Natural Killer Cell Lytic Granule Secretion Occurs through
a Pervasive Actin Network at the Immune Synapse
Gregory D. Rak1,2, Emily M. Mace2, Pinaki P. Banerjee2, Tatyana Svitkina3, Jordan S. Orange1,2*
1University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 2Children’s Hospital of Philadelphia Research Institute, Children’s
Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America, 3Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United
States of America
Accumulation of filamentous actin (F-actin) at the immunological synapse (IS) is a prerequisite for the cytotoxic function of
natural killer (NK) cells. Subsequent to reorganization of the actin network, lytic granules polarize to the IS where their
contents are secreted directly toward a target cell, providing critical access to host defense. There has been limited
investigation into the relationship between the actin network and degranulation. Thus, we have evaluated the actin
network and secretion using microscopy techniques that provide unprecedented resolution and/or functional insight. We
show that the actin network extends throughout the IS and that degranulation occurs in areas where there is actin, albeit in
sub-micron relatively hypodense regions. Therefore we propose that granules reach the plasma membrane in clearances in
the network that are appropriately sized to minimally accommodate a granule and allow it to interact with the filaments.
Our data support a model whereby lytic granules and the actin network are intimately associated during the secretion
process and broadly suggest a mechanism for the secretion of large organelles in the context of a cortical actin barrier.
Citation: Rak GD, Mace EM, Banerjee PP, Svitkina T, Orange JS (2011) Natural Killer Cell Lytic Granule Secretion Occurs through a Pervasive Actin Network at the
Immune Synapse. PLoS Biol 9(9): e1001151. doi:10.1371/journal.pbio.1001151
Academic Editor: Philippa Marrack, National Jewish Medical and Research Center/Howard Hughes Medical Institute, United States of America
Received January 17, 2011; Accepted August 3, 2011; Published September 13, 2011
Copyright: ? 2011 Rak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by US National Institutes of Health (NIH) grants R01AI067946 to JSO, and R01GM070898 and S10RR22482 to TS. GDR was
supported by NIH training grant T32AR007442. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: CMA, concanamycin A; CW, continuous wave; F-actin, filamentous-actin; IS, immunological synapse; LAMP1, lysosomal-associated membrane
protein 1; MFI, mean fluorescence intensity; MTOC, microtubule organizing center; NK, natural killer; PMA, phorbol myristate acetate; STED, stimulated emission
depletion; TIRFm, total internal reflection fluorescence microscopy; WAS, Wiskott-Aldrich Syndrome; WASp, Wiskott-Aldrich Syndrome protein
* E-mail: email@example.com
Natural killer (NK) cells are lymphocytes of the innate immune
system that function in clearance of tumor and virally infected cells
. Elimination of susceptible target cells is tightly regulated and
follows ligation of germline-encoded activation receptors . As
NK cells do not require receptor gene rearrangement, they are
constitutively enabled for cytotoxicity. Thus, NK cell activation
must be tightly regulated to ensure that healthy cells remain
unharmed. Efficient lysis requires the tight adherent formation
between the NK cell and the target cell termed the immunologic
synapse (IS). The formation of a mature, cytolytic synapse between
an NK cell and a target cell occurs in stages that can be thought of
as checkpoints in the activation process [3–5]. Major cytoskeletal
steps that are required in this process include the rearrangement of
filamentous actin (F-actin) and the polarization of the microtubule
organizing center (MTOC) [6–8]. These events culminate in the
directed secretion of lytic granule contents at the IS, which is
prerequisite for NK cell cytotoxicity.
F-actin accumulation at the synapse is the first major cytoskeletal
reorganization event and is critical to subsequent steps and function
of the IS . Inhibiting proper F-actin dynamics in NK cells with
the actin targeting drugs cytochalasin [6,9], latrunculin , or
jasplakinolide  inhibits their cytotoxicity. Furthermore, NK cells
from patients with Wiskott-Aldrich Syndrome (WAS) who have
mutations in the actin regulatoryprotein, WAS protein (WASp), are
poorly cytotoxic . This defect is attributable to improper
reorganization of F-actin at the IS. Additionally, the actin nucleator
Arp2/3 complex, which is enabled by WASp, is also required for
cytotoxicity . Cytochalasin treatment, Arp2/3 complex deple-
tion, or WASp deficiency prevent the normal accumulation of F-
actin at the synapse [5,9,10].
One question that arises from the creation of a dense polarized
network at the IS is how secretion of lytic granules occurs through
a potential barrier. The traditional view of granule delivery
through the actin network holds that granules reach the synaptic
membrane through a void of actin in the center of the network.
