Ancient Egyptians used water clocks to measure the passage of time. Mechanical clocks started ticking away in 14th-century Europe; and pocket watches, in the 17th century. Timex was founded in 1854 and Rolex in 1905. Today, you might use a smartphone to follow your schedule. But before all these timers, there were living cells — themselves impeccable timekeepers.
Cells in the human body follow cycles that repeat anywhere from once a second, in the case of a heartbeat, to once a month, for the female reproductive cycle. Living organisms have biological cycles that span all sorts of other time frames — the fastest-replicating bacterial cells duplicate every 15 minutes, bears hibernate annually, some cicada species emerge from the ground only once every 17 years, and many bamboo plants go for more than 60 years without flowering.
Scientists have watched these cycles with awe, asking what keeps these clocks ticking. Slowly, they’ve revealed many of the molecular gears that let cells stay on schedule. And even among disparate species — and between cycles with drastically different periods — they have uncovered commonalities.
“All these cycles are driven by clocks,” says James Ferrell, MD, PhD, a Stanford professor of chemical and systems biology and of biochemistry. “There’s almost nothing in common between each clock when it comes to the exact genes and proteins involved. But at a fundamental level each type of circuit is the same.”
With that knowledge in mind, scientists have now turned to a new type of question: How can we take advantage of what we know about the clock? Some researchers are exploring how to combat jet lag or treat narcolepsy and insomnia by altering sleep cycles. Some are probing how to administer different medications — from vaccines to drugs that enhance memory — by giving them at the time of day when they’re most effective. And others are working to stall tumor growth by slowing the clock that controls how fast cancer cells divide. At Stanford and in time zones around the globe, scientists are learning not just how to take a clock apart and see its insides, but to reassemble that clock the way they want, prevent it from getting off-kilter or get it back on track when it’s lost track of time.
Just as the same underlying principles of electronic circuits are used to design machines from calculators and radios to cars and cellphones, all biological clocks are governed by the same basic patterns of molecular switches. But unlike electronic circuits that are composed of wires and metals and silicon, cellular circuits are made of molecules that work together in a big game of tag, turning each other on and off.
To understand the molecular circuits that underlie biological clocks, Ferrell studies one of life’s most integral timers: the cell cycle. All living cells on Earth — from bacteria to stem cells — go through a similar cell cycle, which includes getting larger, copying genetic material, divvying it up and forming two new cells. The cell cycle is how an embryo develops, how skin cells are constantly replaced and why you can’t get your hair and fingernails to stop growing.
“You can think of the cell cycle as being driven by a clock in the same way you think of heart rate or sleep patterns being driven by clocks,” says Ferrell.
Human cells go through a cell cycle every 24 hours on average. But many organisms have faster cell cycles: Yeast cells divide every few hours, frog embryos cycle every 25 minutes and bacteria multiply even faster.
Recently, researchers started hypothesizing how sets of molecules might control this timing. One idea, says Ferrell, was that it’s driven by a positive feedback cycle. That would mean that a set of molecules switch each other on in brief pulses, like people passing a hand squeeze around in a circle. If it took a fixed amount of time for the signal to get back to the start of the circle, then a clock — or, in more technical terms, an oscillator — would be born. Every time the signal would hit one molecule in the cycle, it could spur some other biological process, be it cell division or hunger for lunch.
But when Ferrell and others started running computer simulations on how a positive molecular feedback loop could keep a biological clock ticking, their results didn’t click. “It makes complete sense that positive feedback should be able to work as an oscillator,” says Ferrell. “But it just doesn’t work. It turns out that the circuit will either eventually fade out to nothing, or end up with everything turned on all the time. The balancing point between these is just too fine a knife blade.”
Instead, biological clocks seem to always be driven by a negative feedback loop. In retrospect, Ferrell says, although it wasn’t the intuitive answer, this fits with what modeling and theories have been suggesting for a few decades; in fact, mathematician René Thomas conjectured in 1981 that all complex oscillators must contain a negative feedback loop.
Ferrell’s lab has spent the past few decades studying the negative feedback loop that controls the cell cycle. Like a positive feedback loop, a negative feedback loop involves molecules passing a signal in a circle. But in this case, they don’t just turn each other on in pulses: They alternately turn each other on and off. The cell cycle, though, isn’t just a simple loop — it also has all sorts of checkpoints. These ensure, for instance, that a cell doesn’t start copying its genetic material if it hasn’t grown large enough.
