," Dr. Ryan explained.
However, they faced one methodological roadblock.
As Dr. Ryan explained, scientists have long known how to gauge the importance of particular proteins, using a kind of Òon/offÓ comparison. They do this by using Òknock-outÓ mice genetically engineered to either express -- or not express -- the protein under study.
But Dr. Ryan's group wanted to go deeper, and discover what happened to endocytosis when amounts of clathrin increased gradually. "The knock-out mouse model was just too blunt an instrument," he said.
Instead, they came up with a whole new method of inquiry. It combined two pre-existing technologies -- RNA interference (RNAi), where scientists cut down on protein expression by jamming the responsible gene with bits of RNA; and microscopy, which looks at the activity of individual living cells by attaching a fluorescent tag to a specific cell component -- in this case clathrin.
"This means that now we could suddenly look at a much broader dynamic range of activity," Dr. Ryan said. "Instead of it being an 'all or nothing' proposition, we were able to understand how changing different amounts of clathrin changed the speed of endocytosis."
"This had literally never been done before, which is one of the reasons the journal editors are so excited about this study," he said.
The new technology really paid off in terms of results, he added.
"It turns out that clathrin regulates endocytosis in a manner we never suspected before. It's not a linear relationship, where simply doubling the amount of clathrin doubles the speed at which vesicles are shuttled back and forth," Dr. Ryan explained. "Instead, it's what biochemists call a highly 'cooperative' relationship. That means that at a certain moment in this relationship some kind of 'tipping point' occurs, so that a relatively small increase in clathrin kicks endocytosis into high gear."
The study was conducted in fibroblasts
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Source:Cornell University
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