‘Lava-Lamp’ Proteins May Help Cells Cheat
Death
With proteins that reversibly self-assemble
into droplets, cells may control their metabolism — and harden themselves
against harsh conditions.
https://www.quantamagazine.org/phase-separating-proteins-may-protect-and-regulate-cells-20181126/?utm_source=Quanta+Magazine&utm_campaign=fc63c7b840-RSS_Daily_Biology&utm_medium=email&utm_term=0_f0cb61321c-fc63c7b840-389846569&mc_cid=fc63c7b840&mc_eid=61275b7d81
Hibernating animals put
themselves into a largely inert state to survive a hostile winter. Individual
cells may do something similar to cope with stressful conditions by
solidifying and lowering their metabolism with the help of phase-shifting
proteins.
Rachel Suggs for Quanta Magazine
November 26, 2018
“If you make a discovery and at
first people tell you that it can’t be right, and then they eventually switch
to telling you ‘we knew that all along,’ then you are probably on to
something.” It’s a quip that has stuck in Clifford Brangwynne’s mind. For the biophysicist
at Princeton University, that is “exactly what happened with our findings on
intracellular liquid phases.”
Think of liquids with different
properties that don’t really mix but, under specific circumstances, cluster and
separate like the shifting blobs in a lava lamp. That phenomenon, also known as
liquid-liquid phase separation, was once considered to be an exclusively
chemical process. But less than a decade ago, Brangwynne became one of the first
to observe it happening inside cells as well,
and ever since then, biologists have been trying to learn its significance.
Now scientists are beginning to
understand that evolution has tuned certain proteins to act in aggregate like
liquids. Through phase separation, they spontaneously self-assemble into
dynamic, membrane-free, dropletlike structures that can perform needed tasks in cells.
In this ‘solidified’ state, a
cell can survive starvation.
Vasily Zaburdaev, Max Planck
Institute for the Physics of Complex Systems
“Somehow, no one thought that
this kind of ability of molecules could be harnessed by evolution to achieve
functionality or regulate functions,” said Simon Alberti, a biologist
at the Max Planck Institute of Molecular Cell Biology and Genetics (MPICBG) in
Dresden, Germany. “The focus was on the individual molecule and not on the
collective.”
The breakthrough has big
implications for our understanding of cellular organization and function,
said Vasily Zaburdaev, a biophysicist at the Max
Planck Institute for the Physics of Complex Systems, also in Dresden. One of
the latest findings is that phase separation allows certain types of cells to
cheat death when they are deprived of nutrients or otherwise put under stress. Phase separation enables the
cells to turn a large part of their cytoplasm from a liquid to a solid —
essentially putting themselves into a hardy condition of stasis until the
nutrients return.
Organelles Without Membranes
Nineteenth-century cell
biologists coined the term organelle (“little organ” in Latin) to describe the
tiny components they saw inside cells. Even then, pioneers in the field such as
the American cell biologist Edmund Beecher Wilson suspected that the jellylike
cytoplasm filling cells might hold various liquids “like suspended drops … of
different chemical nature.” That early insight found little purchase in biology
for almost a century, however: Researchers simply assumed that any
droplet-shaped cellular organelles must have an encapsulating lipid membrane to
prevent their contents from remixing with the cytoplasm.
Still, electron microscopy by
researchers such as L. Dennis Smith of the University of
California, Irvine, and Edward Mitchell Eddy of the National Institute of
Environmental Health Sciences in the 1960s and early 1970s showed that some
organelles simply didn’t seem to have any membrane at all. More membraneless
structures continued to be found, such as the nucleolus, a dense structure in
the cell nucleus. Yet until 2009, how and why they were forming wasn’t clear.
While
studying the distribution of membraneless organelles called P granules (green)
in the cells of a roundworm, Clifford Brangwynne and his colleagues discovered
that they were liquid droplets of protein, not solid masses.
Courtesy of Clifford Brangwynne/Science
That year, when Brangwynne was a
young postdoc at MPICBG, he, his colleague Christian Eckmann and
his supervisor Tony Hyman saw
something unexpected. They were looking at the uneven and inconsistent
distribution of organelles called P granules inside cells of the
roundworm Caenorhabditis elegans. P granules were widely assumed to
be dense pellets of RNA and protein. But Brangwynne, Eckmann and Hyman saw that
the granules were not solid at all. Instead, they appeared to be droplets of liquid that were coalescingat times
to form bigger drops, like oil in a well-shaken vinaigrette.
