Study on the Dynamics of PTEN Protein and the p53-MDM2 Interaction in Single Cells Using Fluorescence Correlation Spectroscopy
Author:Du Zhi Xue
Supervisor:ren ji cun
As important carriers of biological functions,proteins play extremely important roles in life activities.A large number of important proteins have been found with the development of proteomics.The revelation of their physiological functions and regulatory mechanisms greatly promotes our understanding for the mechanism of life activities.So far,most of the research results about proteins are based on the biochemical methods.These methods are mainly carried out in a simple and controllable solution system,which makes the study much easier.However,as the basic unit of living organisms,cell is far more complexity than our imagination.The results obtained in the solution system may not be able to truly reflect the state of protein in living cells.In addition,most of the results in vitro are based on a cell population,ignoring cell heterogeneity.This will lead to the information obtained from these studies diluted,lost,or even come to a contradictory conclusion.Fluorescence correlation spectroscopy(FCS)is a single molecule detection method developed in recent years.The advantages of high sensitivity,high spatial resolution and noninvasive detection make FCS and its variants quite suitable for studying protein concentration,diffusion coefficient,oligomerization and interaction in living cells.Moreover,FCS is also suitable for dynamic measurement in a single living cell or even a subcellular structure.In this paper,we labeled target proteins with fluorescence proteins,and also constructed a system for cell culture,high-resolution imaging and in situ FCS measurement.And then,we measured the dynamics of PTEN protein and the interaction between p53 protein and MDM2 protein,which are closely related to cancer.We systematically studied their functions and regulatory mechanisms in single living cells both from protein structures and external stimuli aspects.The research work mainly includes:1.PTEN(phosphatase and tensin homolog on chromosome 10)is one of the most important tumor-suppressor proteins,which plays a key role in negative regulation of the PI3K/AKT pathway,and governs lots of cellular processes including growth,proliferation,survival and migration.So far,although PTEN has been studied in depth,it is still not clear about the dynamics of PTEN proteins in single living cells.Here,we established a monoclonal cell line stably expressing EGFP-PTEN fusion proteins,and then studied the dynamics of PTEN both in nucleus and cytoplasm by using FCS method.We investigated the dynamics of PTEN in single living cells under oxidative stress stimulation and the cellular ATP depletion treatment.With oxidative stress stimulation,EGFP-PTEN concentration increased in nucleus,but slightly decreased in cytoplasm;the diffusion coefficient and the anomalous parameter α of EGFP-PTEN decreased significantly both in nucleus and cytoplasm.Under the cellular ATP depletion treatment,the concentration of EGFP-PTEN remained unchanged in nucleus and decreased significantly in cytosol.Meanwhile,the diffusion coefficient of EGFP-PTEN decreased significantly in cytosol,but it showed no significant change in nucleus.The anomalous parameter α decreased significantly both in nucleus and cytoplasm.These results suggest that the concentration and the mobility of PTEN in cells can be regulated by stimulation ways.These stimulations have different effects on the nucleus and the cytoplasm,which reflects the differences in function and regulation of PTEN protein between the nucleus and the cytoplasm.2.The tumor suppressor protein p53 plays a central role in apoptosis,cell cycle arrest and DNA repair,and MDM2 protein is a key negative regulator of p53.Since MDM2-p53 interaction plays a key role in p53 regulation,it has been studied in depth.However,due to the limitation of traditional biochemical methods,it is still not clear about how p53 structures influence the p53-MDM2 interaction in living cells.Here,we developed a direct method for studying the p53-MDM2 interaction in living cells using fluorescence cross-correlation spectroscopy(FCCS).p53 and MDM2 proteins were labeled with EGFP and mCherry in living cells,respectively,using lentivirus infection and stable clone selection.This was the first time for observing the p53-MDM2 interaction in living cells using FCCS method.We then designed various p53 mutants and systematically studied the effects of p53 homo-oligomerization,helicity of p53 transactivation domain(TAD)and the hot-spot mutants in p53 DNA-binding domain on p53-MDM2 binding affinity in living cells.Based on these results,the effects of the alterations in the primary,secondary and quaternary structures of protein on the protein-protein binding affinity in living cells can be studied,and we can draw some major conclusions:(1)The p53 monomers cannot bind to MDM2,while both p53 dimers and tetramers can bind to MDM2,and the affinity of p53 tetramers is higher than for the p53 dimers.This result illustrates that MDM2 can selectively bind to p53 tetramers and regulate the p53 functions.(2)The affinity of p53 to MDM2 increased with the increase in the helicity level of p53 TAD.This directly demonstrates that the helicity level of the p53 TAD determines the affinity of p53 to MDM2 in living cells.(3)Most of the hot-spot p53 mutants in the DNA-binding domain did not alter the affinity of p53 to MDM2,but the R175 H mutation resulted in a decrease in the affinity of p53 to MDM2 in living cells due to its highly unfolded structure.3.MDM2 in the tumor cells with wild-type p53 is usually amplified and overexpressed,which results in down-regulation of wild-type p53 level and its functions.Screening of inhibitors to block the p53-MDM2 interaction has been recognized as a new cancer therapeutic strategy.Although lots of inhibitors have been developed and found effective in vitro,most of them are not effective in living cells.Therefore,it is meaningful for developing methods to evaluate inhibitors of p53-MDM2 interaction in living cells.Here,based on the FCCS system for the detection of p53-MDM2 interactions in living cells,we developed a new method for evaluating the inhibitory effects of inhibitors in living cells.We compared the inhibitory effects of three known inhibitors Nutlin 3α,MI773 and RITA in living cells,and found both of Nutlin 3α and MI773 could inhibit p53-MDM2 interaction,and MI773 was more effective.Meanwhile,RITA showed no inhibitory effect on the p53-MDM2 interaction.At present,the inhibitory activity of RITA is in dispute,and our results further negate the inhibitory effect of RITA.Moreover,to study the inhibitory dynamics of the p53-MDM2 interaction in single living cells,a microfluidic chip was introduced into the FCCS system.Herein,we established the in vivo FCS/FCCS detection systems and methods,and we studied the functions and regulation mechanisms of PTEN and p53-MDM2 interaction from protein structures and external stimulis aspects.The results mainly include: PTEN proteins in nucleus and cytoplasm have different dynamic behaviors under external stimulis,which revealing their different physiological functions and the different effects of external stimulis;We directly proved the influence of p53 protein TAD helicity,homo-oligomerization,and hot-spot mutations of DNA-binding domain on the p53-MDM2 interaction in cells;We introduced microfluidic chip into FCCS,and developed the method to evaluate the inhibitory effect of inhibitor on p53-MDM2 interaction in single living cells.