But the cell cycle — like many other biological clocks — can be complicated by the fact that it sometimes speeds up or slows down. In some cases, this is OK; cells in frog embryos, for instance, begin dividing slowly and speed up as they grow larger. But in other cases, this can lead to disease: Cells that progress through many fast cell cycles in a row can form a cancerous tumor. So, understanding how cells control the pace of the cell cycle is key to understanding one of the most fundamental properties of cancer.
“Every cell type in the body does the cycle a little bit differently; they’re very idiosyncratic,” Ferrell says. “And even within one population of cells, the cycle can speed up and slow down.” And then there are cancer cells: the fastest dividing cells of them all. Cancers cells march through the cell cycle at a faster pace than other cells, and that’s what makes tumors grow so aggressively. “The hope is that if we understand the cell cycle better, we can design more effective therapies for cancer,” he says.
Ferrell’s group has turned to frog embryos, because of their unusually reliable cell cycle length, to learn in more detail what proteins and genes control the speed of this clock. Using frog eggs, he’s shown why the first division cycle of the embryo is long, about 80 minutes, while those following are less than half an hour. The difference, he found, is due to the ratio of two proteins: Having more of one protein leads to the longer cycle. The lesson isn’t directly applicable to cancer cells — tumors don’t contain those same two proteins — but gives scientists hints about how cancer cell cycles might be sped up. Already, researchers have found that many tumor suppressor genes and oncogenes are directly involved in cell cycle checkpoints. Drugs targeting these pathways — and therefore restoring the cell cycle to its normal pace — are in clinical trials.
Sleep’s concert conductor
As cells tick tock through the cell cycle, other rhythms in the human body are progressing at their own paces. For anyone who has ever flown halfway around the globe only to spend days like a zombie and nights wide awake, the steady beat of one clock is obvious: the sleep cycle. Most of us find our bodies sticking to a 24-hour pattern of sleep; we get drowsy around the same time each night.
At the Stanford Center for Narcolepsy, sleep doctor and researcher Emmanuel Mignot, MD, PhD, is using findings he’s made on the human sleep cycle over the past few decades to develop new treatments for sleep disorders, including narcolepsy. Patients with narcolepsy have severe disturbances in their sleep-wake cycles, often characterized by sudden bouts of extreme fatigue during the day. At the end of the 1990s, Mignot and his colleagues identified the first narcolepsy gene, hypocretin receptor 2, in dogs. Since then, they’ve uncovered how a lack of the protein hypocretin in mammals, including humans, can cause narcolepsy. In most people, hypocretin levels peak during the day, when the protein promotes wakefulness and blocks sleep, Mignot has shown. In many people with narcolepsy, hypocretin is missing — or is found at very low levels in the brain — so the sleep pathways aren’t blocked during the day.
But even uncovering hypocretin hasn’t answered some of the most basic questions on why most of us have a regular pattern of alertness and fatigue and what other molecules wax and wane in tune with the sleep cycle. “The hypocretin system is like an orchestra director,” Mignot says. “It’s controlling the music — sleep and wake — but not making it. Right now we don’t even know who’s in the orchestra or what music is being played during sleep or wake.”
There’s another interesting cycle linked to narcolepsy, though, that’s leading to unexpected findings — an annual cycle. “There are always a lot more new cases of narcolepsy during the spring and summer,” Mignot explains. “And there was a huge rise in the number of narcolepsy cases in 2010 just after the winter of the swine flu.”
Mignot’s latest research looks at this intersection between this seasonal cycle and the sleep cycle. The onset of narcolepsy, he’s shown, can likely be triggered by a case of the flu, which may be asymptomatic and tends to happen over the winter. A few months later, narcolepsy appears. Mignot has been among the scientists who have shown over the past decade that most narcoleptics have an overactive immune system that attacks the cells that produce hypocretin, causing the lack of hypocretins. This problem, he thinks, may be triggered by the body’s production of immune cells produced to fight specific strains of influenza.
“It’s turning out to be quite an interesting journey looking at this,” says Mignot.
Through this research, Mignot is illuminating not only ways to treat narcolepsy, but other sleep disorders, like insomnia; an insomnia drug related to the hypocretin system is hitting the market soon.
“The view now is that healthy sleep is as important as diet or exercise to overall health,” Mignot says. “And sleep disorders of any kind are really an important societal problem.”
