The 'mitoflash' probe cpYFP does not respond to superoxide.

Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial. There have been a number of studies suggesting that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of 3-day old correlated inversely with lifespan. The interpretation of ‘mitoflashes’ as ‘bursts of superoxide’ radicals assumes that circularly permuted yellow fluorescent protein (cpYFP) is a reliable indicator of mitochondrial superoxide. This interpretation has been criticised because experiments and theoretical considerations both show that changes in cpYFP fluorescence are due to alterations in pH, not superoxide. We now provide direct evidence that purified cpYFP is completely unresponsive to superoxide. Therefore ‘mitoflashes’ do not reflect superoxide generation and are not evidence for a link between mitochondrial free radical dynamics and lifespan.