JCB: Article
JCB 383
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 215 No. 3 383–399
https://doi.org/10.1083/jcb.201603080
Introduction
Recognition of antigen on the surface of an antigen-presenting
cell (APC) initiates signaling cascades within the T cell that
drive large-scale reorganization of its actin cytoskeleton (Bee-
miller and Krummel, 2013; Yu et al., 2013; Kumari et al., 2014).
This reorganization is essential for the formation of the immu-
nological synapse (IS), the specialized interface between the
two cells (Monks et al., 1998; Grakoui et al., 1999). Initially,
activation of actin polymerization within the T cell at the pe-
riphery of its contact with the APC drives the spreading of the T
cell across the surface of the APC. Once spreading is complete,
continued actin polymerization begins to drive an inward ow
of actin toward the center of the contact site and in the plane of
the IS. By coupling this inward ow with depolymerization at
the center of the IS, the T cell creates an ongoing centripetal
ow of actin that is thought to be a major driving force for the
inward movement of T cell receptor microclusters (TCR MCs)
and integrin clusters in the T cell’s plasma membrane (Bunnell
et al., 2001; Varma et al., 2006; Kaizuka et al., 2007; Babich et
al., 2012; Beemiller et al., 2012; Smoligovets et al., 2012; Yi
et al., 2012). Over the next 5–10 min, the inward movement of
receptor clusters culminates in the formation of a mature IS, in
which TCR MCs are concentrated at the center of the IS (the
central supramolecular activation cluster [cSMAC]), and leu-
kocyte functional antigen 1 (LFA-1), the T cell’s major integ-
rin, is concentrated in a surrounding ring (the peripheral SMAC
[pSMAC]). Importantly, actin assembly and dynamics are inti-
mately linked not just to TCR MC movement, but to virtually
every key event during IS formation, including signalosome
assembly and tuning (Mattila et al., 2016), integrin activation
(Comrie et al., 2015a,b), the mechanical regulation of T cell
signaling (Chen and Zhu, 2013), and effector functions such
as lytic granule release (Brown et al., 2011; Mace et al., 2012;
Basu et al., 2016). Clearly, a full understanding of how actin cy-
toskeletal forces are created and organized at the IS is required
to dene the mechanisms by which they drive T cell function.
Numerous laboratories have used diffraction-limited im-
aging of T cells engaged with planar lipid bilayers containing
freely diffusing activators (e.g., anti-CD3 and intercellular ad-
hesion molecule 1 [ICAM-1]) to correlate the dynamics of actin
Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor
microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination micros-
copy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments
generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamelli-
podia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-
dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and
total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc
formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T–B cell
adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional signifi-
cance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.
Formin-generated actomyosin arcs propel T cell
receptor microcluster movement at the
immune synapse
SricharanMurugesan,
1
JinsungHong,
1
JasonYi,
1
DongLi,
2,3
JordanR.Beach,
1
LinShao,
2
JohnMeinhardt,
1
GreyMadison,
1
XufengWu,
1
EricBetzig,
2
and JohnA.Hammer
1
1
Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
2
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147
3
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No
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Noncommercial–Share Alike 3.0 Unported license, as described at http ://creativecommons
.org /licenses /by -nc -sa /3 .0 /).
Correspondence to John A.Hammer: hammerj@nhlbi.nih.gov
Abbreviations used: ANO VA, analysis of variance; APC, antigen-presenting
cell; cSMAC, central supramolecular activation cluster; dSMAC, distal supramo-
lecular activation cluster; FMNL1, formin-like protein 1; HLA, human leukocyte
antigen; ICAM, intercellular adhesion molecule; INF2, inverted formin-2; IS,
immunological synapse; LFA-1, leukocyte functional antigen 1; MFI, mean fluor-
escence intensity; pnBB, para-nitro-blebbistatin; pSMAC, peripheral supramolec-
ular activation cluster; ROI, region of interest; SEE, staphylococcal enterotoxin
E; SIM, structured-illumination microscopy; SMAC, supramolecular activation
cluster; SMI FH2, small-molecule inhibitor of formin homology 2 domain; TCR
MC, T cell receptor microcluster; TIRF, total internal reflection fluorescence.
