Regulation Of Membrane Phase Separation To Manipulate Cell Behavior

Cell membrane is an important platform for cell signal transduction and regulates various cellular functions.The classic flow mosaic cell membrane model states that cell membranes are heterogeneous.Cell membrane components are continuously remodeled through intracellular and extracellular interactions and cytoskeletal rearrangements.In order to perform corresponding cellular functions in the correct time and space,a variety of complex components in the cell form a certain order in the cell in the form of phase separation,and regulate the physiological function of the cell in an orderly manner.Abnormal phase separation can lead to neurodegenerative diseases,cancer and other diseases.Therefore,understanding the mechanism behind cell are phase separation and using phase separation to regulate cell behavior are very important for the treatment of diseases.Nanodomains generated by two-dimensional phase separation of cell membranes are major platforms for multiple signal transduction,commonly referred as lipid rafts,and play important roles in signal transduction,viral infection,and membrane transport.Membrane modification methods targeting lipid rafts provide a new idea for cell therapy.However,due to the small size and transient heterogeneity of lipid rafts,the localization and functional analysis of lipid rafts are difficult,and the lipid raft hypothesis remains controversial.In addition,many cellular behaviors are controlled by binding to multivalent extracellular ligands that promote membrane phase separation,but extracellular molecular tools capable of manipulating transmembrane receptor aggregation in a controllable manner are currently rare.The functional modification of cell membranes to manipulate and study cellular behavior has been extensively studied.With the development of nanomaterial technology,the commonly used methods of cell membrane engineering include nanoparticles,biopolymers,DNA nanotechnology,etc.Among them,nanoparticles have remote controllability,and the controllable adjustment of nanoparticles can be realized through optical field,electric field,magnetic field,etc.DNA nanotechnology has strong advantages in structural design.For example,compared with other materials such as peptides,proteins,and synthetic macromolecules,the programmability of DNA enables precise bottom-up construction of complex structures,and the reversible regulation of structures can be achieved through strand displacement reactions.Secondly,DNA can be anchored on the cell membrane surface through functional modification and covalent or non-covalent binding,and has excellent biocompatibility.Finally,the DNA nanostructures formed according to the principle of base complementary pairing have precise addressability and programmability,and DNA devices with different shapes and sizes can be designed as needed.The main content of this thesis is to use DNA nanotechnology and nanomotors to realize the regulation of cell membrane phase separation,and then manipulate cell behavior.The main contents are as follows:First,a DNA nanodevice composed of cholesterol-functionalized DNA duplexes was designed to stabilize transient lipid rafts,which facilitates further analysis of lipid raft components and their functions in combination with light microscopy.The DNA nanodevice proposed in this study can induce the aggregation of lipid raft-related components and realize the regulation of lipid raft aggregation.In addition,functionalizing DNA nanodevices on the surface of T cell membrane can not only induce the aggregation of lipid rafts and stabilize the lipid rafts,but also induce the aggregation of T cell-related receptors,prolong the interaction time of T cell signaling receptors,activate T cells,enhance T cell proliferation.Second,a DNA nanodevice generated by DNA hybridization chain reaction was designed to aggregate membrane dynamic lipid rafts to enhance membrane phase separation.The DNA nanodevice can target the lipid raft region of the cell membrane.In the tumor microenvironment,ATP can activate the DNA reaction unit,initiate the hybridization chain reaction on the cell membrane,and form a DNA nanodevice on the cell membrane,which not only enhances the membrane phase separation,but also can act as a barrier between the cell membrane and the extracellular environment,blocking the interaction between the transmembrane surface adhesion receptors and the extracellular environment,thereby inhibiting cell migration.In this paper,we verified the effect of DNA nanodevices on tumor cell migration in vitro and in vivo.Third,a visible light-driven micro-/nano-motor is designed.The motor converts light energy into its own mechanical kinetic energy,which can realize light-controlled autonomous movement.When the micro-/nano-motor acts on the extracellular environment of T cells,the fluid shear force and local pressure generated by the synergistic motion of the motor can disturb the phase separation of T cell membranes.Perturbation of membrane structure can change the conformation of intracellular mechanosensitive ion channel proteins,triggering influx of calcium ions,thereby activating T cells.The flexibility of random-sequence noncoding DNA and the precise controllability of nanomotors provide multiple possibilities for manipulating lipid rafts and membrane phase separation,thereby opening up new avenues for regulating cellular behavior for therapeutic purposes.The use of DNA nanotechnology and micro-nanomotor technology to regulate membrane phase separation to manipulate cell behavior provides new possibilities for regulating various cellular functions by rationally designing DNA structures and functional motor structures.