“It was a serendipitous
discovery,” Brangwynne said. “When we discovered that they were liquids, a
number of quantitative measurements that we had been taking suddenly made
perfect sense.” It also changed biologists’ understanding of how cells work.
That initial work by Brangwynne,
Eckmann and Hyman triggered an avalanche of papers investigating the assembly
and dispersal of various cytoplasmic proteins under various conditions. The
evidence was getting stronger that cells had evolved a fine-tuned mechanism for
organizing some of their internal structure and processes through phase
separation — that is, letting proteins self-assemble into structures that could
perform distinct functions.
Michael Rosen, a structural
biologist and chairman of the biophysics department at the University of Texas
Southwestern Medical Center in Dallas, was the first to reproduce this kind of phase separation in the lab with
certain proteins and RNA molecules that could coalesce into droplets. Phase
separation seemed to give proteins a reversible way to align and separate again
when conditions were right.
In some instances, however,
researchers are learning that the process is not reversible — and that this
failure represents a malfunction of proteins associated with a broad range of
diseases, including neurodegenerative disorders and cancer. For example, Zaburdaev
observed that several mutant forms of a protein linked to certain diseases
showed abnormal phase-separation behavior. “Instead of forming nice
drops, they form very strange hedgehog structures,” he said.
Solidifying for Survival
Intrigued, Zaburdaev and several
of his colleagues, including Alberti, decided to check what happens to proteins
when cells are subjected to stresses such as falling temperatures and the
sudden disappearance of nutrients. The surprising result they uncovered was
that phase separation can be part of a cell’s survival mechanism.
The cells’ behavior could be
likened to hibernation for bears. The animal lays still in a dormant state for
weeks, minimizing its expenditure of energy. At a cellular level, phase
separation helps the gelatinous cytoplasm make a protective transition into
something more solid. “In this ‘solidified’ state, a cell can survive
starvation,” Zaburdaev said.
Clifford
Brangwynne, a biophysicist at Princeton University, was named a MacArthur
Fellow in 2018 for his pioneering work on identifying the role that phase
separation plays in cell regulation.
The researchers studied this
phenomenon by depriving yeast and amoebas of nutrients. No nutrients means no
energy, and yeast cells need energy to pump protons out of their cytoplasm to
maintain the neutral pH essential for their biochemistry. “By starving, cells
acidified,” Zaburdaev said. Under the more acidic conditions, proteins readily
went from a dissolved state to a more condensed and solid one, and the
well-mixed cytoplasm separated into clusters of gelatinous blobs.
Simply by varying the acidity of the
cells’ environment, the scientists could induce them to switch into this survival state, even without
taking away the cells’ nutrients. The cells could rest this way for hours or
even days. “We found that the cells are so rigid that they keep their shape”
instead of being deformable, Alberti said. They “transition into a completely
different material state.”
When their normal pH was later
restored, the cells returned to normal, “dividing and living happily,”
Zaburdaev said.
The scientists found that they
could also trigger phase separation and solidification by completely
dehydrating the yeast through osmosis. Different types of stresses seem to
induce slightly different solid states, however. Exactly how that works is “something
we don’t yet understand,” Alberti said.
Nevertheless, the survival
mechanism that the experiments revealed was very simple, Alberti said: When
there is stress, extensive phase separation leads to the rigidification of the
entire cytoplasm, and the cell turns off its metabolism, like a hibernating
bear settling down for the winter.
The comparison to hibernation may
be more than figurative. “The cells of hibernating mammals may also solidify
inside,” Alberti said. “It’s a perfect way of dealing with these kinds of
environmental changes because solidification comes for free. The energy comes
out of the temperature change or the drop in pH.” However, the hypothesis that
phase separation is involved still needs to be tested, he said.
Immobilized for Metabolic Control
Most recently, Alberti’s team has
been probing the phase-separation response of cytoplasmic proteins to stress at
the molecular level. Their particular interest is in how it relates to control
over cellular metabolism.
The perfect way to turn something
off, Alberti said, is to put it into a solid material that can reversibly
immobilize it until it’s needed again. “It’s a way of protecting molecules from
damage, but also turning them off, storing them for later use.”
The team found that when a protein
has a certain identifiable domain or region, the protein will form easily
reversible gels. In the absence of this domain, the protein forms an
irreversible type of assembly — permanently removing it from further use.
It’s a way of protecting
molecules from damage, but also turning them off, storing them for later use.
Simon Alberti, Max Planck
Institute of Molecular Cell Biology and Genetics
In effect, this domain modifies
the protein’s phase behavior and keeps it reusable. “The domain provides a new
possibility, for that protein to assemble into a benign kind of gel and not
something from which you cannot come back,” Alberti said.