This model is based on the observation from 3-D confocal mic-
roscopy that actin forms a dense peripheral ring around the IS
[5,11]. There is a caveat to the seemingly unobstructed access to
the membrane that this ‘‘ring’’ provides: the actin motor protein,
myosin IIA, is required for secretion and, more specifically, for
granule delivery to the synaptic plasma membrane [12,13]. These
data are at apparent odds with one another as a requirement for
myosin IIA for secretion necessitates a requirement for actin. One
explanation is that granules are secreted at the periphery of the
synapse where the traditional model depicts the location of F-
actin. Another explanation is that the center of the synapse
actually contains F-actin but does so at a level that has been
undetectable by conventional 3-D confocal microscopy.
PLoS Biology | www.plosbiology.org1 September 2011 | Volume 9 | Issue 9 | e1001151
Here we use microscopy techniques that provide enhanced
sensitivity and resolution over those used previously to investigate
the NK cell IS. We show that F-actin is present throughout the
synapse and that lytic granules likely navigate and are secreted
through the filamentous network by accessing minimally suffi-
ciently sized clearances. These data demonstrate a previously
unappreciated distribution of F-actin at the NK cell IS and
redefine granule access to the synaptic membrane and functional
Actin Accumulates throughout the Activated
Visualization of the synaptic actin network has relied on 3-D
reconstructions of confocal slices [5,11]. Here, we took advantage
of the superior resolution of imaging in the XY plane to investigate
the polarization and distribution of actin. First, we evaluated GFP-
actin expressing NK-92 cells conjugating with the susceptible and
adherent cell line, mel1190. These cells conjugated in a manner
that afforded us the ability to image the synapse in the XY plane.
GFP-actin was polarized toward the contact site (Figure 1A) and
surprisingly displayed a diffuse distribution across the synapse
(Figure 1B). This distribution was quantitatively analyzed and
confirmed using a radial intensity profiling algorithm, which de-
monstrated that the intensity throughout the contact site was
substantially above the background.
To more directly image the cortical region of the NK cell
immunologic synapse, we used total internal reflection fluores-
cence microscopy (TIRFm), which has the benefit of an improved
signal to noise ratio over confocal microscopy and is limited to
visualization within the first membrane proximal 100 nm .
Cells were activated via crosslinking of NKp30, a natural
cytotoxicity receptor whose ligand is expressed on tumor cells
, and CD18, a member of the heterodimeric integrin
lymphocyte function-associated antigen-1 (LFA-1). Both integrin
receptor and activation receptor activation are critical for
polarized secretion of granule contents . This combination
of signals resulted in robust activation, which was demonstrated by
degranulation as measured by enzymatic activity of granzyme A in
the supernatant (Figure S1). TIRFm imaging of activated NK-92
cells demonstrated a distribution of F-actin throughout the synapse
(Figure 1C). Quantitative analysis using radial profile plotting
confirmed the presence of F-actin throughout the cell contacts
(Figure 1E). To ensure that these findings were not particular to
the NK-92 cell line, we activated and imaged freshly isolated ex
vivo NK cells. Similar to NK-92 cells, synaptic F-actin in ex vivo
NK cells was identified throughout the contact (Figure 1D,F).
These results demonstrate that the NK cell synapse is defined by
an abundant, diffuse F-actin network.
To evaluate the kinetics of actin accumulation at the activated
synapse, NK-92 cells expressing GFP-actin were imaged using
TIRFm after contacting an activating surface. Actin accumulated
quickly, within 5 min, and was sustained over the period of
observation (50 min) (Figure S2A, Video S1). There was an initial
paucity of actin at the synapse followed by a rapid filling in, as
demonstrated by the separation of peak contact area and mean
fluorescence intensity (MFI) of GFP-actin in that region (Figure
S2B). The decrease in MFI over time was due to photobleaching
as separate imaging of fields at 10 and 40 min did not show MFI
differences (unpublished data). Importantly, actin was diffusely
accumulated prior to timepoints at which granule contents were
detected in the supernatant (Figure S1). Thus, actin was present as
a potential barrier to lytic granule access to the plasma membrane.
Lytic Granules Approximate the Synapse in Areas of Actin
Because there was abundant actin present at the synapse, we
wanted to determine if lytic granules might utilize relative
clearances in the actin network to access the synaptic membrane.