THE JOURNAL OF CELL BIOLOGY
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384
ow and receptor cluster movement in an ideal imaging plane
(Dustin, 2009). Importantly, these studies revealed robust, po-
lymerization-driven, actin retrograde ow in a ring surrounding
the pSMAC now known as the distal SMAC (dSMAC; Kaizuka
et al., 2007; Babich et al., 2012; Beemiller et al., 2012; Yi et al.,
2012). Moreover, the rate of centripetal TCR MC movement
in this radially symmetric dSMAC roughly correlated with the
rate of inward actin ow (Kaizuka et al., 2007), and elegant
biophysical studies demonstrated frictional coupling between
the TCR MCs and actin ow (DeMond et al., 2008; Yu et al.,
2010). Less clear, however, is what propels TCR MC movement
across the pSMAC, especially as GFP-actin, the reporter typi-
cally used to image actin dynamics at the IS, does not reveal
obvious actin organization there (Kaizuka et al., 2007). Using
F-Tractin, an indirect reporter for F-actin, we, in contrast, iden-
tied concentric actin arcs in the pSMAC that are decorated
with myosin II (Yi et al., 2012). Additionally, we showed that
the lamellipodial-like dSMAC and lamella-like pSMAC exhibit
distinct rates of inward actin ow and that the rates of centrip-
etal TCR MC movement across these two zones matched their
distinct actin ow rates (Yi et al., 2012). Nevertheless, the exis-
tence of these arcs has been questioned (Beemiller and Krum-
mel, 2013; Le Floc’h and Huse, 2015), and they have never
been observed in primary T cells. Moreover, an alternate mech-
anism to drive TCR MC movement across the pSMAC has been
proposed that involves dynein-dependent, microtubule-based
transport (Hashimoto-Tane et al., 2011). Finally, the role of
myosin II in TCR MC movement has been controversial (Ham-
mer and Burkhardt, 2013).
In this study, we sought to determine how the actin arcs
are created and how they become organized into concentric
structures. Beyond that, we sought to dene the spatial relation-
ship between the arcs and receptor clusters during IS matura-
tion and to quantitate the contributions made by arcs to receptor
cluster distribution, T cell–APC adhesion, and proximal TCR
signaling. Finally, we sought to directly image the arc-depen-
dent translocation of TCR MCs. Key to accomplishing these
goals was the use of total internal reection uorescence–struc-
tured-illumination microscopy (TIRF-SIM), which, by virtue of
its high spatiotemporal resolution (Kner et al., 2009; Beach et
al., 2014; Li et al., 2015), proved pivotal in reaching several
of our main conclusions.
Results
SIM reveals the architecture and dynamics
of F-actin at the IS
To characterize the architecture of actin networks at the IS at
higher resolution, we imaged activated, phalloidin-stained Jur-
kat T cells with 3D-SIM to reveal endogenous F-actin structures
at ∼120-nm lateral and ∼250-nm axial resolution. Consistent
with our previous work at lower resolution (Yi et al., 2012),
3D-SIM demonstrated that the IS is comprised of three dis-
tinct zones of F-actin: an outer, dense ring corresponding to
the branched actin network in the dSMAC, a middle ring com-
prised of concentric actin arcs corresponding to the pSMAC,
and a central, relatively actin-depleted zone corresponding to
the cSMAC (Fig.1A, rst and second panels). Color coding the
3D projection of this cell according to z position (Fig.1A, third
panel) shows that these actin structures are largely conned to
the plane of the IS, indicating that their dynamics will occur in
close association with the plasma membrane at the IS. Also con-
sistent with previous work, 3D-SIM images of Jurkats stained
for actin and endogenous myosin IIA, the dominant myosin II
isoform in T cells, showed that the concentric actin arcs in the
pSMAC are highly enriched for this contractile motor (Fig.1B).
We next sought to visualize the dynamics of IS actin net-
works using a live-cell compatible SIM technique. Given that
these networks are close to the plasma membrane, we used
TIRF-SIM, which merges the power of SIM (in this study,
∼100-nm xy resolution) with TIRF (enhanced signal-to-noise
ratio for structures close to the coverslip) in a format that dis-
plays little bleaching or phototoxicity while providing high
temporal resolution (Kner et al., 2009; Li et al., 2015). Fig.1C
(rst three panels) shows still images taken from a video of a
Jurkat T cell expressing GFP–F-Tractin, an indirect, dynamic
reporter for F-actin (Johnson and Schell, 2009), 180, 210, and
240 s (Fig. 1 C) after engagement with an activating surface
(Video1). It is immediately apparent that F-Tractin faithfully
reports the two actin networks and three IS zones identied in
xed images of phalloidin-stained cells. This is most clearly
seen in Fig.1C (fourth panel), as well as in the corresponding
video. Additionally, TIRF-SIM imaging of Jurkats expressing
GFP–myosin IIA and tdTomato–F-Tractin revealed in dy-
namic fashion the enrichment of myosin on the actin arcs in the
pSMAC (Fig.1D, rst and second panels). Importantly, close
examination of these images, stills from Video2 (Fig.1D, third
and fourth panels), and Video2 itself revealed the presence of
individual, 300-nm-long myosin IIA bipolar laments decorat-
ing the actin arcs and moving inward with them. Together, these
observations conrm and enhance our previous understanding
of actin architecture at the Jurkat T cell IS and provide addi-
tional support for a radially symmetric contractile actomyosin
network within the pSMAC.
