INSIGHTS | P E R S P E C T I V E S

CELL SIGNALING

Liquidity in immune cell signaling

Phase separation of proteins into liquid-like microclusters promotes T cell receptor signaling
By Michael L. Dustin1,2 and James Muller2 Dispersed bulk phase other while maintaining a roughly round
Unphosphorylated LAT proteins span the membrane shape and allow rapid exchange of pLAT be-
but favor ordered membrane regions (yellow)

T
lymphocytes of the immune system
need to integrate myriad signals to
decide on life and death matters in de-
fending the host from pathogens and
in distinguishing normal from trans-
formed cells, all while minimizing im-
munopathology. On page 595 in this issue,
Su et al. (1) provide insight into how phase
separation of signaling proteins in the two-
dimensional (2D) liquid of the plasma mem-
brane enables these critical decisions.
tween a cluster and surrounding unclustered
pLAT. Su et al. expanded the system of pu-
LAT Membrane rified proteins to include kinases and phos-
phatases that act downstream of the TCR
signaling cascade and generate pLAT. The
Ordered authors also overlaid an actin polymerizing
lipids
Cytosol system on the pLAT clusters, which consisted
of globular actin (G-actin) and five proteins:
Grb2-related adapted downstream of Shc
Signaling G-actin (Gads), SH2 contains leukocyte protein of 76
proteins kDa (SLP-76), noncatalytic region of tyrosine

Downloaded from http://science.sciencemag.org/ on February 25, 2018

Phase separation in live cells has become a
useful concept in explaining the formation of
µm-scale spherical, liquid-like compartments
that are held together by weak, rapidly re-
versible interactions and that exchange com-
ponents with the surrounding bulk phase
(2). Phase separation is also well studied in
2D systems such as biological membranes,
where coexisting liquid disordered and liquid
ordered phases are important for signaling
by many transmembrane receptor systems
(3), including the T cell receptor (TCR) sig-
naling process. In response to binding an-
tigen peptides, the TCR triggers a series of
biochemical events orchestrated by various
enzymes and adaptor proteins. This signal-
ing cascade impinges on the nucleus and the
actin cytoskeleton to alter T cell behavior. Su
et al. examined how soluble and membrane-
anchored adapter proteins downstream of
the TCR form a 2D phase–separated system
that depends fully on protein-protein inter-
actions on top of a liquid disordered bilayer.
Su et al. looked at the behavior of a signal-
ing efector of the TCR called linker of acti-
vated T cells (LAT), and two of its binding
partners—growth factor receptor–bound pro-
tein 2 (Grb2), which binds to phosphorylated
LAT; and son of sevenless (Sos), which binds
to the N- and C- termini of Grb2 (1). This pro-
tein trio is critical for activating the signal-
ing protein Ras as part of the TCR signaling
pathway. The authors found that physiologi-
cal amounts of bilayer-anchored prephos-
phorylated LAT (pLAT), plus soluble Grb2
and Sos proteins, formed micrometer-scale
T cell receptor activation, kinase adapted protein 1 (Nck), actin-related
LAT phosphorylation protein 2/3 (Arp2/3), and neuronal Wiskott-
Aldrich syndrome protein (N-WASP). The
SLP-76 2D pLAT clusters could interact with the
Liquid phase separation Gads actin-nucleating factor N-WASP to trigger
pLAT clusters phase
formation of an elongated filamentous actin
separate from bulk
dispersed LAT and pLAT (F-actin) gel (see the figure). The F-actin gel
appears to act like a solid container that ab-
sorbs liquid-phase pLAT and takes the shape
of the F-actin.
The pLAT clusters are likely to be compo-
nents of TCR “microclusters,” where antigenic
major histocompatibility complex-peptide li-
gand (antigen) are engaged and signaling re-
actions occur on the cytoplasmic face of the T
cell plasma membrane. These microclusters
are highly dynamic and display fusion events
to form larger clusters (4). TCR microclus-
ter formation in response to physiological
Enhanced signaling to
nucleus and cytoskeleton ligands depends on F-actin remodeling. It
is possible that the complex interaction be-
tween the pLAT-rich liquid phase and F-actin
Solid phase scafold is related to this requirement—that is, initial
The pLAT cluster triggers, then takes TCR triggering could induce pLAT phase sep-
the shape of, an F-actin gel
aration and signal actin remodeling, which
would feed forward to promote further TCR
interaction with antigen.
Concepts of 2D phase separation can also
be applied to reconstituted cell adhesion sys-
tems. The adhesion of T cells that express
the receptor CD2 to an artificial lipid bilayer
F-actin bearing its ligand CD58, displays characteris-
tics of a phase-separated system with the pre-
cisely apposed membranes contributing the
necessary dynamic multipoint attachments
(5). Furthermore, diferent size receptor sys-
ILLUSTRATION: V. ALTOUNIAN/SCIENCE

