|Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
Gelsolin, an actin-regulatory protein has at least three different activities, nucleating, severing and capping. These activities are tightly regulated by calcium ions (Ca^<2+>), pH, and polyphosphoinositides. Gelsolin regulates actin dynamics in cells by interaction with both filamentous (F-) and monomeric (G-) actins and controlling the length of actin polymers through a variety of mechanisms. In this study, we sought to obtain an image of spatio-temporal activation of gelsolin in the living cells by means of FRET (Fluorescence Resonance Energy Transfer).
For bioimaging of gelsolin activities in cells, mouse gelsolin cDNA were inserted between ECFP and EYFP, derivatives of green fluorescence protein, to create a probe (CGY) for FRET, and then the mammalian expression vector for CGY was constructed. The CGY expression vector was transfected in fibroblast derived from gelsolin knock-out mice and expression of CGY was confirmed by western blot analysis. The expression of CGY in G- cells resulted in increased rate of cell crawling, indicating CGY, chimeric gelsolin protein was functional in cells. Fluorescence intensity of EYFP was decreased at cell peripheral, whereas it was higher around nulei, suggesting FRET was occurred at the central region of cells, and gelsolin was activated at the edge of cells, because FRET was lost.