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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.01117

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Designing biosensors for Rho family proteins — deciphering the dynamics of Rho family GTPase activation in living cells

The Scripps Research Institute, Department of Cell Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

View larger version (52K): [in a new window]   Fig. 1. Example of specificity and sensitivity of FRET in a dorsal membrane ruffle. (A) GFP-Rac localization in a Swiss 3T3 fibroblast stimulated with serum. (B) GFP-Rac, Alexa-PBD and processed FRET images of the same ruffle. GFP-Rac localization illustrates the presence of Rac in the ruffle but does not provide information about its activation status. PBD localization cannot be used to quantify Rac activity because of the high background from unbound PBD in the cytoplasm, and because of lack of specificity. All images are color-coded with warmer colors representing higher concentrations or higher levels of FRET. Bar, 22 µm. N, nucleus. Figure reproduced with permission from the American Association for the Advancement of Science (Kraynov et al., 2000).

View larger version (28K): [in a new window]   Fig. 2. Design of different fluorescent probes for detection of Rho family GTPase activity in living cells. (A) Bimolecular Rac FRET probe (Kraynov et al., 2000). Red pentagon represents Alexa-546 dye. (B) Unimolecular `GTPase-effector fusion' Rac and Cdc42 FRET probes (Itoh et al., 2002). Non-natural isoprenoid moiety due to K-Ras4B CAAX-box is depicted in red. (C) `Effector domain only' probes (Itoh et al., 2002; Seth et al., 2003). (D) Biosensor based on covalently labeling a domain with a solvatochromic dye. Red pentagon represents the dye. Black dashed arrows represent excitation light. Black solid arrows represent emission light. Red arrows represent direction of FRET.