Quantitatively Imaging Induced Activation Of Rac1, Cdc42 Signaling Pathways

Rho guanosine triphosphate enzyme(Rho GTPases),including Rac and Cdc42,is a kind of proteins molecules that have been studied well in cellular signal transduction.Rho GTPases mainly regulate a variety of cellular processes such as cell transcription,migration,morphology and gene expression.In the course of cellular signal transduction,Rho GTPases usually transduce the upstream stimulating signals to the downstream effectors or target proteins,and produce the corresponding biological outcome as molecular switches.Three kinds of proteins namely GEF(guanine nucleotide exchange facotors),GAP(GTPase-activating proteins) and GDI(guanine nucleotide dissociation inhibitors) regulate the change from active to inactive for the Rho GTPases.In this paper,based on fluorescence resonance energy transfer(FRET) single-molecule probes which contained Discosoma sp.red fluorescent protein gene DsRed1,Rac1 or Cdc42,the GTPase binding domain of the effector that is Pak1 or N-WASP,and enhance cyan fluorescent protein(eCFP) or enhance green fluorescent protein(eGFP) gene constructed by our laboratory to transfect the cells.Several single nucleotide molecules to probe,respectively, in several animal cell transfection,and then to insulin(Insulin),bradykinin (Bradykinin) as inducer,respectively,induced activation of Rac1,Cdc42 signal transduction pathway in different time periods living cells to detect the fluorescence intensity and imaging.Quantitative imaging induced activation of Rac1,Cdc42 signal transduction pathway in transfected cells of the animals produced a FRET phenomenon.Before the non-induced activation of different signal transduction pathway have a certain level of FRET efficiency,induced by activation,the FRET efficiency significantly,the rapid increase in the activation induced by 5～8 min later,reached the maximum.But different signal transduction increase pathway were significantly different.With the passage of time-induced,FRET efficiency progressively decreased.The increase rate of FRET efficiency by induced activation is high than the decreased rate,but differed significantly in the decreasing speed for the signaling pathways.More than 30min after,FRET efficiency is similar to the original level.Protein activation test confirmed that the induced activation of transfected cells,Rac1,Cdc42 both are active in different time-induced activation of the extent of relative performance in line with the FRET efficiency.Induced activation of Rac1,Cdc42 signal transduction pathway, monitoring the transfer of living cells activated sheet pseudopodium emerged and filopodia obvious morphological changes,such as measured values or fluorescence imaging of fluorescence intensity over time to continuously reduce until almost see the determination or observation.Different non-directional cell migration,but migration of cells in the same direction is constant.This shows that these single-molecule FRET probe to monitor the cells can be directly activated signal transduction pathway and its spatial and temporal changes in the cytology of the corresponding effect.Using the single-molecule probe,analyze and determine the number of regulatory proteins of Rac1,Cdc42 guanosine monophosphate of conversion factors or characteristics of activated protein GTPases,which can provide an methods to effective test of the identification of protein molecules,to identify intracellular signaling pathway between protein the role of living cells to monitor the intracellular signal transduction.