In his first year of graduate school, a biochemist discovered something hidden inside a common piece of cellular machinery that's essential for all higher order life from yeast to humans.
What the student saw in 2015 -- subcompartments inside organelles called peroxisomes -- is described in a study published in Nature Communications.
"This is, without a doubt, the most unexpected thing our lab has ever discovered," said study co-author. "This requires us to rethink everything we thought we knew a bout peroxisomes."
Peroxisomes are compartments where cells turn fatty molecules into energy and useful materials, like the myelin sheaths that protect nerve cells. In humans, peroxisome dysfunction has been linked to severe metabolic disorders, and peroxisomes may have wider significance for neurodegeneration, obesity, cancer and age-related disorders.
Much is still unknown about peroxisomes, but their basic structure -- a granular matrix surrounded by a sacklike membrane -- wasn't in question in 2015. That's one reason the discovery was surprising.
"We're geneticists, so we're used to unexpected things. But usually they don't come in Technicolor," the senior author said, referring to another surprising thing about the find: beautiful color images that show both the walls of the peroxisome subcompartments and their interiors. The images were possible because of bright fluorescent reporters, glowing protein tags that the authors employed for the experiments. Biochemists modify the genes of model organisms -- the lab uses Arabidopsis plants -- to tag them with fluorescent proteins in a controlled way that can reveal clues about the function and dysfunction of specific genes, including some that cause diseases in people, animals and plants.
The authors were testing a new reporter in 2015 when he spotted the peroxisome subcompartments. "I never thought Zach did anything wrong, but I didn't think it was real," the senior author said and thought the images must be the result of some sort of artifact, a feature that didn't really exist inside the cell but was instead created by the experiment.
"If this was really happening, somebody would have already noticed it," the senior author recalled thinking.
"Basically, from that point on, I was trying to understand them," the grad student said and checked the instruments, replicated the experiments and found no evidence of an artifact.
"I revisited the really old literature about peroxisomes from the '60s, and saw that they had observed similar things and just didn't understand them," the author said. "And that idea was just lost."
There were a number of references to these inner compartments in studies from the '60s and early '70s. In each case, the investigators were focused on something else and mentioned the observation in passing. And all the observations were made with transmission electron microscopes, which fell out of favor when confocal microscopy became widely available in the 1980s.
"It's just much easier than electron microscopy," the senior author said. "The whole field started doing confocal microscopy. And in the early days of confocal microscopy, the proteins just weren't that bright."
The grad student was also using confocal microscopy in 2015, but with brighter reporters that made it easier to resolve small features. Another key: The author was looking at peroxisomes from Arabidopsis seedlings.
"One reason this was forgotten is because peroxisomes in yeast and mammalian cells are smaller than the resolution of light," the author said. "With fluorescence microscopy, you could only ever see a dot. That's just the limit that light can do."
The peroxisomes in the experiments were up to 100 times larger. Scientists aren't certain why peroxisomes get so large in Arabidopsis seedlings, but they do know that germinating Arabidopsis seeds get all of their energy from stored fat, until the seedling leaves can start producing energy from photosynthesis. During germination, they are sustained by countless tiny droplets of oil, and their peroxisomes must work overtime to process the oil. When they do, they grow several times larger than normal.
"Bright fluorescent proteins, in combination with much bigger peroxisomes in Arabidopsis, made it extremely apparent, and much easier, to see this," the author said.
But peroxisomes are also highly conserved, from plants to yeast to humans, and the senior author said there are hints that these structures may be general features of peroxisomes.
"Peroxisomes are a basic organelle that has been with eukaryotes for a very long time, and there have been observations across eukaryotes, often in particular mutants, where the peroxisomes are either bigger or less packed with proteins, and thus easier to visualize," the author said. But people didn't necessarily pay attention to those observations because the enlarged peroxisomes resulted from known mutations.
The researchers aren't sure what purpose is served by the subcompartments, but the lead author has a hypothesis.
"When you're talking about things like beta-oxidation, or metabolism of fats, you get to the point that the molecules don't want to be in water anymore," the author said. "When you think of a traditional kind of biochemical reaction, we just have a substrate floating around in the water environment of a cell -- the lumen -- and interacting with enzymes; that doesn't work so well if you've got something that doesn't want to hang around in the water."
"So, if you're using these membranes to solubilize the water-insoluble metabolites, and allow better access to lumenal enzymes, it may represent a general strategy to more efficiently deal with that kind of metabolism," the author said.
The senior author said the discovery also provides a new context for understanding peroxisomal disorders.
"This work could give us a way to understand some of the symptoms, and potentially to investigate the biochemistry that's causing them," the author said.
Peroxisomes in plants play a role in fatty acid oxidation and protein compartmentalization
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