Biology Reference
In-Depth Information
information about the turnover of actin in dendritic lamellipodial networks
and thus increased our knowledge of cytoskeletal dynamics at the leading edge
of motile cells.
Fluorescence localization after photobleaching (FLAP)
Conventional fluorescence microscopy can reveal non-steady-state processes
such as the distribution of a molecular species within a cell. In the previous
two sections we have seen how, by detecting single molecules or small groups
of molecules, we can extend our knowledge of dynamics to reveal the flow of
molecules through a structure in the steady state. Many fundamental cell
biological processes, such as the turnover of actin during cell motility, have a
large steady-state component. For example, a smoothly gliding cell such as a
fish keratocyte would reveal
little internal
redistribution of actin by
conventional fluorescence methods.
Although single-molecule methods can reveal some of the underlying
steady-state processes, they can only track the fate of relatively immobilized
molecules. A more general approach requires the ability to label specific
molecules at a given locality and subsequently follow their fate whether they
are anchored, polymerized or freely diffusing. Traditionally, this has been
done by photoactivation of fluorescence (PAF), which requires the construc-
tion of caged fluorescent probes that can be locally activated to fluoresce by
uncaging them using ultraviolet light. Unfortunately, the PAF approach is not
applicable to biofluorescent proteins expressed by the cells but GFP variants
have been produced recently that can be directly photoactivated in
mammalian cells without the requirement for harmful UV light (Patterson
and Lippincott-Schwartz, 2002; Chudakov et al., 2003). In one particular
GFP variant, a threonine at position 203 has been mutated to a histidine
residue, a change that results in poor emission when excited at 488 nm.
However, following intense irradiation with 413 nm light, GFP emission
increases 100-fold. This is due to conversion of non-fluorescent phenolic acid
into the fluorescent anionic derivative, phenolate. This results in photo-
activated molecules that can be tracked by conventional means. The recently
developed FLAP technique also enables effective photolabelling of specific
molecules using standard fluorescent probes (Dunn et al., 2002). The FLAP
method retains important advantages of the photoactivatable GFP - that it
can be used with fluorescent fusion proteins expressed by the cells and that no
harmful UV radiation is required - but it has the additional advantage that
ratiometry between labelled and unlabelled molecules of the same species is
possible (Zicha et al., 2003).
In FLAP, the conventional photobleaching process is used to label a specific
pool of fluorescent molecules. While fluorescence recovery after photo-
bleaching (FRAP) has long been used to study the repopulation of a bleached
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