The Molecular Mechanisms Of Paxillin In Leading Edge Of Directed Migrating Cells

Rac activation is strongly regulated spacially and temporally in migrating cells, leading to the formation of specific cell protrusion-lamellipodia in leading edge of the cells and generating the pushing force to put the cells forward, but the molecular mechanism of positive regulation of localized Rac activation remains unclear.This project proposes that the phosphorylation of the intracellular α4 integrin inhibits the interaction of α4 with paxillin, thus the signaling pathway paxillin-GIT1-PIX-PAK would be formed, leading to localized Rac activation in leading edge of the migrating cells.Western blotting and flurescent colocalization confirmed that the molecular complex composed of paxillin,G1T1 and PIX could be formed in leading edge of the migrating cells.Sh RNA of Paxillin was designed to reduce the expression of Paxillin.Immunfluorescence test showed that Paxillin was strongly inhibited when sh RNA was transfection.Rac activation test proved Rac activation is strongest in the degree when fibronectin stimulate the cells for 5 minutes.FRET assay proved that the situation of Rac activation when the expression of paxillin was inhibited or in nomal.Rac activation assay and FRET assay showed that Rac activation was clealy inhibited when the expression of paxillin was inhibited. Random migration assay indicated that the signaling pathway had great effect on cell migration.The results elucidated the molecular mechanism of positive regulation of localized Rac activation in leading edge of migrating cells by α4 integrin through the signaling pathway paxillin-GIT1-PIX-PAK.