Marvels of complexity, cells host many thousands of simultaneous
chemical reactions. Some reactions happen inside specialized
compartments, called organelles.
Certain organelles, however, lack any
membrane to wall themselves off from the rest of the matter floating
within cells. These membraneless organelles somehow persist as
self-contained structures amidst a cellular sea of water, proteins,
nucleic acids and other molecules.
‘A new tool - optoDroplet - offers unprecedented access to manipulating and understanding the chemistry that allows membraneless organelles to function.’
Scientists at Princeton University have developed a new tool -
dubbed optoDroplet - that offers unprecedented access to manipulating
and understanding the chemistry that allows membraneless organelles to
"This optoDroplet tool is starting to allow us to dissect the rules
of physics and chemistry that govern the self-assembly of membraneless
organelles," said Clifford Brangwynne, an assistant professor of chemical and biological engineering at Princeton and senior author of a paper published online in Cell
"The basic mechanisms underlying this process are very
poorly understood, and if we get a handle on it, there might be a hope
for developing interventions and treatments for devastating diseases
connected with protein aggregation, such as ALS."
Previous research has demonstrated that membraneless organelles
assemble within the cell by a process known as a phase transition:
examples of familiar phase transitions include water vapor condensing
into dew droplets or liquid water freezing into solid ice. Studies over
the last several years by Brangwynne and colleagues have revealed that
altering the concentration of certain proteins, or modifying their
structure, appears to trigger a phase change that allows proteins to
condense into droplet-like organelles.
To date, though, most studies have used purified proteins studied in
test tubes, and researchers have had few methods to study phase
transitions in the frenetic dynamos that are living cells. OptoDroplets
will help scientists learn about when phase transitions go awry,
yielding solid-like gels and crystalline aggregates of proteins
implicated in diseases including Alzheimer's and amyotrophic lateral
OptoDroplet relies on a technique called optogenetics, involving
proteins whose behavior can be altered by exposure to light. (Cells are
mostly water and thus essentially transparent.) The researchers showed
that they could induce phase transitions and create membraneless
organelles by switching on the light-activated proteins.
They also could
undo the transitions by simply turning the light off. Increasing the
light intensity and protein concentrations allowed the researchers to
further control the transition. By changing those inputs, they can
determine when condensed liquid protein droplets form, as well as
solid-like, protein aggregates, possibly linked to diseases.
"OptoDroplet provides us a level of control we can use to precisely
map what we call the phase diagram in living cells," said Brangwynne.
"With that, we're beginning to understand how cells use their natural
machinery to move through this intracellular phase diagram to assemble
different types of organelles."
The lead author of the paper is Yongdae Shin, a postdoctoral fellow
in Brangwynne's Soft Living Matter Group, part of Princeton's Department
of Chemical and Biological Engineering. Co-authors Joel Berry and Mikko
Haataja of the Department of Mechanical and Aerospace Engineering
helped develop mathematical models for understanding the intracellular
phase behavior, while Nicole Pannucci and Jared Toettcher of the
Department of Molecular Biology are experts in optogenetics and helped
guide the molecular design of the optoDroplet proteins. The work was
supported in part by the National Institutes of Health and the National
Using mouse and human cells, the research team spliced in a gene for
a light-sensitive protein from a plant called a mouse-ear cress (or
Arabidopsis thaliana,) a relative of cabbage and mustard that is a
mainstay of genetics research. Blue light exposure causes the protein to
self-associate, scrunching up on itself.
The light-sensitive tag was fused to protein components thought to
drive phase transitions in living cells. Using the light, the
researchers found that they could induce the proteins to huddle up,
mimicking the condensation process that naturally occurs in cells. "To
use the analogy of water vapor, you can think of what we did as using a
laser to locally change the temperature of some area of the air so that
water droplets would condense out of it," said Brangwynne.
The team repeatedly prompted the proteins to condense and then
dissolve by turning the light on and off. The process proved fully
reversible, even after many cycles. However, with high-intensity light
or high concentrations of proteins, the researchers created semi-solid
gels. Those gels were initially reversible, but over time they
solidified to form irreversible lumpy aggregates, similar to those found
in some diseases.
"We've shown with optoDroplet that we can readily assemble and
disassemble phase-separated liquids, and they do not appear to cause any
problem for the cell," said Brangwynne. "But the gel-like assemblies
appear to be more problematic, since over many cycles, they develop into
persistent aggregates that the cell can no longer deal with and that
can start to gum up healthy biological processes."
One example is the protein called FUS. The FUS protein is critical
for the cell's operations; it helps produce other proteins and repair
damaged DNA. But scores of genetic mutations can cause the FUS protein
to become too sticky, leading to ALS, also known as Lou Gehrig's
A neurological condition in which patients lose the ability to
voluntarily control their muscles, ALS is marked by clumps of protein
accumulating in nerve cells. Those clumps might stem from FUS or other
proteins pathologically aggregating, instead of staying as dynamic fluid
droplets. Huntington's disease and Alzheimer's also involve clumps of
proteins clogging up cells, again suggesting that abnormal phase
transitions in cells are closely connected with these conditions.
Edward Lemke, a researcher at the European Molecular Biology
Laboratory in Heidelberg, Germany, who was not involved in the Cell
study, noted the promise of optoDroplet.
"The proteins targeted by optoDroplet are an important constituent
of phase-separating proteins, many of which are also associated with
infamous diseases," Lemke said. "The optoDroplet system gives access to
modulating the state of these proteins inside the cell in a minimally
invasive and highly controlled fashion, so it can provide new insights
on how they carry out their function."
Brangwynne and colleagues look forward to continuing to experiment
with optoDroplet to better understand cells' complex behaviors.
"This is fundamental science we're doing, answering basic questions
about phase transitions in cells," Brangwynne said. "But we're hoping
these insights will reveal not only how healthy cells work, but also how
they can become diseased, and maybe eventually cured."