Visualization Study Of Stress Transfer Between Cell Membrane And Cytoskeleton

Tumor cell migration is the initial step of tumor metastasis,and this process is affected by various stresses.Current researches generally convinced that when stress acts on cells,the cell membrane can sense stress signals and transduce them into intracellular chemical signals to regulate cell functions.However,uniform diffusion is the main intracellular transmission of chemical signals,which makes it difficult to explain the changes in cell polarity under directional stress.The reason for this polarity may be the existence of stress mechanical transmission pathways in cells,and the stress transmission between the cell membrane and the skeleton is the first step of the stress transmission mechanism.Due to the lack of relevant research tools,the regulatory mechanism of the stress state and related stress transmission between the cell membrane and the skeleton is still unclear.A visualized biosensor for stress transfer between cell membrane and skeleton was constructed based on fluorescence resonance energy transfer(FRET).The main structure of the probe is composed of the fluorescent protein pair ECFP/Ypet linked by the spider silk protein(GPGGA)×8,with one end be linked to the lipid raft on the cell membrane by Lyn protein,and another end is directly embedded in the cytoskeleton microfilament structure by β-actin protein.The pc DNA3.1(+)vector was used to construct the LESYB probe plasmid and transfected into cancer cell U-2 OS,and its functional properties such as stability,linearity and reversibility were verified by changing the osmotic pressure of the cell culture medium.Drugs were used to interfere with the cytoskeleton structure,cell membrane fluidity and lipid raft structure of the cell transfected with the biosensor,or change the substrate stiffness,then the FRET fluorescence images of the upper and lower surface cell membranes were obtained by confocal laser scanning microscopy,and the inherent stress between the upper and lower surface cell membranes and the cytoskeleton of cancer cells in the resting state were analyzed.Afterwards,a parallel-plate flow chamber was used to apply directional shear stress to the cell,while drugs are used at the same time to influence the inherent stress state between the cell membrane and the cytoskeleton in the resting state.Fluorescence images of cells were also obtained by fluorescence microscope,and Matlab software was used to analyze stress differences in different regions of cells under external stress.The experimental results show that the biosensor LESYB can be accurately located between the cell membrane and the skeleton with good functional properties.In the resting state,the inherent stress between the upper surface of cell membrane and the cytoskeleton is significantly smaller than that of the lower surface.By reducing membrane fluidity or changing matrix stiffness,differences in stress between the upper and lower surfaces of the membrane still exist,while disruption of lipid rafts,cytoskeletal structure can eliminate this difference.Under directional shear stress,the stress between the cell membrane and the cytoskeleton increases,resulting in a polarity difference between the upstream and downstream,while the destruction of the inherent stress difference in the cell leads to the enhancement of the stress and the disappearance of the polarity.It can be concluded that the visualization biosensor LESYB for stress transfer between cell membrane and cytoskeleton is successfully constructed in this project.The experimental results show that inherent stress differences exist in different regions of the stress between the cell membrane and the cytoskeleton in the resting state of cancer cell,and the cell membrane fluidity,lipid rafts and cytoskeleton integrity are important factors to maintain this difference.Directed shear stress can produce a polar distribution of stress transfer between the cell membrane and the cytoskeleton,and this polarity is regulated by differences in the inherent stress of the cancer cell.This study provides a tool for the study of cell biomechanics to detect local stress changes in cells,demonstrating that the polarity change of the cancer cell first occurs in the stress transfer process between the cell membrane and the cytoskeleton.It is suggested that this polarity may be the premise of stress-induced directional migration of cells,and the differences of intrinsic stress in different regions may mediate the migration of tumor cells by regulating the polarity of stress transmission.