Biology Reference
In-Depth Information
discovered in other marine coelenterates and reef corals have provided a
particularly rich hunting ground. The research impact of these living probes
derives chiefly from the ability of cells of widely differing species to express
them as fully functional fluorescent proteins fused to proteins under study.
This feature has sparked the development of new methods, not only for
locating specific proteins in living cells but also for investigating their
interactions and dynamics. The focus of this review is to describe how these
methods are being used to study molecular translocation, post-translational
modifications and changes in protein activity and conformation. The
techniques discussed include fluorescence (or Fo¨ rster) resonance energy
transfer (FRET), fluorescence lifetime imaging microscopy (FLIM),
(frustrated) total internal reflection fluorescence microscopy (TIRF), fluor-
escence speckle microscopy (FSM), and the more recently developed
fluorescence localization after photobleaching (FLAP), which will be
discussed in most detail. Particular reference will be made to the use of
these methods in studies on cytoskeletal dynamics and cellular signalling.
Fluorescence resonance energy transfer (FRET)
FRET is a quantum mechanical process that has been used as a biochemical
tool to study protein structure, conformational change and protein-ligand
interactions. Excitation of a donor fluorophore can give rise to non-radiative
transfer of the absorbed energy to an adjacent acceptor fluorophore provided
that the emission spectrum of the donor overlaps suciently with the
excitation spectrum of the acceptor. This is not a simple process of emission
and reabsorption but direct energy transfer dependent on coupling of the
respective dipole moments. Since the probability of transfer is proportional to
orientation and inversely proportional to the sixth power of the distance
(1/R
6
) between the donor and acceptor, FRET can be used to deduce the
respective positional or rotational contexts of the two molecules. The
positional constraints limit e
cient FRET to distances of less than 10 nm
and therefore su
ciently close to infer interaction. An example of FRET is
shown in Figure 7.1A, which shows the biochemically well characterized
interaction of paxillin with vinculin occurring in the focal adhesions of live
fibroblasts (Ballestrem and Geiger, unpublished).
Two different approaches to obtain FRET using GFP and variants have
been followed. The first uses cells expressing chimaeric GFP that are then
microinjected with protein, e.g., a specific primary antibody, or peptide,
conjugated with an acceptor dye, usually the red AlexaFluor-546. The second
approach relies on multicoloured variants of GFP. A suitable GFP-variant
pair for FRET, and by far the most widely used (Pollok and Heim, 1999), is
the combination of cyan and yellow fluorescent proteins (CFP and YFP). CFP