To address this, GFP-actin expressing cells were loaded with
LysoTracker Red dye, which enables tracking of lytic granules and
definition of their position relative to actin, and followed in real
time after activation. Numerous granules were identified in the
synaptic actin network using two-color TIRFm. Although some
relative hypodensities were apparent in the synaptic actin network
(Figure S3A–C), the LysoTracker labeled granules did not
necessarily appear in these relative voids of actin (Figure 2A,
Video S2). To quantitatively analyze this observation across all
synaptic granules in an NK cell, the actin intensity in the region of
the synaptic granule was compared to that of the entire synapse by
dividing the MFI of the respective intensity values to produce a
ratio measurement. This ratio, when compared to minimum and
maximum potential ratios, demonstrated that on average granules
approached the membrane in areas of actin (Figure S4A,B).
Combining measurements of all granules in the synapse over 1 h
from 14 cells defined the mean granule ratio value as 1.0 (Figure
S4C). Although there was a range of actin intensities present
throughout the synapse as measured by the ratios of minimum and
maximum intensity values to the MFI, few granules were pre-
sent in areas of particularly low or high actin content. Thus, the
colocalization of lytic granules with mean actin signal suggested
that granules access the synapse in close proximity to the actin
The MTOC Delivers Granules to the Synaptic Actin
The MTOC is known to deliver lytic granules to the
immunological synapse in NK cells . To investigate the
relationship among granules, the MTOC, and the synaptic actin
network, we imaged the synapse using both confocal microscopy
and TIRFm. The MTOC was present in the plane of the synapse
The immune system’s natural killer cells eliminate diseased
cells in the body. They do so by secreting toxic molecules
directly towards the diseased cells, so causing their death.
This process is essential for the host organism to defend
itself against infectious diseases. The interface between
the natural killer cell and its target—the lytic immunolog-
ical synapse—forms by close apposition of the surface
membranes of the two cells. It is characterized by
coordinated rearrangement of proteins to allow lytic
granules, which contain the toxic molecules, to fuse with
the cell surface at the synapse. Given the large size of the
granules, one challenge the natural killer cell faces is how
to contend with network of actin filaments just under the
cell surface, which potentially could pose a barrier to
secretion. The current model proposes large-scale clearing
of actin filaments from the center of the immunological
synapse to provide granules access to the synaptic
membrane. By using very high-resolution imaging tech-
niques, we now demonstrate that actin filaments are
present throughout the synapse and that natural killer
cells overcome the actin barrier not by wholesale clearing
but by making minimally sufficient conduits in the actin
network. This suggests a model in which granules access
the surface membrane by means of specific and facilitated
contact with the actin cytoskeleton.
Granule Secretion through an Actin Network
PLoS Biology | www.plosbiology.org2 September 2011 | Volume 9 | Issue 9 | e1001151
Figure 1. Actin distribution at the activated NK cell IS. (A) 3-D projection of NK-92 GFP-actin expressing cell (green) conjugated to mel1190
target cell (yellow). Scale bar = 5 m. (B) X–Y projection of a synapse taken from a conjugate similar to (A). (C,D) X–Y projections of a representative
NK-92 (left) or ex vivo (right) cell that had been activated for 30 min at 37uC on immobilized antibody to NKp30 and CD18, fixed, stained with 568
phalloidin (red), and imaged by TIRF microscopy. Above each X–Y projection is a plot of the mean fluorescent intensity (MFI) of concentric circles at a
given distance beginning at the periphery of the cell and moving inward to the center (radial intensity profiles). (E,F) Plot of radial intensity profiles
Granule Secretion through an Actin Network
PLoS Biology | www.plosbiology.org3 September 2011 | Volume 9 | Issue 9 | e1001151
construct. (A) Model of the construct depicting relative locations of
sequences: endoplasmic reticulum targeting signal sequence (SS),
flexible glycine-serine linker (GS), transmembrane domain (TM).
(B) Diagram depicting fluorescent state of pHluorin depending on
intralumenal versus surface location.
Model and implementation of pHluorin-LAMP1
approximations. (A) pHluorin-LAMP1 expressing cells were
loaded with LysoTracker Red and imaged for approximately
60 min at a rate of 1 frame per minute. (B) To count events, all
frames from the acquisition were merged into a single image. (C)
Number of Lysotracker positive and pHluorin positive events for
each cell are plotted (n=27; *** p,0.0001, paired t test).
Degranulation events are less abundant than granule
minimum and maximum potential ratios. MFI ratio of actin
intensities at the point of degranulation to that of the respective
footprints (black) is plotted relative to minimum (blue) and
maximum (red) potential values for 52 events.
