Download Starfish ApDOCK protein essentially functions in larval

Immunology and Cell Biology (2012) 90, 955–965
& 2012 Australasian Society for Immunology Inc. All rights reserved 0818-9641/12
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ORIGINAL ARTICLE
Starfish ApDOCK protein essentially functions in larval
defense system operated by mesenchyme cells
Ryohei Furukawa1,2, Hiromi Funabashi2, Midori Matsumoto1,2 and Hiroyuki Kaneko1
In larvae of the starfish, Asterina pectinifera, mesenchyme cells operate in the defense system through various behaviors.
We have investigated mesenchyme cell dynamics during the immune response by identifying ApDOCK, a new member of the
DOCK180 superfamily protein. In 4-day-old bipinnaria larvae processed for morpholino oligonucleotide-mediated knockdown of
ApDOCK, injection of inorganic foreign substances revealed that (1) mesenchyme cells fail to undergo either directed migration
toward a large oil-droplet or persistent spreading on the oil-droplet after contact; (2) neither uptake of micro-beads nor cell-tocell fusion on the large oil-droplet differed from that of mesenchyme cells from control larvae. Similar behaviors were also
recorded in experiments where bacteria were injected. Under culture conditions, the expression level of ApDOCK mRNA was
significantly associated with the immunological behavior of mesenchyme cells. Apparently, the mesenchyme cells from ApDOCK
loss-of-function larvae exhibited insufficient lamellipodium formation via lack of fibrous form of actin organization at the leading
edge. These results suggest that the migratory congregation and persistence of encapsulation of larval mesenchyme cells are
intracellularly regulated by ApDOCK protein, and this regulation is associated with organization of cytoskeletal actin.
Immunology and Cell Biology (2012) 90, 955–965; doi:10.1038/icb.2012.37; published online 17 July 2012
Keywords: Cell migration; DOCK180 superfamily; encapsulation; lamellipodium formation; larval mesenchyme cells; starfish
The starfish Asterina pectinifera is an echinoderm that is phylogenetically close to the origin of the deuterostome. We have previously
reported that the larva of A. pectinifera is equipped with a simple
defense system in which a single type of mesenchyme cell operates as
an immunocompetent cell.1 The structural and functional aspects of
the defense system are as follows. At the 4-day-old bipinnaria larval
stage, the number of mesenchyme cells ranges from 150 to 190 per
larva, corresponding to B1% of the total cell number. Most of these
cells are distributed beneath the ectodermal and endodermal walls,
which are composed of an epithelial monolayer. Each mesenchyme
cell develops cellular processes to contact other mesenchyme cells. The
location of cells fluctuates in time in the immunological naive
situation. Thereby, the overall appearance of mesenchyme cells is
viewed as a three-dimensional and dynamic network similar to a
basket-like structure (see also Figure 2). In the pathological and
physiological situations of larvae, the mesenchyme cells phagocytose
cell debris effluxed from the epithelial layers and foreign substances
taken into the blastocoel through the body wall, respectively.
With regard to defense cell behaviors, the functions of larval
mesenchyme cells have been uncovered through injection experiments
with various foreign substances.1 When a relatively large amount
of bacteria is injected into the blastocoel of the larval anterior
portion where mesenchyme cells are scarcely distributed, multiple
mesenchyme cells undergo migratory congregation from several
directions. Then, they activate concomitantly to form an aggregate
and phagocytose the bacteria. During this process, the mesenchyme
cells frequently undergo cell-to-cell fusion with one another to form
multinucleated giant cells by an ‘encapsulation’ strategy that
completely seals off the foreign body. They are finally dispersed
from the region injected with bacteria, thereby cleaning the blastocoel.
