How the
Body’s Trillions of Clocks Keep Time
Cellular clocks are almost
everywhere. Clues to how they work are coming from the places that they’re not.
Veronique Greenwood
[[The message of science over the past 125 years is that everything is far more complex and mysterious than it seems. Here is another sterling example. DG]]
Individual
proteins, like this protein found in blue-green algae, help
regulate circadian rhythms.
Laguna Design/Science Source
Carrie Partch was
at the tail end of her postdoc when she made the first discovery. The
structural biologist was looking at a database of human proteins, noting those
that shared a piece with the ones she’d been studying. “I was just sort of
flipping through it thinking, ‘I should know all of these,’” she recalls. “Then
this one came up, and it had a different domain architecture than I’d ever
seen.” She looked further into the protein, called PASD1, whose function was
unknown. She found that among the few proteins it resembled was one called
CLOCK. And that made her sit up straighter — because CLOCK is at the heart of a
very large, mysterious process.
Not that long ago, as Partch
knew, it had become clear that nearly every cell in nearly every
tissue in the body keeps time. Every 24 hours, responding to a
biochemical bugle call, a handful of proteins assembles in the cell’s nucleus.
When they bind to each other on the genome, they become a team of unrivaled
impact: Under their influence, thousands of genes are transcribed into
proteins. The gears of the cell jolt into motion, the tissue comes alive, and
on the level of the organism, you open your eyes and feel a little hungry for
breakfast.
These timekeeping protein
complexes, which take some of their cues from a part of the brain that responds
to light and darkness, are known as circadian clocks. By some estimates, they
regulate the expression of 40 percent of the genes in the body. Researchers are
accumulating evidence that circadian clocks have deep effects on everything
from fetal development to disease. Circadian clocks are so ubiquitous, and so
important to the function of individual cells, that biologists whose research
doesn’t overtly connect to a clock are becoming aware of how it might impact
their work. “More and more they are stumbling into clock components,”
said Charles Weitz, a molecular biologist at Harvard Medical
School. “It doesn’t surprise me.”
The Clock the Cow Found
The daily cycling of plants and
animals has been a source of fascination for millennia, but it wasn’t until
about 50 years ago that research into the underlying biochemistry began to take
off. Many people trace the field’s founding to a
meeting at Cold Spring Harbor in the summer of 1960, where researchers
brainstormed ideas about what might cause circadian rhythms and devised
experiments to test their theories. Over the ensuing three decades, researchers
identified mutant creatures with abnormal daily cycles — fruit flies, hamsters,
yeasts and others — and began to uncover the genes required for a normal
rhythm. Studying flies whose natural cycle was 19 or 28 hours, or who had no
discernible rhythm, led clock pioneers Ronald Konopka and Seymour Benzer
to discover
the first family of key clock genes, which they named per, in
1971, and whose levels we now know to rise and fall through the day. Just a
year later, researchers reported that a tiny patch of cells in the brain called
the suprachiasmatic nucleus was necessary
for a 24-hour circadian rhythm in mammals.
Carrie
Partch has discovered a protein that interferes with circadian rhythms.
Courtesy of Carrie Partch
Yet for many years it was not
clear how pervasive the rhythm’s effects were — how deeply it affects
everything in the body. In 1988, Ueli Schibler, now a professor of molecular biology at the
University of Geneva, was studying transcription factors, cellular actors that
control the transcription of genes into proteins. One factor in particular,
isolated from rats by a Canadian postdoc, seemed to be quite powerful. Together
they published their discovery in the journal Cell. Three months
later, however, a student named Jérôme Wuarin took over the project. He soon
approached Schibler with some disturbing news.
“You’ve got to retract this
paper,” Schibler recalls Wuarin saying. “It’s all fake. It doesn’t exist.” When
Wuarin performed the isolation, the transcription factor had failed to appear.
Schibler, taking his concerns seriously, tried the procedure himself. He found
the transcription factor easily.
After a number of weeks, Wuarin
realized why he couldn’t find it himself: He and the postdoc had been
performing the isolation at different times of day. [[Think about that – would you
hve guessed that the operations of the cell are governed by time? DG]] The
postdoc, a late riser, usually arrived around 11 a.m., killed the rats, and had
the transcription factor in hand by midafternoon. “But [Wuarin] was a farmer’s
son,” Schibler explains. “He got up at 5, milked the cows, then came to lab and
killed the rats at 7. And at that time, this protein’s just not there.”
It’s now known that every day,
this transcription factor’s levels start at almost nothing, making it
impossible to detect in the morning, and then rise 300-fold, making it easy for
the postdoc to find in the middle of the day. Schibler notes wryly, as an
aside, that in all the years since, no one has ever found a protein that
oscillates more wildly. It was just their luck.
After this discovery — that the
circadian rhythms of the researchers and the circadian rhythms of the rats interacted
to make the protein appear to disappear — Schibler turned to studying the daily
rhythm and its control of transcription more closely. In 1998, he and
colleagues found something unexpected. For years, the cells of the
suprachiasmatic nucleus were thought to be alone in having their own clock,
controlling all the rhythms in the rest of the body remotely. But Schibler and
colleagues found that the brain wasn’t required for a rhythm; nor really was
a body. Two kinds of rat cells, grown in dishes for generations, would
rhythmically express genes all on their own. The team’s work joined a handful
of other studies suggesting that body clocks were more widely
distributed than people had thought.