pLAT clusters on a supported lipid bilayer in
vitro. The pLAT clusters have characteristics
of a liquid phase in that they fuse with each
1
Kennedy Institute of Rheumatology, The University of
Oxford, Oxford, UK. 2Skirball Institute, New York University
School of Medicine, New York, NY, USA.
Email: michael.dustin@kennedy.ox.ac.uk
Fluid signals. Signaling molecules (blue) form
liquid-like phases, instead of static aggregates, in a
reconstituted system using an artifcial lipid bilayer.
Shown is a model of clustered pLAT and associated
signaling molecules that phase separate into a liquid-
like droplet in response to TCR activation. The cluster
induces actin polymerization, which is a solid phase gel
that changes the shape of the liquid-like droplet.
tems that bridge the gap between the T cell
and artificial lipid bilayer further subdivide
the interface into TCR and adhesion mol-
ecule–rich domains of the immunological
synapse (the interface between the antigen-
presenting cell and the T cell) (6), the natural
setting for the reactions studied by Su et al.
It may be that the actinomyosin–based trans-

516 29 APRIL 2016 • VOL 352 ISSUE 6285 sciencemag.org SCIENCE

Published by AAAS
port system, which drives centripetal trans-
port in the immunological synapse, moves
the TCR and pLAT clusters by using the F-
actin–based “container” that was observed
by the authors in their reconstituted system.
Su et al. focused on phenomena largely
confined to the 2D supported lipid bilayer
surface by tethering key components to the
bilayer’s surface. It is not clear whether 2D
phase separation of pLAT could also nucleate
a 3D phase–separated structure that would
be released into the cytoplasm. Such a struc-
ture appears to be formed during T cell stim-
ulation by immobilized antibodies to TCR,
where foci (containing SLP-76) are released
as particles from TCR microclusters and traf-
ficked toward the microtubule-organizing
center (7). These particles seem to dissipate
in the center of the immunological synapse,
consistent with reversible phase separation.
The findings of Su et al. provide insights
into characteristics of TCR signaling that are
otherwise hard to reconcile. Recent super-
resolution microscopy studies suggest that
substrate-adherent T cells display basal seg-
regation of TCR and pLAT into distinct “pro-
tein islands” on a ~100- to 300-nm length
scale (8). It is possible that interaction be-
tween layered dynamic liquid phases could
govern signal integration. Segregation of
TCR clusters from pLAT may be mediated
by the tendency of the TCR to reside in liq-
uid disordered bilayer phases, whereas LAT,
which is palmitoylated, resides in liquid or-
dered domains. These interactions between
phases may also govern important regulators
like the transmembrane tyrosine phospha-
tase CD45. The large extracellular domain
of CD45 excludes it from TCR microclusters,
which is important for the phosphorylating
tyrosine kinase cascades proximal to the TCR
(9). Su et al. found that CD45 is also excluded
from pLAT clusters, likely due to charge re-
pulsion between the phosphatase domain
and pLAT cluster components. It may be
important that CD45 is excluded in this way;
segregation of TCR clusters and pLAT clus-
ters suggests that extracellular domain size
does not exclude CD45 from the pLAT clus-
ters. The tools generated by Su et al. have
the potential to further address underlying
principles governing the dynamic, layered
circuitry of transmembrane signaling. j
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10.1126/science.aaf8179
SCIENCE sciencemag.org
NEUROSCIENCE
Ionic control of sleep
and wakefulness
The ionic composition of brain fluid is linked to
neuronal activity and sleep
By Hans-Peter Landolt1,2 and
Sebastian C. Holst1,2
B
rain electrical activity difers mark-
edly between wakefulness and sleep.
Concomitant shifts in the ion com-
position of brain extracellular fluids
were thought to be a consequence
rather than a cause of the sleep-wake–
dependent changes in neuronal activity. On
page 550 of this issue, Ding et al. (1) report
