Thursday, 23 October 2014

'Mitoflashes' indicate acidity changes rather than free radical bursts

As we've written about before, mitochondria generate the energy required by our cells through respiration that involves using an "electrochemical gradient" as an energy store (a bit like pumping water up into a reservoir for energy storage to then harness it flowing down the gradient of a hill to turn a turbine), and produces superoxide (free oxygen radicals) as a by-product (a bit like sparks when the pumps are running hot). The fundamental importance of this machinery which not only delivers energy, but is also involved in disease and aging  has led to its investigation in great molecular detail (comparable to taking the turbines and generators apart to learn about their function). Much less is known about how mitochondria actually behave when they are fully functional in their natural environment inside our cells (comparable to looking at the fully intact and running turbine), and progress has been difficult since suitable `tools' are scarce.

A debate exists in the scientific literature about one of the key "tools" used in the investigation of living cells. A particular fluorescent sensor protein called cpYFP (circularly permuted yellow fluorescent protein) is used in biological experiments, ostensibly as a way of measuring the levels of superoxide/free oxygen radicals  in a mitochondrion. Our colleagues, however, have cast doubt on the ability of cpYFP to measure superoxide, providing evidence that it instead responds to pH, part of the above electrochemical gradient. This debate was complicated by the fact that in biology, pH and superoxide can vary together, as the amount of "driving" and amount of "sparks" might be expected to.

As another analogy: If we found an unknown measuring device and we did not know how it works, but we saw that it responds during sunny weather, we may conclude that it measures warm temperature. However, it may in fact measure high atmospheric pressure which is, like warm temperatures, often correlated with good weather.  
The protein cpYFP changes its fluorescence in response to pH changes, but is unaffected by superoxide changes.

A recent and fascinating paper in Nature observed that "flashes" of the cpYFP sensor during early development of worms (as a model for other animals and humans) were correlated with their eventual lifespan. However, despite the debate about what it is exactly that the  cpYFP sensor measures, the paper interpreted it as responding to superoxide: looking at the correlation in the light of the so called “free radical theory of aging". This long-standing and much debated theory hypothesizes that the cause of why we age and eventually die is related to the constant production of free oxygen radicals in our mitochondria causing a steady increase in damage to our cells weakening their energetic machinery more and more and making them prone to illnesses.

In response to this, our colleagues decided to settle the question about what the sensor actually measures chemically, removing biological complications from the system. In the analogy of the unknown measurement device, the device was now tested under controlled temperature and controlled pressure to clearly distinguish between the two. They produced an experimental setup where a mix of chemicals was used to generate superoxide in the absence of any pH change. cpYFP in this mix did not show any signal, showing that it remains unresponsive to superoxide. In concert, they showed that even small changes in pH produced a dramatic response in cpYFP signal. Finally, they investigated the physical structure of cpYFP, showing that a large opening in the barrel-like structure of the protein exposes a pH-sensitive chemical group to its environment (comparable to showing how exactly the inner mechanics of the unknown measurement device can pick up pressure changes). We thus concluded, in a recent publication "The ‘mitoflash’ probe cpYFP does not respond to superoxide" (in the journal Nature here) that the cpYFP sensor reports pH rather than superoxide, and that results using cpYFP (including the above Nature paper, which remains fascinating) should be interpreted as such. Iain, Markus and Nick

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