Biology Reference
In-Depth Information
Figure 7.1 Examples of FRET and FLIM. (A) Ballestrem, C., and Geiger, B.,
unpublished material: FRET demonstrates interaction of paxillin and vinculin in live
cells. NIH3T3 cells were co-transfected with cDNA encoding CFP-paxillin and YFP-
vinculin. Image shows (i) FRET in focal adhesions at the cell periphery and (ii) a spectrum
colour scale representation of the region highlighted in (i). Red corresponds to high FRET
eciency, blue corresponds to low FRET eciency. FRET values were calculated as
previously described (J. Cell Sci. 108, 1051-1062. 1995). Bar, 2 mm. (B) Edme et al.,
unpublished material: Spatially variant interaction between ezrin and PKCa V1V3 shown
by FRET in multi-photon FLIM measurements. MCF-7 cells were dually transfected with
both a GFP-PKCa (aa1-337) and a VSVG-tagged ezrin construct. After 15 h, cells were
fixed in 4% PFA for 15min at room temperature. Cells were permeabilized with 0.1%
Triton-X100 and then stained with a Cy3-labelled anti-VSVG antibody. The GFP-intensity
images were acquired and fluorescence GFP-lifetimes in cells were determined by multi-
photon FLIM. The fluorescent images of the donor (GFP-PKCa aa1-337) (i) and the
acceptor (anti-VSVG-Cy3) (ii) are shown. Both ezrin-positive cells exhibit a shortening of
average GFP-fluorescence lifetimes in their peripheries from 2.3 ns to 1.8 ns (iii). The
population map of interacting species is shown in (iv). (A colour reproduction of this figure
can be found in the colour plate section)