Cycles of learning
If anyone knows how surprisingly different the body can be at different points in its rhythmic cycles, it’s biologist Craig Heller, PhD, who co-directs the Stanford Down Syndrome Research Center. Mice with the genetic mutation that causes Down syndrome in people usually have trouble learning and on memory tests. They quickly forget objects they’ve seen and can’t remember how to complete a maze. But when Heller gives these mice a dose of pentylenetetrazole each day as the sun rises, before these nocturnal animals go to sleep, he can reverse their deficits. For months after receiving a two-week morning regimen of PTZ, the mice score better on memory tests. When Heller switches the timing of the daily PTZ dose, though, giving the drug at night instead of during the day, suddenly its effects completely disappear.
“This is becoming more and more appreciated in medicine,” says Heller. “The body is not the same at all hours of the day, and some drugs should be given at particular times to be most effective.”
The brain, it turns out, goes through daily cycles of learning and memory storage, coinciding with when a mouse (or a person) sleeps. So at different times of day, PTZ interacts differently with the brain.
When Heller made the observation that his Down syndrome treatment wasn’t as effective at night, when mice are active, as it was during the day, when mice are asleep, he began trying to make a link between circadian rhythms and sleep cycles and learning and memory. The crux of his research rests on the basic idea that the brain has two opposing functions: turning on neurons so they can communicate, and — at other times — blocking neurons from communicating. While people are sleeping, Heller has shown, this mandatory quiet time in the brain is especially vital. When the circadian rhythms of hamsters are obliterated, the animals no longer remember things from day to day.
“You’d think that inhibiting brain activity would always be contrary to our ability to learn and remember,” Heller says. “But while a person or animal is sleeping, memories from their daily experience are being translated into long-term memories, and as these memories are being moved from one part of the brain to another, they’re vulnerable to being altered.” During that process, he says, it’s important for most of the brain to not have any new activity, which could change the memory. PTZ, though, helps ensure that the activity ban isn’t too harsh; some areas of the brain need to function to store the memory. Having shown that PTZ treatment before sleep can lead to memory improvements in mice with Down syndrome and in hamsters lacking circadian rhythms, Heller is investigating whether the drug can treat other neurological conditions as well.
To further understand the role of this daily activity cycle in the brain, Heller’s group has studied what happens when memories are, incorrectly, reactivated during sleep. He trained mice to associate a particular smell with a shock. Then, during the day while the mice were sleeping, he piped the odor back into some of their cages. The mice who had re-experienced the smell had a much stronger fear response to the smell the next day. And, on the flip side, when the scientists blocked the whole brain from making new connections during the night, the mice didn’t remember the smell at all the next day.
Resetting the body’s clock
In collaboration with Jamie Zeitzer, PhD, associate professor of psychiatry and behavioral sciences at the medical school and at the Veterans Affairs Palo Alto Health Care System, Heller has also been studying more basic questions about circadian rhythms in people — and how to change these rhythms. The easiest way to alter the circadian clock, scientists know, is by exposing someone to light during their normal sleeping hours. This more quickly shifts the body’s clock than exposure to darkness during the waking hours.
“Typically, researchers thought someone had to be exposed to at least half an hour of constant light to shift the clock,” says Heller.
But if you’re on a plane with the lights out, working nights or arrive in a new time zone after the sun has set, it might not be possible to get this half-hour of light to get your clock on the right schedule. So Heller and Zeitzer started investigating whether shorter bursts of light could do the trick. In both human and mouse studies, they’ve now shown that 2 millisecond flashes of light every 30 seconds for an hour during the night — while it doesn’t interrupt sleep — can make people wake up earlier in the morning, shifting their circadian clock by almost an hour. The finding could lead to the development of new devices to help people avoid jet lag or adjust to a new shift at work.
“You could build these timed light pulses into glasses or travel alarm clocks or the cabins of airplanes to prevent jet lag,” says Heller. By exposing someone to a series of flashes during a flight, for instance, Heller thinks he could shift their clock enough to at least ease the transition to a new time zone, although the flashes of light wouldn’t help you sleep in if you’re traveling in the other direction.
For world travelers, preventing jet lag might be the ultimate biological clock hack. But even if you don’t jet around the globe on a regular basis, the ways scientists are learning to take advantage of your body’s cycles could help you recover faster from an illness, sleep more effectively, adjust to a new schedule or get better at learning new things. And as researchers continue to learn more about how cycles drive the rhythm of life, they’ll surely realize new ways to use this information.