Single-particle Tracking Microscopy Enables Investigation Of Receptor-ligand Interaction In Live Cells

For a long time,the interaction between cell membrane receptor and ligand molecules has been a research hotspot in various fields such as molecular biology,biophysics and nanobiology.Owning to the capability of in-situ monitoring the ongoing biological events at the unprecedented resolution,single-molecule approaches have provided new opportunities for investigating receptor-ligand interactions in live cells.In the past years,several single-molecule techniques such as fluorescence cross-correlation spectroscopy and fluorescence resonance energy transfer have been developed for the measurement of receptor affinity towards the corresponding ligands.These single-molecule affinity anal ysis methods relied on the determination of receptor-ligand complexes at the equilibrium state.Such techniques also required fluorescent labeling of receptor and ligand simultaneously.In contrast,single-particle tracking technique is capable of exploring the molecular dynamics and interactions of individual membrane receptors over extended periods in live cells.However,quantitative detection of receptor-ligand interaction kinetics and receptor affinity via single-particle tracking techniques remain challenging due to the difficulty of extracting transient binding and unbinding properties from the complicated mobility observations.Therefore,in this thesis,we performed the research in the following three parts:In the second chapter,we have proposed a robust single-particle tracking microscopy?SPTM?for the in-situ determination of receptor-ligand interaction kinetics and receptor affinity simultaneousl y in live cells.We linked the cyclic RGD peptide?cRGD?with gold nanoparticles using polyethylene gl ycol to construct bioactive and biocompatibile nanoprobes.We used the dark field microscopy to observe the interaction between the probe and the integrin receptor on cell membrane in real time.The data was automati call y identified by using a robust two-state hidden Markov model,and we can obtain the dwell time of nanoprobes at the integrin-ligand bound and unbound states respectively.Moreover,on the basis of theoretical derivations of receptor-ligand binding and unbinding reactions,we obtain the equations of the time-dependent association and disassociation probability,and the rate constants can be then extracted by fitting the corresponding probabilit y with above equations.With the association and disassociation rate constants,we determined the affinity constant(².5?mol·dm^-3)of the cRGD peptide to the integrin receptor at the nonequilibrium state.In the third chapter,we investigated the effect of Mn²⁺on the meditation of integrin-ligand interactions.Using the established approach for in-situ detection of receptor-ligand interaction kinetics,we analyzed binding and unbinding processes respectively.We found that Mn²⁺activator can promote the integrin-ligand engagement and lower the dissociation of integrin-ligand complexes.We further validated the results obtained by SPTM by using conventional flow-cytometry-based affinity assay.In the fourth chapter,we investigated the interaction between aptamers and protein molecules on target cells.By using the aptamer?TLS11a?targeting hepatocellular carcinoma cells as a model,we tracked interaction between the aptamer-functionalized nanoprobe and hepatocellular carcinoma cells in real time.We validated the capability of SPTM for the anal ysis of aptamer-protein interactions on target cells.