single Particle Tracking(SPT)technology to track the uptake of DNA/PEI nanoparticles into the cells.We used total internal reflection fluorescence microscope and successfully observed the process of DNA / PEI nanoparticle uptake into cells by imaging 5-tamra-se(red fluorescent dye)labeled DNA /PEI nanoparticles and DIO(green fluorescent dye)labeled HeLa cells.Then we quantitatively analyzed the trajectory of nanoparticles,and found that the nanoparticlesdid not separate or divide when entering the cell,and the speed of nanoparticles in cells was slightly faster than that out of cells.This suggests that SPT can be utilized to track and analyze the delivery,movement and distribution of nanoparticles in cells.In chapter 6 of the thesis we briefly described that the inhibition of the activity of epigenetic factor G9 a can induce apoptosis in bladder cancer cells.G9 a is a histone methyltransferase that specifically mediates mono-and di-methylation of histone H3 lysine 9(H3K9).Western Blotting results showed that knockdown of G9 a by small interfering RNAs(siRNAs)or suppression of G9 a via a small molecule inhibitor UNC0642 can induce apoptosis in bladder cancer cells,suggesting the possibility of G9 a as a potential therapeutic target for bladder cancer.