The Visualization Of Shear Stress-induced RhoGDI? Activation In Tumor Cells

INTRODUCTION: Rho GDP-dissociation inhibitor ?(Rho GDI?)could affect cell migration and metastasis through Rho GTPases,while some studies indicated that Rho GDI? might be regulated in some pathway independent of Rho GTPases.The mechanism of shear stress-induced RhoGDI? activation is still not clear since the lack of effective tools which can detect RhoGDI? activation in living cells directly.METHODS: A biosensor was designed based on Fluorescence Resonance Energy Transfer(FRET),named sl-Rho GDI?.It consisted of the whole Rho GDI? sequence,switch II sequence,linker sequence,and ECFP/Ypet fluoresce nce protein pairs,which can show the affinity changes of RhoGDI? and Rho GTPases dynamically,and then been modified with Kras and Lyn sequence respectively to link the biosensor onto different location of cell membrane.Biosensors were transfected into Hela to visualize the real-time affinity of RhoGDI?-Rho GTPases complex under fluorescence microscope in a flow chamber with different shear stress,and cell membrane fluidity and cytoskeleton were modified to explore the mechanism.The ratio images were divided into 50 parts averagely along the direction of shear stress after morphological processing and background subtraction with Matlab software,and the percentage of ratio in each part accounting for the whole cell was calculated and combined with continuous time points to achieve the spatial and temporal affinity changes of RhoGDI?-Rho GTPases complex.The affinity differences between upstream and downstream,the same as affinity polarity,was calculated by statistical methods.RESULTS : The result showed firstly that the sl-Rho GDI? biosensor could reflect the bounding situation of RhoGDI? and Rho GTPases in living cells.The affinity declined variously on different subcellular localizations under different magnitudes of shear stress,and the complex dissociated less at downstream along the direction of shear stress.C hanging the membrane fluidity had obvious effects on the affinity polarity and dissociation of RhoGDI?-Rho GTPases complex.Destroying cytoskeleton could enhance the affinity polarity of the complex.Depolymerizing microfilaments or inhibiting force transmission through microfilaments,but not microtubule depolymerization,had no obvious effects on the promoted dissociation of the complex.In addition,inhibiting Src could depress the complex dissociating and cause the the position of affinity polarity changing.CONCLUSION : These results showed that the biosensor designed in the study can show the bounding of Rho GDI? and Rho GTPases in living cells successfully.Upon shear stress,there existed polarity in the affinity of RhoGDI? bounding Rho GTPases in Hela,which was mediated by membrane fluidity and cytoskeleton,especially the microfilaments,with the help of Src.O ur findings indicated that there is a relatively independent regulatory mechanism for RhoGDI? activation under shear stress,which is independence of Rho GTPases.