In another injection experiment where the bacteria are replaced by an
oil-droplet with a size similar to that of the injected bacteria, the
encapsulation behavior of the mesenchyme cell is more clearly
observed. Encapsulation of the oil-droplet is accomplished by an
extraordinary extension of the lamellipodium of each mesenchyme
cell, and is not cancelled thereafter. In this way, the defense system of
larva is underpinned by a variety of different mesenchyme cell
dynamics to achieve migratory congregation, encapsulation,
phagocytosis and multinucleated giant cell formation. Therefore, the
larval mesenchyme cell is functionally similar to the vertebrate
macrophage.
To improve our understanding of the larval defense system of
A. pectinifera, we have undertaken studies to elucidate the molecular
mechanism(s) that regulates mesenchyme cell dynamics during the
immune response to foreign bodies. We recently reported that the
starfish ApSRCR1 protein, the orthologue of a vertebrate scavenger
1Department of Biology, Research and Education Center for Natural Sciences, Keio University, Yokohama, Japan and 2Center for Chemical Biology, School of Fundamental
Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama, Kanagawa, Japan
Correspondence: Professor H Kaneko, Department of Biology, Research and Education Center for Natural Sciences, Keio University, Kohoku-ku, Hiyoshi 4-1-1, Yokohama,
Kanagawa 223-8521, Japan.
E-mail: [email protected]
Received 6 June 2012; revised and accepted 19 June 2012; published online 17 July 2012
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receptor cysteine-rich-domain-containing protein, serves as an opsonin against bacteria in the larval defense system of this animal.2
ApSRCR1 protein is extracellularly released by mesenchyme cells
and promotes their phagocytosis and aggregate formation. On the
other hand, little is known about how the mesenchyme cell is
regulated by intracellular molecule(s) to undergo its many defense
cell behaviors.
On the basis of previous studies, DOCK180 superfamily proteins
are expected to be the candidates for critical intracellular regulators in
the defense cell behaviors of larval mesenchyme cells. DOCK180related proteins are known to be guanine nucleotide exchange factors
and have a shared intramolecular structure, wherein two protein
domains, termed DOCK homology region-1 (DHR-1) and DHR-2,
are arranged.3 These domains are involved in the recruitment of
DOCK180 superfamily proteins toward the plasma membrane to
rearrange cytoskeletal actin in concert with other types of molecules,
such as ELMO, Crk, and Rac proteins.3,4 They have also been shown
to regulate several important cell behaviors, such as the spreading and
migration of mammalian epithelial cells,5 phagocytosis of apoptotic
cells and cell migration of the distal tip cells in C. elegans,6 cell
migration of epithelial cells and cell-to-cell fusion of myoblast cells in
D. melanogaster,7,8 cell-to-cell fusion of myoblast cells in zebrafish9
and axon growth of hippocampal neurons in rat,10 among others.
Thus, DOCK180-related proteins have been structurally conserved in
a wide variety of animals during evolution, and function as
intracellular regulators of cell dynamics. Interestingly, the DOCK2
protein, which is a member of the DOCK180 superfamily proteins, is
exclusively expressed in hematopoietic cells including B- and
T-lymphocytes,11 neutrophils,12 natural killer cells13 and
macrophages.11 Significant efforts have been made to identify the
regulatory mechanism of DOCK2 in these hematopoietic cells, with a
focus on chemotactic migration.4,12,14
In the present study, we report the first identification of a
DOCK180 superfamily protein in the starfish, which we have called
ApDOCK protein after the species name of A. pectinifera. We show
that ApDOCK is a new member of the DOCK-A subfamily of
DOCK180-related proteins. Following the molecular identification of
ApDOCK, we attempted to prepare ApDOCK loss-of-function
bipinnaria larva by using antisense morpholino oligonucleotides
(MOs). By means of foreign body injection into the ApDOCK lossof-function larvae, we show that ApDOCK regulates larval mesenchyme cells only during the immune response, and especially in
migratory congregation and persistence of encapsulation. Some of
these results have also been captured in live specimens under timelapse microscopy (Supplementary Movies 1–7).