Actomyosin arcs are also prominent
structures at the IS of primary
mouse T cells
We performed 3D-SIM on activated primary mouse CD8
+
T
cells stained with phalloidin and an antibody to myosin IIA to
reveal endogenous structures. Fig.2A shows that the IS of a
CD8
+
T cell is essentially indistinguishable from that of a Jurkat
T cell. Also, like Jurkats, the two major F-actin networks at the
CD8
+
T cell IS are very close to the IS membrane (Fig.2C).
Strikingly, enlarged images of typical CD8
+
T cell synapses re-
veal an almost sarcomere-like pattern for myosin IIA embedded
in the actin arcs comprising their pSMAC (Fig. 2 B, arrow-
heads). Perhaps most importantly, rather than being difcult to
detect or absent, actomyosin arcs are an even more prominent
component of the mouse CD8
+
T cell IS than the Jurkat T cell
IS (Fig.2D). Finally, still images from TIRF-SIM imaging of
primary mouse CD8
+
T cells expressing GFP–myosin IIA and
tdTomato–F-Tractin demonstrated the formation of the actomy-
osin arcs in the pSMAC (Fig. S1, A and B). Together, these
results demonstrate in unequivocal fashion the existence of ac-
tomyosin arcs at the IS of primary mouse T cells.
3D-SIM and TIRF-SIM reveal linear actin
filaments/bundles embedded within the
dSMAC that appear to give rise to the
actin arcs in the pSMAC
Closer examination of xed, phalloidin-stained Jurkat synapses
using 3D-SIM revealed linear actin laments/bundles embed-
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Formin-generated actomyosin arcs at the immune synapse
ded within the branched actin network of the dSMAC. These
structures appear to originate at the distal edge of the IS, tra-
verse the dSMAC, and bend upon exiting the dSMAC to give
rise to the actin arcs of the pSMAC (Fig.3 A, yellow arrow-
heads in the bottom panels mark the origin of these structures
and red arrowheads mark their exit from the dSMAC). Further
support for their existence came from live-cell TIRF-SIM im-
aging of Jurkat T cells expressing GFP–F-Tractin. Specically,
Fig.3B shows still images taken from Video4 of a quadrant
of a Jurkat T cell synapse 180, 240, 300, and 360s after en-
gagement with an activating surface. Close examination reveals
the progressive formation of several linear actin laments/bun-
dles (Fig.3B, arrowheads) at the distal edge of the IS that are
oriented perpendicular to the plasma membrane, traverse the
dSMAC, and then bend and splay upon exit from the dSMAC to
form arcs (Video3). Fig.3C shows four clear examples of such
bending upon exit from the inner aspect of the dSMAC (see red
arrowheads), and Video4 shows all of the events described in
this section occurring across a broad section of a Jurkat IS.
The linear actin filaments/bundles, and
the actin arcs they give rise to, are
formin generated
From a topological standpoint, the linear actin laments/bun-
dles observed in Fig.3 are consistent with actin structures built
by formins (Kovar, 2006; Breitsprecher and Goode, 2013).
Specically, they originate at the plasma membrane, the site
where formins are typically active, and project inwards, one
of two fates typically exhibited by formin-generated laments
(the other being projection outward to make structures like lo-
podia). Additional indirect evidence that these structures are
formin generated stems from the observation that the arcs are
very poorly labeled by GFP-actin (Kaizuka et al., 2007) because
formins are known to incorporate modied actins into laments
very ineffectively (Chen et al., 2012). This is completely con-
sistent with our previous observations (Yi et al., 2012), which
we conrmed and extended in this study using colocalization
analyses (Fig. S1, C–E).
