Characterization Of FRET-based Single Molecule Probes Of Rac, Cdc42 Signaling Pathways

Rho GTPases,including Rac and Cdc42,are a kind of proteins that have been studied well in signal transduction.Rho GrPases mainly regulate a variety of cellular processes such as cell transcription, migration,morphology,and gene expression.In the course of signal transduction,Rho GrPases 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(GrPase-activating proteins) and GDI(guanine nucleotide dissociation inhibitors)regulate the change from active to inactive for the Rho GrPases.In this thesis,several FRET-based single-molecule probes which contained Dfscosoma sp.red fluorescent protein DsRedl,Rac1 or Cdc42,the GTPase binding domain of the effector that is Pak1 or N-WASP,and cyan fluorescent protein CFP constructed by our laboratory to transfect the cells.Rac1 and Cdc42 signaling pathways were activated in the transfected cells by the inducer,insulin or bradykinin respectively.In vitro fluorescent spectroscopy assays showed that FRET phenomenon was observed in the transfected NIH3T3 cells.For all three signaling pathways,the values of FRET efficiency were realtively high before the induction, and reached the highest after induced for 5 min,but the increasing extents of the values of FRET efficiency varied in three signaling pathways.The values of FRET efficiency decreased with the extention of the induction time,but differed significantly in the decreasing speed for the signaling pathways.Rac1 and Cdc42 activation assays indicated that Rac1 and Cdc42 were in the activated state in the induced cells.Their relatively activated activities in the cells induced for different time were consistent with the values of FRET efficiency of In vitro fluorescent spectroscopy assays.The activated Rac1 and Cdc42 signaling pathways led to the formation of lamelliopodia and filopodia in the transfected cells respectively.The results showed that these single-molecule probes could be directly used to monitor the spatial and temporal imaging and the corresponding cellular effect of the induced activation of the signaling pathways in living cells.With these single-molecule probes,the GEF or GAP activities of putative regulatory proteins for Rac1 and Cdc42 will be analyzed and judged,thus provided an effective method for identifying the regulatory proteins for Rho GTPases.