Diffusion Dynamics Of Integrins And Their Roles In Regulating Cell Migration

Integrin is a transmembrane protein that plays a key role in the connection between cells and extracellular matrix(ECM).Integrins regulate cell-ECM interactions by dynamically participating in the assembly and disassembly of focal adhesions.Thus integrins can influence cell morphology,migration and related biological functions,including embryonic development,wound healing,cancer invasion and metastasis.Therefore,it is crucial to investigate how integrins dynamically regulate cell-ECM adhesion to affect cell migration.In this paper,we have studied the diffusion behaviors of integrins on living cells by single-particle tracking,and explored the regulation mechanism of cell migration.First,we have developed a single-molecule labelling method for integrins on living cells.In this method,the integrin is labeled by quantum dot through the linking strategy of antibody-biotin-streptavidin.With the single-molecule tracking experiment,we have analyzed the temporal-spatial dynamics of integrins on living cell and obtained the dynamic characteristics of integrin diffusion on the membrane.We have investigated the diffusion dynamics of integrins,and the mechanisms of cell migration behaviors regulated by epithelial-mesenchymal transition and substrate rigidity,respectively.Epithelial-mesenchymal transition(EMT)is a process that significantly enhances cell migration and plays an important role in embryonic development and tumor metastasis.During this process,the cells acquired mesenchymal phenotype,frontback cell polarity and loss of cell-cell contact,to acquire promoted migration.At present,the study of epithelial-mesenchymal transition mainly focuses on intracellular and intercellular biological characteristics,but lack research on cell-ECM adhesions,especially how integrin participates in cell-ECM adhesions and affects cell migration is still unclear.By single-molecule fluorescence tracking of β1-integrins on living cell membranes,we reveal the temporal-spatial dynamics of integrins during EMT.During EMT,β1-integrins exhibit significantly enhanced dynamics,which temporally behave more diffusive and less immobilized,and spatially become distributed asymmetrically with front regions being more dynamic.By studying the influence of cytoskeleton,we have found that the change of integrin diffusion is resulted from microtubule remodeling in EMT: the appearance of MTs at the front protrusive region makes the integrins more dynamical compared to the rear cell region.The spatial polarity of integrin dynamics could generate bias adhesions in a cell,driving the bias migration.Our results demonstrate that cell-ECM adhesions are play roles in EMT and reveal the mechanism that how integrins regulate cell migration during EMT.Furthermore,enhanced integrin diffusion may represent as a new hallmark of EMT.The stiffness substrate affects the cell migration.For example,cells on the substrate with stiffness gradients will migrate towards the stiffer substrate,that is,the durotaxis.Integrins,as an important part of the connection between cells and the substrate,can sense the substrate stiffness by regulating focal adhesions.At present,the response of integrin diffusion to substrate stiffness and the mechanism of regulating cell migration are not clear.We have investigated the diffusion of integrins on substrates with different stiffness by tracking individual integrins.With the increase of stiffness,the diffusion rate of β1 integrins on the membrane is enhanced,and the proportion of immobilizations is increased.Next,we make a substrate of high and low rigidity and focus on the diffusion of integrin in single cell on transition area between high and low rigidity.We have found that the diffusion rate of β1 integrins in the single cell shows spatial polarity,with the diffusion rate being enhanced on the side of the stiffer substrate.We believe that the spatial polarity of diffusion rate of β1integrins affects the focal adhesions in single cell and contributes to the directional migration of cells.Our study reveals that diffusion dynamics of integrins is related to substrate stiffness,contributing to the understanding of the durotaxis.In this paper,we quantitatively study the temporal-spatial dynamics of integrin diffusion on cell membrane through the single-molecule tracking experiment.To study of diffusion behaviors of β1-integrins shed new light on the mechanism of cell migration regulated by EMT and substrate rigidity.The temporal enhancement in integrin dynamics may facilitate the translocation of integrins to the leading edge,which could facilitate the formation of initial adhesions at the protrusion.The spatial polarity of integrin dynamics could generate bias adhesions in a cell,driving the bias migration.The characteristics of integrin diffusion and its regulation mechanism on cell migration have improved our understanding of the connection between molecular dynamics and cell behaviors.