Degranulation MFI actin ratios plotted relative to
following treatment with actin inhibitors. NK-92 cells were
activated for 10 (A) or 20 min (B) before addition of DMSO or
inhibitor. Following 5 min of incubation, cells were fixed, stained
for actin with phalloidin, and imaged by TIRFm using a 1006
objective. Radial intensity profiles were generated and averaged
for 30 cells/condition over 3 experiments. For latrunculin A
treated cells, DIC images were used for spatial reference since
actin fluorescent signal was undetectable.
Radial intensity profile plots for synaptic actin
timepoints of activation. GFP-actin expressing NK-92 cells were
activated and imaged at a rate of 2 frames per minute after 10 min
and 30 min of activation for 5 min. Scale bar=5 mm. (A) Images
from the first 2.5 min of the 10 to 15 min timeframe are shown. (B)
Corresponding intensity surface plots from the timepoints shown in
(A). Overlay of line profiles through the centroid of the cell contact
from images taken between 10 and 15 min of activation (C), or 30–
35 min (D) of activation. (E) To compare variation in multiple cells
(n=10) between the two timeframes, the standard deviation of
mean intensity over 5 min for each pixel along the measured line
was calculated for each cell. The mean standard deviation for each
cell was calculated and plotted for the 10–15 min and 30–35 min
timeframes. (F) The standard deviation of pixel intensity change
2 experiments following DMSO or sequential jasplakinolide and
latrunculin A treatment after 10 min of activation (*** p,0.0001).
The actin network is dynamic at early and late
and their relation to clearance area. (A) NK-92 cells were activated
on glass, fixed, and stained for perforin. 104 granules were
measured. (B) The mean clearance area for each cell (defined as
any area large enough to accommodate a 250 nm in diameter
granule) was divided by the mean granule equatorial area derived
from (A) and plotted according to interval. The mean granule
diameter of 333 nm corresponds to a mean equatorial area of
Diameters of granules imaged by STED microscopy
magnification image of filaments at the activated synapse using
Branched networks at the activated IS. (A) High
platinum replica electron microscopy. (B) Image from (A) with
pseudocolored region indicating examples of branching filaments.
Scale bar=100 nm.
replica electron microscopy. (A–C) Comparative measurements of
the synapse include: contact area (A); filament density (B); and
distance from the cell centroid of individual clearances that would
be greater than or equal in size to the equatorial area of a 250 nm
granule (C) (* p,0.05, unpaired t test).
Additional analyses of cells imaged by platinum
F-actin network. Colored regions indicate appropriately sized
clearances that were identified.
Algorithm-based identification of clearances in the
92 cells. Cells were imaged at 2 frames per minute and are shown at
6 frames per second starting at time zero relative to activation.
LysoTracker Red loaded NK-92 cells. Cells were imaged at 1
frame per minute and are shown at 4 frames per second starting
2 min after activation.
Live cell imaging of activated GFP-actin expressing,
pHluorin-LAMP1 expressing cell undergoing degranulation and
attaining pHluorin fluorescence. Images were acquired at 2 frames
per minute and are shown at 6 frames per second.
Live imaging of a Lysotracker Red loaded granule in a
expressing, LysoTracker Red loaded NK-92 cells. Cells were
imaged at 1 frame per minute and are shown at 4 frames per
second starting at time zero relative to activation. LysoTracker
Red events outnumber pHluorin events.
Live cell imaging of activated pHluorin-LAMP1
mCherry-actin expressing NK-92 cells. Cells were imaged at 6
frames per minute and are shown at 6 frames per second. Video
begins 25 min after activation.
Live cell imaging of activated pHluorin-LAMP1 and
activated GFP-actin expressing NK-92 cells. Cells were imaged
at 2 frames per minute and are shown at 2 frames per second
starting at 10 min after activation.
Live cell imaging and intensity surface plots of
The authors thank P. Kumar, R. Pandey, and L. Monaco-Shawver for
technical assistance; K. Campbell for gifts of cell lines; W. Pear, G.
Miesenbo ¨ck, R. Balice-Gordon, M. Marks, and R.Y. Tsien for gifts of
reagents; K. Rak for valuable suggestions; and C. Yang and F. Korobova
for technical training.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: GDR TS JSO.
Performed the experiments: GDR EMM PPB. Analyzed the data: GDR
EMM PPB JSO. Contributed reagents/materials/analysis tools: TS. Wrote
the paper: GDR JSO.
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