In one test-tube experiment, the
researchers took a solution containing a single type of protein and lowered its
pH. They saw molecules of the protein phase-separate from the solution and form
gel-like blobs. Then they brought the pH back to neutral, getting the gels to
dissolve, “showing exactly what we saw in cells,” Alberti said.
Such results imply that nature
has designed the domain sequences to tune the proteins’ material properties.
That’s very beneficial, said Dustin Updike, a biologist at the MDI Biological
Laboratory in Bar Harbor, Maine, because it gives cells “a mechanism to respond
to abrupt stress, such as heat shock, pH or osmotic stress.” Regulatory
mechanisms in cells often work at the genetic level, he explained, meaning that
they depend on signals reaching the nucleus, initiating gene transcription and
the manufacture of an appropriate enzyme. But those events take
time. In contrast, phase separations are very rapid — and can provide an
almost immediate response to stress.
Does It Really Matter?
Understanding the precise
mechanism and effects of phase separation in cells could be highly relevant for
a whole range of big biological challenges —
from organ preservation and aging research all the way to space travel,
according to Zaburdaev.
Recently, for example, the
neuroscientist Pietro De Camilli at
Yale University and his colleagues found evidence that phase separation might
be involved in the controlled release of
neurotransmitters at synapses. It had been observed that vesicles containing
neurotransmitters routinely hover in clusters near the presynaptic membrane
until they are needed. De Camilli’s team showed that a scaffolding protein
called synapsin 1 condenses into a liquid phase, along with other proteins, to
bind the vesicles into these clusters. When the synapsin is phosphorylated, the
droplet rapidly dissipates and the vesicles are freed to spill the
neurotransmitters into the synapse.
Lucy Reading-Ikkanda/Quanta
Magazine
It’s still early days, though.
When Brangwynne and his colleagues published their paper a decade ago,
biologists reacted with either total incredulity or hope for a brand-new
direction of research. As Updike noted, it can be hard for cell biologists
to go from thinking about a phenomenon in terms of protein aggregation to the
more complex problem of liquid phase separation, which requires fluid dynamics
to describe.
“To me, Cliff’s work was a huge
advance that better described the nature of P granules and what we were
seeing,” Updike said, in part because it also explained why P granules had
evaded biochemical purification for over two decades. “You can purify a
granule, but purifying something more similar to an oil droplet is much more of
a challenge.”
As ever more scientific papers
back up the concept of phase separation as a cellular mechanism, the number of
skeptics keeps on dropping, according to Updike and Brangwynne. Questions still
remain, though.
When we discovered that [the P
granules] were liquids, a number of quantitative measurements that we had been
taking suddenly made perfect sense.
Clifford Brangwynne, Princeton
University
“One of the criticisms is that
some people say every protein can do this,” Alberti said. It’s common knowledge
in science that concentrating proteins under various conditions can sometimes
make them solidify or liquefy. “But there was never this idea that this could
actually be used by cells, that evolution would actually act and use this
ability of biomolecules to achieve a functional change such as down-regulating
metabolism.”
Susan Wegmann, a biologist
at the German Center for Neurodegenerative Diseases (DZNE), said, “So far it
has not been shown that phase separation of proteins actually occurs in a
living multicellular organism.” The relevance of phase separation in cells to
complex problems in neuroscience and other areas is therefore uncertain. “We
and others are trying to make that link, but it is of course very difficult and
technically challenging. And if it turns out that protein condensation is
linked to human diseases such as neurodegeneration, then we have to find smart
ways to interfere in a specific manner with it.”
Tim Mitchison, a professor of systems biology at
Harvard Medical School, is skeptical about whether phase separation is a
generally important concept in biology. “I haven’t seen much evidence for phase
separation in the cytoplasm of cells except for a few specific examples, like
stress granules,” he said. The concept has seemingly not found much of an
audience outside of cell biology: Many researchers either have not yet heard of
phase separation or are ignoring the research.
“Maybe [they’re] waiting until there is more
functional evidence,” Mitchison said. He noted that with enough of the right
solvent, almost any protein or RNA can be made to phase separate. “But it’s not
clear how much of this is physiologically relevant. I’m totally convinced phase
separation is a thing, perhaps especially in RNA-protein biology,” he said.
“I’m less clear how general it is.”
Brangwynne seems unperturbed by
that reservation. He thinks that some skeptics “are asking very valid questions
about what this all means for cell function and dysfunction, which is still not
well understood.” Others might still be warming up to the idea of predictive
quantitative models, he said, “but that is the
future of biology.”
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