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Researchers at the Whitehead Institute say they have developed a method to isolate quickly and systematically measure metabolite concentrations within mitochondria. Prior attempts at such measurements have yielded unreliable results, either by taking too long to isolate mitochondria or by contaminating mitochondrial metabolites with contents from other cellular components, according to the scientists.
“The advantage of this new method is that it offers a combination of both increased speed and specificity,” says Whitehead Member David Sabatini, Ph.D., who is also a Howard Hughes Medical Institute investigator and a professor of biology at MIT. “We are quite excited about applying this workflow in vivo and to other organelles such as lysosomes.”
Through precisely controlled chemical reactions, the mitochondria produce energy in the form of adenosine triphosphate (ATP) and play a critical role in cellular homeostasis. Mitochondrial dysfunction is found in several disorders, including Parkinson’s disease, cardiovascular disease, and mitochondrial diseases. Until now, peering into the inner metabolic workings of these vital organelles has been challenging at worst and inaccurate at best, noted Dr. Sabatini.
One conventional method of profiling mitochondrial metabolites involves purifying mitochondria using several rounds of centrifugation, a process that can take more than an hour to complete. According to Walter Chen, a graduate student in Whitehead Member Sabatini’s lab, time is a significant issue when studying metabolites.
“Even if you keep your sample at 4oC or 0oC to slow down any reactions, you’re still gradually getting distortion of the mitochondrial metabolite profile because the enzymes are still going and so are the transporters,” says Chen, who is also a third-year medical student at Massachusetts General Hospital. “As time goes on, the mitochondria are getting less happy outside the cell.”
The other commonly used method for profiling mitochondrial metabolites relies on abbreviated forms of centrifugation to isolate mitochondria. Although faster, this protocol also brings down nonmitochondrial material and other organelles, thereby distorting the true mitochondrial signal with metabolites from extramitochondrial sources.
To reduce the time needed to isolate mitochondria and increase the accuracy of the metabolite analysis, Chen took a completely different approach—rapid immunopurification. He coated the exterior of mitochondria with epitope tags and added tiny beads covered in antibodies specific for the tags. By locking onto the tags, the antibodies link the mitochondria to the beads, allowing Chen to isolate the mitochondria easily, break them open, and stop all enzymatic activity within 10 minutes.
According to his analysis, this quicker method yields results that better reflect the actual mitochondrial metabolite levels found within a living cell. The study (“Absolute Quantification of Matrix Metabolites Reveals the Dynamics of Mitochondrial Metabolism”) by Chen and colleagues is published in Cell.
“From the data we have so far, profiling mitochondria with this method definitely gives you greater resolution than what you would obtain using traditional methods to profile whole cells,” says Chen, who is a co-author of the Cell paper. “On the in vivo front, I think this is going to be quite powerful, and that’s what I’m most excited about. But I can already see that this can lead people in new and interesting directions.”
Chen says the method is potentially very versatile and could be adapted to analyze the metabolite contents of other organelles and to compare mitochondria in cells affected by mitochondrial dysfunction—such as neurons damaged by Parkinson’s disease—with normal cells or other cell types seemingly unaffected by disease.