Since then, liver cells, heart cells, lung cells — in the words of
Charles Weitz, “just about every tissue we’ve looked at” — have turned out to
beat their own time, in addition to taking cues from the suprachiasmatic
nucleus. “Almost every cell in our body has a circadian clock,” said Satchin Panda, a clock
researcher at the Salk Institute. “It helps every cell figure out when to use
energy, when to rest, when to repair DNA, or to replicate DNA.” Even hair
cells, for instance, divide at a particular time each evening, Panda has found.
Give cancer patients radiation therapy in the evening rather than in the
morning and they might lose less hair.
[[So register this: for yours it
was thought there was only one cell type with its own clock. Now they find that
almost every cell type has one. They were surrounded by clocks and missed
almost all of them. How could they have been so wrong? One factor: they drew a
general conclusion without any testing….DG]]
Researchers have spent the past
15 years untangling the molecular components of these peripheral clocks, as
they’re known. A big step forward came in 2004, when a team led by Joseph
Takahashi, now a professor at the University of Texas Southwestern,
developed mice with a glowing PER protein. When PER is expressed, cells from
these mice are bright; when it’s not, they’re dark. This advance has
enabled studies that track the clock’s cycling in myriad
different tissues and circumstances.
Researchers have found that
peripheral clocks are based on CLOCK and a protein called BMAL1, as is the
clock in the suprachiasmatic nucleus. Clasping each other tightly, this pair
attaches to the genome and recruits other proteins to start the transcription
of nearby genes, including per. Many of these genes are behind
certain physiological rhythms — the production of liver enzymes around mealtime,
for instance, and the daily rise and fall of blood pressure.
But some proteins, including PER,
serve as counterbalances. As PER and its partners gradually build up in
the cell over a period of 12 hours, they inhibit the activity of CLOCK and
BMAL1. Over the next 12 hours, the counterbalances are slowly degraded, and
CLOCK and BMAL1 surge back. Just before dawn and just before dusk, John Hogenesch, a
chronobiologist at University of Pennsylvania, has found, there are “rush
hours” of gene expression, perhaps the body preparing for the different demands
of surviving in the light and in the darkness.
It’s a tidy, self-governing
system, and it’s tempting to call it ubiquitous. But these studies have revealed
too that not everything has a clock. Embryonic stem cells, which can develop
into almost any cell type, don’t keep time. The testes, almost alone among the
organs that have been tested, don’t seem to have a clock either. And many
cancer cells do not keep a regular rhythm. What could these things have in
common? This is where Partch’s discovery comes in.
How the Clock Stops
One of the first things Partch
learned about PASD1 was that it appears in very few tissues. But the ones where
it does are intriguing: the testes and cancers. When Partch became a professor
at the University of California, Santa Cruz, she and her students began adding
PASD1 to cells equipped with glowing PER. They found the cells’ usual light was
damped down to a faint glimmer, indicating that PASD1 was interfering with the
normal operation of the clock. And the more PASD1 they added, the dimmer the
cells were.
Next, Partch and her students
grew cells with glowing PER and got all the cellular clocks synchronized. The
glow would get brighter and dimmer like a sine wave with a 24-hour period, with
defined peaks and troughs for as long as the cells stayed synchronized. Partch
then caused some of those cells to produce PASD1. In these cells, the glow
became more of a wobble than a wave — the peaks low and the troughs shallow —
and very soon it faded away. The cells could not maintain their rhythm.
The team is still working to pin
down exactly how PASD1 calls a halt to the cells’ cycling. But one specific
part of the protein gives them a hint. This section of PASD1 looks like a part
of CLOCK that is absolutely essential for circadian rhythms. “But no one still
to this day knows quite exactly what it does,” Partch said. She hopes that by
understanding how the key piece of PASD1 works — perhaps, for instance, it
binds to BMAL1 itself and keeps CLOCK from doing so — they can learn the role
of this key piece of CLOCK.
So far, the work has confirmed
Partch’s initial hunch that PASD1 would stop the clock. And it suggests that in
the tissues where PASD1 is present, it is part of the reason why the cells
don’t oscillate. That finding opens the door to deeper questions: With the
clock directing so many aspects of cellular behavior, and with mutations in
clock genes leading to illness — they’ve been fingered in cancers and metabolic
disorders — why would some types of cells lack a clock or have a weakened one?
“It seems like there’s some
really interesting and still unexplored connection between some perfect
pluripotency,” meaning the cellular ability to develop into any cell type, “and
running a clock,” Partch said. She recounts experiments from Kazuhiro Yagita’s
lab, in which embryonic mouse stem cells are spurred into development. “At
first, it’s like come on, come on, no ticking, no ticking… and then, at some
point in the differentiation of these cells, the clock comes on.” When the
process is reversed, the clock turns off.
In cancers, the protein’s other
known hangout, the reasons for its presence are likely to be different. “It may
be the reason why the clock is not operational in most solid tumors,” said
Hogenesch, who was not involved in the work. “If you’re a tumor, and you want
to keep dividing and dividing and dividing, maybe you don’t want to be confined
to dividing at one time of the day. Maybe there’s an evolutionary advantage —
to tumors at least — to disrupt the clock so they can divide whenever they have
sufficient resources rather than being nudged and nuanced to divide at a
particular time of day.” Partch’s group found that interfering with PASD1’s
production in two cancer cell lines made their oscillations stronger and more
regular. That suggests that future work should look to see whether knocking
PASD1 down might also rein in the cancer cells’ out-of-control reproduction.
Ultimately, the research should
illuminate something more fundamental. “Understanding how PASD1 interferes with
clock function lets us know how the clock is working,” Partch said. She and her
team are also realizing that just as the clock affects far more processes than
were at first evident, PASD1 may be doing more than just interfering with CLOCK
and BMAL1. But that work will come, with time.
This article was reprinted
on Wired.com.
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