RESULTS
Molecular structure of ApDOCK
A DOCK180 orthologue was identified in A. pectinifera from the
complementary DNA (cDNA) library of cultured mesenchyme cells
and termed ApDOCK (GenBank Accession No. AB669902). The
length of the whole cDNA sequence was B5800 bp, and the open
reading frame encoded a polypeptide of 1866 amino-acid residues.
The deduced amino-acid sequence showed the characteristic domain
architecture of an ApDOCK protein. As represented in Figure 1a,
ApDOCK protein has an intramolecular arrangement comprising two
regions of high sequence homology, named DHR-1 and DHR-2; these
regions are functionally important domains of the DOCK180 superfamily protein (see the earlier section), and are evolutionarily
conserved throughout this superfamily (Figures 1b and c). ApDOCK
protein was also characterized by the presence of an N-terminal Src
Immunology and Cell Biology
Homology 3 domain (yellow) and by a C-terminal proline-rich region
(black) containing one Crk-binding consensus motif (PPxLPxK
motif), which has been previously reported in mammalian
DOCK180.15
Figure 1d shows a phylogenetic tree of the DOCK180 superfamily
including ApDOCK protein. ApDOCK protein was classified into the
DOCK-A subfamily (green group in Figure 1d). In the DOCK180
subfamily group, ApDOCK protein exhibited high similarity to the
congeneric proteins of other animals; this similarity is shown to not
only human DOCK180 (B70% similarity), DOCK2 (67% similarity)
and DOCK5 (66% similarity) in vertebrates, but also to sea urchin
DOCK (84% similarity) and Drosophila myoblast city (MBC) (55%
similarity) in invertebrates. In particular, the Src Homology 3
domain, DHR-1 and DHR-2 domains of ApDOCK protein showed
55%, 59% and 49% similarity to the respective domains of human
DOCK180.
ApDOCK-MO does not affect the spatio-temporal arrangement of
mesenchyme cells formed by normal developmental migration
To determine the effect of loss-of-function of ApDOCK, three
different concentrations (1.2, 12, 120 mM) of ApDOCK-MO or
control-MO were injected into discharged follicle eggs and fertilized
with sperm. At the respective concentrations, both types of experimental egg showed a similar time course of normal development,
during which mesenchyme cells started to ingress into the blastocoel
from the tip of the archenteron at the mid-gastrula stage, and
subsequently migrated to the ectodermal and mesendodermal walls
(n4100, respectively). By using MC5 monoclonal antibody (mAb)
(see Methods), we examined the spatial distribution of mesenchyme
cells from thirty 4-day-old ApDOCK-MO and control-MO larvae;
these larvae were obtained from eggs that had been injected with
MO at a concentration of 120 mM in each of the three experiments. In
both the samples all mesenchyme cells were highlighted, indicating
that ApDOCK-MO did not disrupt the synthesis of MC5 antigens
by mesenchyme cells (Figures 2a and b). The majority of mesenchyme
cells in both samples were identically distributed along the ciliary
band in the ectodermal wall and in the internal organs derived
from the mesendodermal walls, such as the coelomic pouches and
the digestive tract. Close-up views of individual mesenchyme
cells revealed that the morphology was indistinguishable between
the ApDOCK-MO and control-MO samples (Figures 2c and d):
each cell had developed branched cellular processes in multiple
directions. Thus, there were no marked differences in spatio-temporal
distribution or morphology of mesenchyme cells between the two
samples, and these characteristics were also comparable to those
of mesenchyme cells during normal development, as reported
previously.1,16
ApDOCK loss-of-function suppresses the directed migration of
larval mesenchyme cells toward a large oil-droplet in a dosedependent manner
As described in the earlier section, multiple mesenchyme cells
vigorously undergo migratory congregation against foreign bodies.
