Single Particle Imaging And Tracking Microscopy For Studying The Dynamics And Function Of Membrane Proteins

Receptor proteins on cell membrane will form clusters after activated in the presence of specific ligands or extracellular stimulis,which play critical roles in different biological processes such as molecular recognition,signal transduction,and material exchange.Therefore,investigatin g the organization and dynamics of receptor clusters is fundamental for the understanding of receptor function and regulation in live cells.Single-molecule/single-particle imaging and tracking microscopy enables the visual analysis of the dynamic behavior s of single molecules,and provide real-time quantitative information about the molecular interaction at single molecule level.It provides a powerful tool for scientific researchers to reveal complex biochemical reactions and biological events on cell mem branes.By using the single-particle imaging and tracking microscopy,this thesis focus on the analysis of dynamic interaction of the protease-activated receptor 1(PAR1)receptor clusters and the biological consequences in single live cells.The detailed researchs are described as follows:In Chapter 2,the active peptide ligand SFLLRN and gold nanoparticles were used to construct the nanoprobes for PAR1 receptor cluster labeling.With the single-particle tracking microscopy,the diffusive characteristics of nanoprobes before and after binding to single receptor cluster were determined in single live cellsIn Chapter 3,with the prepared nanoprobes in Chapter 2,we monitored the dynamic interaction process between PAR1 receptor clusters by single particle darkfield tracking microscopy.Furthermore,the effect of the inhibitor Parmodulin 2(PM2)on the dynamic cluster-cluster interactions was investigated,which suggested that PM2 could achieve the regulation of downstream signaling pathways by inhibiting the effective binding between receptor clusters.In Chapter 4,by using the quantum dots coated with sulfhydryl polyethylene glycol phospholipid as probes,we explored the changes in membrane fluidity in cells after the PAR1 signaling pathway was activated.Our results suggested that the diffusion rate of single probes on the leading edge of cell membrane was increased significantly,as compared to that of probes in the center region.These findings implied that the membrane fluidity characteristics was heterogeneous.