the surprising observation that ionic changes
in the extracellular fluid are a potent control
of sleep-wake–dependent neuronal activity.
Although the biological functions of sleep
are still elusive, sleep is essential for optimal
brain functioning and general physiology
that varies in synchrony with the sleep-wake
cycle. In wakefulness, cortical brain activa-
tion is driven by wake-promoting neuromod-
ulators (acetylcholine, hypocretin, histamine,
serotonin, noradrenaline, and dopamine)
produced in the basal forebrain, hypothala-
mus, and brain stem (2). These regions are
interconnected in an excitatory network
that sends projections to the thalamus and
cerebral cortex. Together with neurons that
produce glutamate and g-aminobutyric
acid (GABA), this ascending arousal system
maintains conscious and alert wakefulness,
characterized by low-voltage, fast-frequency
activity in an electroencephalogram (EEG).
In deep non–rapid eye movement (NREM)
sleep, the EEG of animals and humans is
characterized by large, slow waves of electri-
cal activity with frequencies of roughly 1 to
4 Hz. Synchronous oscillations between “up
states” and “down states” in the membrane
potential of cortical neurons underlie these
slow waves (3, 4). Sleep deprivation increases
the number, amplitude, and slope of EEG
slow waves in recovery sleep (5), reflecting
their robust homeostatic regulation.
Neuronal activity is controlled by ion flow
across the plasma membrane. It has been
thought that during wakefulness, the break-
down of energy-rich compounds increases
1
Institute of Pharmacology and Toxicology, University of
Zürich, Zürich, Switzerland. 2Zürich Center for Interdisciplinary
Sleep Research, University of Zürich, Zürich, Switzerland.
Email: landolt@pharma.uzh.ch
Published by AAAS
the transmembrane conductance of potas-
sium (6). This may play a role in restoring
brain energy metabolism during sleep, and
in the homeostatic regulation of sleep need
in response to prolonged wakefulness. In-
creased transmembrane conductance of
potassium underlies membrane hyperpolar-
ization in cortical neurons that produces
Downloaded from http://science.sciencemag.org/ on February 25, 2018
EEG slow oscillations in NREM sleep (6).
Furthermore, increases and decreases in
the extracellular concentration of potassium
ions ([K+]e) parallel the alternating periods of
depolarization and hyperpolarization of neu-
rons and glial cells during the natural slow
oscillation and when under anesthesia (7).
The idea that positively charged ions af-
fect the sleep-wake cycle was proposed in
the 1930s, and calcium contained in breast
“…that positively charged ions
afect the sleep-wake cycle
was proposed in the 1930s…”
milk was hypothesized to promote sleep in
infants (8). Injecting calcium chloride (CaCl2)
into the pituitary of cats could induce sleep
for up to 3 hours (9). Additionally, CaCl2 and
magnesium chloride (MgCl2) injected near
the hypothalamus of diferent animal models
promoted sleep, whereas potassium chloride
(KCl) promoted wakefulness (8). Analyses of
brain tissue from dogs and rabbits revealed a
slight increase in brain calcium during sleep
when compared to wakefulness (10).
Ding et al. found that alterations in the
ion composition of the brain’s extracellular
fluid are sufcient to control sleep-wake–
dependent changes in neuronal activity.
Noting that modifications of ionic concen-
trations in the bathing solution of cortical
slices of adult mice induced stable changes
in neuronal excitability, they determined
that a “wake-up cocktail”—an artificial ce-
rebrospinal fluid (CSF) containing acetyl-
choline, hypocretin, histamine, serotonin,
noradrenaline, and dopamine—altered [K+]e
in brain-slice preparations. Micromolar con-
centration of this cocktail increased [K+]e,
even when neuronal activity was silenced
29 APRIL 2016 • VOL 352 ISSUE 6285 517
Liquidity in immune cell signaling
Michael L. Dustin and James Muller

Science 352 (6285), 516-517.

DOI: 10.1126/science.aaf8179

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