Abstract
This chapter discusses fluorescence photoactivation as an alternative technology for probing cytoskeleton dynamics. In this approach, the protein is tagged with a caged fluorochrome. This probe molecule is nonfluorescent until illuminated with a brief pulse of ultraviolet light. Such illumination leads to photolysis of the caging groups and generation of a fluorescent species. In principle, fluorescence photoactivation can avoid the problem of generation of local oxidative damage inherent to photobleaching and can also produce a more favorable signal-to-noise ratio for imaging. Photoactivation can also produce toxic by-products in the form of the nitrosoaldehyde or nitrosoketone side products from photolysis. The chapter discusses the progress in caged fluorescent probes. The first caged fluorescent probe that was used for a biological experiment was a fluorescein derivative, C2CF-sulfo-N-hydroxysuccinimide. This probe, attached to tubulin, led to the discovery of poleward flux in mitotic spindles. C2CF is highly hydrophobic, and most proteins other than tubulin tend to aggregate if they are labeled with it.
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