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Localized Rac Activation Dynamics Visualized in Living Cells

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Science  13 Oct 2000:
Vol. 290, Issue 5490, pp. 333-337
DOI: 10.1126/science.290.5490.333

Figures

  • Figure 1

    The Rac nucleotide state biosensor. (A) Cells expressing GFP-Rac are injected with a fragment of p21-activated kinase (PBD) labeled with Alexa-546 dye (PBD-A), which binds selectively to GFP-Rac-GTP. The Alexa and GFP fluorophores undergo FRET when brought close together. FRET produces a unique fluorescence signal because excitation of GFP leads to emission from Alexa as energy is transferred from the excited GFP fluorophore to the nearby Alexa dye (30). This FRET can be measured within a living cell to map the distribution and amount of Rac-GTP binding. By imaging the cell with different wavelengths, both the distribution of Rac and Rac activation can be studied in the same cell. GFP excitation and emission are used for overall Rac distribution, whereas GFP excitation and Alexa emission are used for FRET. (B) Fluorescence emission from solutions containing 100 nM GFP-Rac bound to GTPγS at different concentrations of Alexa-PBD. Excitation at 480 nm was used for selective excitation of GFP, and direct (nonFRET) excitation of Alexa was subtracted from these spectra (9). In the absence of Alexa-PBD, the emission from GFP (peak at 508 nm) is maximal and no Alexa emission (peak at 568 nm) is observed. Binding of Alexa-PBD to Rac-GFP leads to FRET, producing increasing emission at 568 nm and a decrease at 508 nm. The inset shows variation of the 568-nm/508-nm emission ratio as a function of Alexa-PBD concentration for GFP-Rac bound to GTPγS (circles) or to GDP (open squares). Addition of increasing concentrations of unlabeled PBD blocks FRET (open triangles). (C) Variation of this same emission ratio with changes in the nucleotide state of Rac. All data points were the average of three independent experiments.

  • Figure 3

    Specificity and sensitivity of FLAIR. (A) Swiss 3T3 fibroblasts were transfected with GFP-RhoQ36L, a constitutively active mutant of Rho (bar, 22 μm), and cells were prepared and imaged as described for GFP-Rac (18), with comparable concentrations of GFP-RhoQ63L and Alexa546-PBD. Despite a constitutively high proportion of GTP-bound protein, the localization of GFP-Rho showed no corresponding FRET. The GFP images show intensities ranging from 10 to 185, while those in the FRET image range from 0 to 10. (B) Simple localization of Alexa-PBD is inferior to FRET in quantifying and localizing Rac-GTP binding (bar = 8 μm). The ruffle in Fig. 2D is shown here in close-up, visualized using FRET, or using simple Alexa-PBD localization (18). Even though scaling in the Alexa-PBD image is optimized for detection of the ruffle, the high background due to unbound PBD cannot be eliminated, and binding to other target proteins is not eliminated as it is in the highly specific FRET signal. Without prior knowledge of the ruffle's location, this localization would have been difficult to discern. Color scale for the intensity of FRET or GFP fluorescence is the same as in Fig. 2C.

  • Figure 4

    Rac nucleotide state in motile cells. (A) Two examples of Rac activation and localization in motile Swiss 3T3 fibroblasts (bar = 24 μm). Cells were induced to move by scraping a wound in a cell monolayer (20). The highest concentration of activated Rac1 was seen in the juxtanuclear region, and a gradient of Rac activation was also observed, highest near the leading edge and tapering off toward the nucleus. Color scale for the intensity of FRET or GFP fluorescence is the same as Fig. 2C. FRET intensities are 0 to 18 (top image) and 0 to 32 (bottom image). In the GFP images, intensities range from 98 to 700 (top image) and 100 to 1100 (bottom image). (B) Example of a cell in the monolayer, away from the wound. In such cells, FRET was either not detected or found around the cell edge. (GFP intensities = 0 to 1023, FRET intensities = 0 to 10).

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