Visualization Of Shear Stress-induced Alternation Of Cell Membrane Surface Tension

Objective:The effect of blood flow shear stress on tumor cells may be one of the important causes of tumor cells metastasis. Shear stress affects the upper surface of cell membrane and causes the change of cell membrane surface tension firstly. This physical change may be the base of directional migration of tumor cells, but there is not research about the distribution model of tumor cell membrane surface tension under the shear stress at present. The FRET technique will be adopted in this paper to explore the change model of the cell membrane surface tension and the related mechanism under shear stressMethods:The common subcloning technology was used firstly in this paper to design FRET probe that can visually detect the change of cell membrane surface tension using a molecular spring-like spider silk protein sequence (GPGGA)8 and a pair of fluorescent proteins ECFP/YPet that can occur FRET. Next, different laminar shear stress were loaded on HeLa cells using the parallel plate flow chamber, and the cell membrane and cytoskeleton were treated by a variety of agents. Under the FRET fluorescence microscope, the cell membrane surface fluorescence FRET changes of the single cell could be recorded dynamically.Then the fluorescent images were processed by MetaFluor software to get the overall trend of cell FRET ratio image. Then the ratio images were divided into 50 equal parts along the direction of flow using Matlab software to get the local change data of cell membrane surface tension. The FRET data were fitted with Hill curve along the flow direction to explore the local distribution change rule of cell membrane surface tension under shear stress.Results:The FRET ratio change of the probe could reflect the dynamic changes of the cell membrane surface tension. Upon the application of laminar shear stress, the cell membrane surface tension increased obviously with distribution model of higher tension at midstream and lower at upstream and downstream along the direction of flow, and its amplitude was positively related with the magnitude of shear stress. The location of peak point shifted towards cell upstream with the increasing of shear stress. The cell membrane fluidity was changed artificially before loading shear stress, and it was found that the cell membrane surface tension increased more significantly after benzyl alcohol pretreatment, and the overall declined obviously after cholesterol pretreatment. But the non-uniform distribution mode of upstream and downstream still displayed similar to that of the control group. Nocodazole, Cytochalasin D and ML7 were used to destroy cytoskeletal microtubule, microfilament or inhibit microfilament contraction, and the cell membrane surface tension under shear stress still appeared the model of higher at midstream and lower at upstream and downstream. The destruction of microtubules could significantly inhibit the shear stress-induced increasing of overall cell membrane surface tension, and change the local distribution, including the decrease of peak amplitude and shifting towards downstream, and the change range of upstream and downstream tended to accordance. After the microfilament depolymerization, no significant influence was found on the increasing of whole surface tension, but the amplitude reversal of the surface tension increase at upstream and downstream was induced. The inhibition of microfilament contraction suppressed the increasing of the shear stress-induced cell membrane surface tension to a certain extent, and the non-uniform distribution mode of upstream and downstream was affected significantly.Conclusion:A FRET biological probe that can visually detect the change of cell membrane surface tension has been successfully designed and prepared in this paper. Upon the application of shear stress, the change of cell membrane surface tension on tumor HeLa showed a non-uniform distribution at upstream and downstream, and it is likely to be the base of the directional migration of tumor cells under fluid. The change of cell membrane fluidity affects significantly on the magnitude of cell membrane surface tension, but not the non-uniform distribution rule of the shear stress-induced-cell membrane surface tension.The integrity of the cytoskeleton plays a decisive role on the non-uniform distribution of the shear stress-induced-cell membrane surface tension, in which the microtubule system plays a leading role, and the influence of the microfilament system is weak relatively.