Recent, paradigm-shifting studies in both yeast (Burke et
al., 2014) and mammalian cells (Lomakin et al., 2015; Rotty
et al., 2015) have demonstrated that the Arp2/3 complex and
formins are competing for a limiting pool of actin monomer, such
that when one actin nucleator is inhibited, the network created by
the other becomes more robust. We reasoned, therefore, that if
the linear actin laments/bundles embedded within the dSMAC
are formin generated, they should be augmented upon Arp2/3
inhibition. Consistently, Video5 and the still images taken from
it (Fig.4A) show that the addition of the Arp2/3 inhibitor CK666
to activated Jurkat T cells expressing GFP–F-Tractin resulted in
the dramatic collapse of the Arp2/3-dependent, branched actin
network comprising the dSMAC and the progressive enhance-
ment of the linear actin laments/bundles, which took on the
Figure 1. SIM imaging reveals concentric ac-
tomyosin arcs in the pSMAC region of the Jur-
kat T cell IS. (A, first and second panel) 3D-SIM
image of an activated Jurkat T cell stained with
phalloidin. (A, third panel) Cell in first panel
color-coded by z position. Lighter colors are
closer to the coverslip. (B) 3D-SIM image of a
Jurkat T cell stained with phalloidin (red) and
anti–myosin IIA antibody (green). Individual
channels and the merged image are shown.
(C) Still images from a TIRF-SIM video of a Jur-
kat T cell expressing GFP–F-Tractin (Video1).
(D) Still images from two color TIRF-SIM videos
of two different Jurkat T cells expressing GFP–
myosin IIA and tdTomato–F-Tractin (Video 2,
corresponding to third panel). SMAC zones
bracketed in A–C at top. Bars, 5 µm.
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appearance of radial actin “spikes” in which actin polymeriza-
tion and retrograde ow continued (Fig.4A, compare second
panel [+CK666] to rst panel [control]; note that this effect was
completely reversible in third panel [washout]; see also Video5
and Gomez et al., 2007). 3D-SIM imaging of xed, phalloidin-
stained Jurkat T cells treated with CK666 for 1, 2, and 4 min
revealed these spikes in greater detail as they emerged from the
shrinking lamellipodial network and increased in robustness over
time (Fig.4B, top). Importantly, actin arcs were still present in
these Arp2/3-inhibited cells. Moreover, the corresponding insets
in Fig.4B (bottom) show that the CK666-enhanced actin la-
ments/bundles in the dSMAC bend and reorient into actin arcs at
the dSMAC/pSMAC boundary (the red arrowheads mark bend
points where the spikes marked by yellow arrowheads give rise to
arcs). This bending and reorientation process could be observed
in real time in Jurkats expressing GFP–F-Tractin and imaged
using TIRF-SIM (Video6 and the still images taken from it in
Fig.4C). Together, these observations indicate that arc assembly
is largely Arp2/3 independent and argue that the arcs are created
by one or more formins acting at the distal edge of the IS.
To provide more direct support for the hypothesis that the
actin spikes observed upon Arp2/3 inhibition are formin gen-
erated, we compared cells treated with CK666 alone to cells
treated with both CK666 and the pan-formin inhibitor small-
molecule inhibitor of formin homology 2 domain (SMI FH2;
Rizvi et al., 2009). As before, 3-min incubation in the presence
of CK666 resulted in a considerable diminution in the size of
the branched actin array comprising most of the dSMAC and
the appearance of actin spikes at the cell edge (Fig.4D, com-
pare second panel [+CK666 −SMI FH2] to rst [DMSO con-
trol] panel; the red bracket in the second panel marks a typical
actin spike). Importantly, addition of CK666 to cells that had
been pretreated with SMI FH2 resulted in a dramatic reduc-
tion in the formation of spikes (Fig.4D, compare third panel
[+CK666 +SMI FH2] to second panel [+CK666 −SMI FH2]).
This reduction is indicated by signicant decreases in both the
mean length of spikes (Fig.4D, bottom left) and the number
of spikes per unit of cell perimeter (Fig.4D, bottom right) for
cells treated with both inhibitors relative to cells treated with
CK666 alone. These results provide additional support for the
Figure 2. Actomyosin arcs are also a very prominent feature of the primary mouse CD8
+
T cell IS. (A) 3D-SIM image of a primary mouse CD8
+
T cell
stained with phalloidin (green) and anti–myosin IIA antibody (red). (B) Magnified views of actomyosin arcs in a separate CD8
+
T cell stained as in A.Yellow
arrowheads mark sarcomere-like pattern of myosin IIA. (C, left) Cell in B color-coded by z position; each channel is separated in middle and right panels.
Lighter colors are closer to the coverslip. (D) Area occupied by pSMAC actin arcs (bracketed by yellow traces) in typical Jurkat IS (first panel) and primary
mouse CD8
+
T cell IS (second panel). The ratio of pSMAC to dSMAC area (third panel) and pSMAC to total IS area (fourth panel) for Jurkat and mouse
CD8
+
T cells. n = 17–24 cells/condition. Data are means ± SEM. SMAC zones bracketed in A at top. Bars, 5 µm. ***, P < 0.001; ****, P < 0.0001.
