Study On Visualization Of Paxillin Mediated Stress Transfer

Introduction: Paxillin is one of the structural proteins in focal adhesions(FAs).It is connected to the cytoskeleton at one end and to FAs and integrin-related proteins at the other end,so it is likely to be a key node in the intracellular pathway of mechanotransduction.However,due to the lack of probe tools for detecting stress changes within paxillin protein in living cells,the change of stress and the related mechanism of signal transduction within paxillin protein are still unknown.Methods: A probe DSMP was designed and constructed based on fluorescence resonance energy transfer(FRET),which could detect the stress within paxillin protein in living cell.One end of the probe was LD12 motif,that could be linked to cytoskeleton related proteins.The other end was LIM1234,which targeted to FAs.A pair of fluorescent proteins ECFP/Ypet were embedded in the middle of the probe.The sequence of molecular spring(GPGGA)×8 deriving from spider silk was inserted between the fluorescence protein pairs.The probe was transfected into the tumor cell U-2 OS,firstly,to verify the stability,specificity and linearity of the probe DSMP.Then shear stress was loaded on the cell with a classical parallel plate flow chamber system,and drugs were used to interfere with the cell membrane,cytoskeleton,and change the stiffness of the matrix.Finally,fluorescence microscope was used to get FRET images and the time dependent FRET efficiency changes were obtained combining with Matlab programming for image analysis.Cells were divided into upstream and downstream according to the direction of stress action,and the difference of stress within paxillin at upstream and downstream FAs were analyzed.Results: The functional verification proved that the DSMP probe could accurately embedded into FAs,and its FRET efficiency could represent the stress changes in paxillin with good stability,specificity and linearity.Upon shear stress application,the stress within paxillin increased,and there was a significant difference between the upstream and downstream,which meaned polarity changes of stress in cell.When increasing the cell membrane fluidity,the polarity disappeared.On the contrary,reducing the fluidity of cell membrane,and there were still differences between upstream and downstream.However,the polarity was opposite to that of loading stress only.Destroying the microfilament structure would also inhibit the polarity,while the stress polarity still remained when the microtubule structure was destoried.Conclusion: A DSMP probe was successfully constructed in this project that can reflect the stress changes of paxillin protein in FAs.The stress of paxillin in cellular FAs was dependent on the integrity of cytoskeleton,while the stress polarity was directly related to the fluidity of cell membrane and microfilament system.This paper demonstrated the stress transmission pathway of cell membrane-cytoskeleton-FAs in cells,and the difference between upstream and downstream in stress transmission might be the direct cause of the change of cell polarity.At the same time,this paper provided a visual tool to detect the intracellular stress for the mechanobiological research of cancer cell migration.