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interactions has revealed a spatio-temporal pattern of signalling events at
the single-cell level (Verveer et al., 2000). FLIM-based measurements of
FRET interactions between ErbB1-GFP donor and a Cy3-conjugated
phosphotyrosine-specific acceptor antibody were used to measure the
activation state of the receptor. The antibody only recognized the active
phosphorylated form of the receptor and thus only the active ErbB1-GFP
population could undergo FRET. The study revealed rapid lateral propaga-
tion of receptor activation within the plasma membrane following only
localized stimulation with EGF-coated beads. This finding suggests that a
relatively small and localized pool of ligand-occupied receptor can initiate a
transactivation cascade that ultimately leads to the activation of the remaining
ligand-free receptor population within the plasma membrane.
A similar approach has been used to image protein kinase Ca (PKCa)
activation upon TPA treatment (Ng et al., 1999). In this study a Cy3.5-
labelled acceptor antibody specific only to the active, phosphorylated form of
the protein was microinjected into living cells and used as a dynamic indicator
of GFP-PKCa activity. An example of the data produced from FLIM
experiments can be found in Figure 7.1B. Here, GFP-PKCa (V1V3 domains
only) and a Cy3-labelled anti-VSVG antibody (to locate VSVG-tagged ezrin)
showed that interaction between PKCa and ezrin only occurs at the cell
periphery (Edme et al., unpublished).
More recently, FLIM has been used to reveal interactions between the
CD44 hyaluronan receptor and the actin binding protein ezrin in a PKC-
dependent manner (Legg et al., 2002). Activation of PKC with phorbol ester
resulted in the loss of a basal level of FRET between GFP-CD44 and ezrin-
VSVG indirectly labelled with Cy3-conjugated anti-VSVG antibody. The
authors concluded that PKC-mediated disruption of CD44-ezrin interaction is
necessary for directional cell motility.
Total internal reflection fluorescence (TIRF)/
evanescent wave microscopy
TIRF, also known as evanescent wave microscopy is not a new technique but
recent developments have increased its range of application. While TIRF has
traditionally been used for studying cell surface features, such as focal
adhesions or endo/exocytosis, it is also useful for the study of single molecule
When light travelling in a high refractive index medium (such as a glass
coverslip) strikes an interface with a material of lower refractive index (such as
water or culture medium) above a critical angle of incidence, the result is
known as total internal reflection. However, some energy penetrates the distal
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