Just as a pocket watch requires a complex system of gears and springs to keep it ticking precisely, individual cells have a network of proteins and genes that maintain their own internal clock—a daily rhythm that, in humans, regulates metabolism, cell division and hormone production, as well as the wake-sleep cycle. Studying this “circadian” rhythm in fruit flies, which have genes that are homologous to our own, scientists have constructed a basic model of how the cellular timekeeper works.
But now, a new report in this week’s issue of Science turns the old model on its head: By providing a glimpse into living cells, Rockefeller University researchers have uncovered a previously undetected clock inside the circadian clock. At the most basic level, an organism’s sleep-wake rhythms are governed by 10 known genes. In the fly, two of those genes—called period and timeless—produce proteins that fluctuate in a negative feedback loop that takes about 24 hours to complete.
At night, two other genes, clock and cycle, stimulate production of Period and Timeless proteins, which begin to accumulate in the cell’s cytoplasm. After about six hours, the two proteins move into the nucleus; their presence there turns off the genes, which then remain inactive until Period and Timeless degrade and the whole cycle begins anew.
Michael W. Young, the Richard and Jeanne Fisher Professor at Rockefeller University and head of the Laboratory of Genetics, isolated period, the first circadian gene, in 1984. He and his peers have been piecing together the cellular circadian puzzle ever since, and thought they had some of the basics figured out. Prior studies, which examined the placement of Period and Timeless during different stages of the cycle, seemed to indicate that the two proteins idle in a cell’s cytoplasm until they bump into each other and then, bound together, enter the nucleus. But Young and Pablo Meyer, who was then a graduate student in Young’s lab, used a novel method to show that this scenario was far too simple.
Meyer, a physicist by training, found himself frustrated by how little he could see of what was occurring in a cell. “The truth is, we really don’t know, mechanistically, what happens in the cytoplasm, and how things are being done in such a precise way,” Meyer says. So he turned to a technique invented in 1948, called fluorescence resonance energy transfer, or FRET. The method makes it possible to gauge interactions between proteins by fluorescently tagging them and measuring how they react to different wavelengths of light. But no one had ever thought to use it to follow proteins in a single cell for an extended period of time.
“This begins to measure all these biochemical interactions inside the cell,” Meyer says. For the first time, Period and Timeless could be tracked within a cell for eight hours or longer. “No one had ever labeled the components to follow them over time, to see one clock as it ticks away in a single cell,” Young says. “All the biochemistry and molecular biology that had been done on this had been piecing together information from dead flies.”
Now, instead of freeze-frames, the researchers had a movie that allowed them to follow the interactions between Period and Timeless with a resolution never before possible. They discovered that, rather than randomly colliding, the two proteins bind together in the cytoplasm almost immediately and create what Young and Meyer refer to as an “interval timer.” Then, six hours after coming together, the complexes rapidly break apart and the proteins move into the nucleus singly, all of them within minutes of each other. “Some switch is thrown at six hours that lets the complex explode. The proteins pop apart and roll into the nucleus,” Young says. “Somehow, implanted within the system is a timer, formed by Period and Timeless, that counts off six hours. You have a clock within a clock.” He notes that this precise timer shows how carefully orchestrated interactions between proteins really are.
He and Meyer, who is about to begin his postdoctoral research at Columbia University, have yet to figure out exactly how the timer works, but its discovery opens up the door to a whole new suite of questions. “How does this interval timer tick? Is it made from additional proteins? Is this the only such timer in the circadian clock? Each of these questions are ahead of us,” Young says. “A couple of years ago, we had identified lots of genes and had this sweeping picture of how circadian clocks work. But this indicates that there are much more formidable properties of the system that were overlooked.”