Construction Of Single-molecule Probes Of Rac,Cdc42 Signaling Pathways And Detection Of Fluorescence Spectrum

Rho GTPases, including Rac1, RhoA,Cdc42, function as molecular switches cycling from an inactive GDP-bound state to an active GTP-bound state. After Rho GTPases are activated by the stimulating signals outside of the cells, they initiate a diverse of intracellular signaling pathways by interacting with downstream effectors, thus regulating much cellular processes, such as cell morphology, migration, growth, proliferation, apoptosis and transcriptional activation, and directly influencing some diseases, like tumourigenicity and metastasis. Fluorescence resonance energy transfer (FRET) is highly sensitive to the relative distance and spatial orientation between two fluorphores. Using this technique we are able to observe the conformation change of the interaction between bio-macromolecules by measuring FRET efficiency. More importantly, using this technique, it is unnecessary to crash or damage the cells, thus keeping the living cells intact. The protein-protein interactions in living cells, like legend-receptor, signaling molecules-effectors could be dynamically studied in a real-time and quantitative way.In this thesis , we report that full-length Rac1,Cdc42 cDNA genes and GTPase binding domains (GBD) of their effectors genes, Pak1, N-WASP were amplified from human brain cDNA library by PCR.The amplified genes or gene fragments were cloned into pECFP-N1 vector, and FRET-based single-molecule probes containing dsRed1, GBD of Pak1 or N-WASP, Rac1 or Cdc42 and ECFP were constructed. Based on these probes, by adding farnesylated motif of CAAM to the C-terminal of dsRed1, the probes containing EGFP, GBD of Pak1 , Rac1 or Cdc42, dsRed1 and CAAM motif , which could be specifically expressed in plasm membrane were constructed. In vitro fluorescent spectroscopy assays showed that FRET phenomena were observed in two kinds of transfected cells, NIH3T3 and Hela. FRET efficiency was highest in the cells induced for 5 min, and gradually decreased with the extention of the induction time .These two kinds of probes could be used to localize and trace the 3D spatial and temporal imaging of induced activation for Rac1, Cdc42 singaling pathways in living cells, and identify GEF or GAP activities of putative regulatory proteins for Rho GTPases.