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Fluorescence resonance energy transfer (FRET) between coupled fluorescent proteins is a promising tool for studying caspase activation during apoptosis. Unfortunately, current FRET systems are acutely sensitive to changes in pH and chloride ion concentration, and since these fluctuate considerably during apoptosis the results of FRET experiments are often difficult to interpret. Takemoto et al., whose report appears on page 235, developed a new FRET system that is resistant to acidification and chloride, and used it to obtain a detailed view of caspase-3 and caspase-9 activation in apoptosis. The new technique should also enable studies of caspase activity in vivo that were previously impossible.To make a caspase-sensitive FRET system, two fluorescent proteins, a donor and an acceptor, are linked with a cleavage site that can be cut by a particular caspase. Caspase activity is then detected as a change in fluorescence. The authors used a new variant of enhanced yellow fluorescent protein called Venus as the acceptor, and linked it to a donor protein through either a caspase-3 or caspase-9 cleavage sequence. Both systems are resistant to acidification and high chloride ion concentrations.
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The new systems show that, when apoptosis is induced, caspase-3 is activated rapidly in the cytosol and nucleus, reaching full activation before apoptotic morphological changes begin. This suggests that activated caspase-3 may enter the nucleus to induce the early structural changes. Caspase-9 is initially activated at the same time as caspase-3, but does not become fully activated until the morphological changes are well underway.7 e) C0 u2 M, @' Y3 y) \( }' d
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The authors have now developed a line of transgenic flies expressing the caspase-3 indicator system, and hope to have a transgenic mouse system soon. The ability to monitor caspase activity in whole tissues should make it possible to study the early steps of this important process, before the apoptotic cells are engulfed by their neighbors.(Caspase 3 is activated first in the cyto) |
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发表于 2009-3-6 00:39
发表于 2015-7-14 07:15