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Formin-generated actomyosin arcs at the immune synapse 387
idea that the linear actin laments/bundles embedded within the
dSMAC and contiguous with the actin arcs in the pSMAC are
formin-generated structures.
As a more direct test for the role of formins in actin
arc creation, we imaged arc formation with and without
SMI FH2. To accomplish this, we allowed Jurkats to activate
for 2 min before SMI FH2 addition and then asked if its addi-
tion prevented further actin arc formation. We observed that
5-min incubation in the presence of SMI FH2 (both 10 and
25 µM) resulted in a dramatic decrease in the presence of
actin arcs within the pSMAC, as revealed by 3D-SIM im-
aging of xed, phalloidin-stained cells (Fig.5 A, compare
second and third panels and the corresponding insets in the
fth and sixth panels to the DMSO control in the rst and
fourth panels). To quantitate arc loss, we measured total
phalloidin uorescence in the pSMAC area of control cells
and cells treated with 10µM SMI FH2. Fig.5A (bottom left)
shows that SMI FH2-treated cells exhibited signicantly less
F-actin in their pSMACs, consistent with the loss of actin
arcs after formin inhibition. Importantly, SMI FH2-induced
arc loss is likely attributable to formin inhibition rather than
off-target effects because treatment with 10µM KVSM18,
a closely related but inactive analogue of SMI FH2 (Rizvi et
al., 2009), does not cause arc loss (Fig.5A, bottom middle).
Interestingly, formin-inhibited cells also exhibited a dra-
matic increase in the number of F-actin foci in the dSMAC
(Fig.5A, arrowheads). We believe these foci correspond to
the Arp2/3-dependent F-actin foci described recently (Ku-
mari et al., 2015) and that they become more robust upon
formin inhibition as a result of an increase in actin monomer
available for Arp2/3-dependent nucleation. On the ip side,
the inhibition of Arp2/3-dependent nucleation using CK666
increased the content of arcs in the pSMAC (Fig.5A, bottom
right), presumably because of an increase in actin monomer
available for formin-dependent nucleation.
Importantly, the SMI FH2-mediated inhibition of actin
arc formation was reversible, as washout of SMI FH2 re-
sulted in the rapid resumption of actin arc formation. This
is apparent in still images taken from a TIRF-SIM video
(Video7) 30 (Fig.5B, top) and 180 s (Fig.5B, bottom)
after SMI FH2 washout. Careful examination of the mag-
nified insets in Fig.5B (right panels) show that SMI FH2
washout was accompanied not only by the appearance of
newly formed arcs in the pSMAC (Fig.5B, bottom right),
but also by the disappearance of the actin foci (Fig.5B, yel-
low arrowheads in top right), and the reappearance of linear
actin filaments/bundles embedded in the dSMAC (yellow
arrowheads in bottom right). Within 5 min of washout, the
content of F-actin in the pSMAC was restored to control
levels (Fig.5A, bottom left).
We next tested if one or more formins could be detected
at the tips of CK666-enhanced actin spikes by immunouor-
escence staining for endogenous proteins. Previous studies
(Gomez et al., 2007; Ramabhadran et al., 2011) have pro-
vided evidence for the expression of four formins in Jurkat
T cells: mDia1, mDia2, formin-like protein 1 (FMNL1), and
the non-CAAX version of inverted formin-2 (INF2). Of these,
we were able to detect clear signals for mDia1, FMNL1, and
non-CAAX INF2 at the tips of CK666-enhanced actin spikes
(Fig. S2 A, micrographs). Moreover, the fraction of spike tips
that stained for these three formins, which ranged from ∼35 to
∼55%, was signicantly higher than the fraction that stained
for mDia2 or with preimmune sera (Fig. S2 A, right panel),
arguing that the tip staining seen for mDia1, FMNL1, and
Figure 3. SIM imaging reveals linear actin filaments/
bundles embedded in the branched actin network of
dSMAC. (A) 3D-SIM images of three representative phal-
loidin-stained Jurkat T cells. (B) Successive still images
from a TIRF-SIM video of a Jurkat expressing GFP–F-
Tractin (Video3). (C) Four still images from TIRF-SIM vid-
eos of Jurkats expressing GFP–F-Tractin. Yellow arrow-
heads mark the origin of linear actin filaments/bundles
embedded in the branched actin network of the dSMAC,
whereas red arrowheads mark where they bend upon
exit from the dSMAC. See also Video4.Bars, 5 µm.
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