To examine the regulatory function of ApDOCK protein in mesenchyme cell dynamics, a large oil-droplet (diameter, 20 mm) was injected
into the blastocoel of 4-day-old control-MO or ApDOCK-MO larvae
(MO concentration, 120 mM). Figure 3 shows an experiment in which
the injection site was defined at the central region of the blastocoel in
the larval anterior portion (Figure 3a, red circle), wherein few
mesenchyme cells were distributed. In the control-MO larvae, the
mesenchyme cells showed a typical response to the oil-droplet at 2 h:
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Figure 1 Sequence analysis of ApDOCK protein. (a) Schematic diagram of the structure of ApDOCK protein. ApDOCK protein contains an Src Homology 3
domain domain (yellow) at the N-terminus and proline-rich region (black) at the C-terminal flanking region in addition to DHR-1 (blue) and DHR-2 (red) domains.
(b, c) Multiple alignment of the DHR-1 (b) and DHR-2 (c) domain. Each domain in ApDOCK protein, DOCK180 (BAA09454), DOCK2 (BAA13200), MBC
(NP_477144) and CED-5 (AAC38973) was aligned with ClustalW. Amino-acid residues that are conserved in more than 50% of the sequences are colored blue.
(d) Phylogenetic tree of the DOCK180 superfamily. The scale bar corresponds to 0.1 changes per amino acid. The four subfamilies of the DOCK180 superfamily
were previously denoted by Cote and Vouri.15 National Center for Biotechnology Information protein accession numbers are provided except for ApDOCK protein.
approximately six to eight mesenchyme cells had congregated around
the oil-droplet from a number of different directions, and some of
them had spread over the surface of the oil-droplet while extending
lamellipodium (‘encapsulation’, control-MO in Figure 3b and
Supplementary Movie 1). The migratory congregation of mesenchyme cells was activated after approximately 30 min. Subsequently, the
‘encapsulation’ behavior of the cells became prominent at and after
60 min, and persisted all of the time. In contrast, in the ApDOCKImmunology and Cell Biology
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Figure 2 Spatial distribution and morphology of mesenchyme cells from
4-day-old bipinnaria larva obtained from eggs injected with 120 mM controlor ApDOCK-MO. (a, b) Spatial distribution of mesenchyme cells in the
control-MO (a) and ApDOCK-MO (b) larvae. (c, d) A single mesenchyme cell
from the control-MO (c) and ApDOCK-MO (d) larvae. Both cell samples were
immunofluorescently stained with MC5 mAb, and are shown by means of
the stacking function of the confocal microscope. Scale bars: 50 mM (a and
b), 5 mM (c and d).
MO larvae, the blastocoelic region around the oil-droplet remained
the same in appearance at 2 h as it did before 2 h, although a few of
the mesenchyme cells were occasionally sensitive to the oil-droplet
(ApDOCK-MO in Figure 3b). In many cases, no mesenchyme cells had
migrated toward the oil-droplet until 2 h (Supplementary Movie 2).
To confirm the specificity of ApDOCK-MO for the immune
behavioral suppression of the mesenchyme cells, we examined the
dose-dependent effects of ApDOCK-MO on the numbers of mesenchymal cells that congregate to encapsulate the oil-droplet at 2 h after
injection (Figure 3c). Ten-fold serial increases in ApDOCK-MO
caused a significant decrease in the number of mesenchyme cells that
encapsulated the oil-droplet (Figure 3c). On the other hand, the
number of encapsulating mesenchyme cells in control-MO larvae
remained constant in the range of six–eight cells (Figure 3c). These
analyses were performed on 35 oil-droplet-injected samples for each
of the three concentrations of ApDOCK-MO and control-MO in each
of two experiments. We did not examine the effects of concentrations
of ApDOCK-MO stronger than 120 mM.
ApDOCK loss-of-function inhibits larval mesenchyme cells from
persistent spreading on the large oil-droplet even when the cells
can contact the oil
In the 4-day-old ApDOCK-MO bipinnaria larvae, an oil-droplet with
a size equal to that in Figure 3b was injected in the vicinity of the
Immunology and Cell Biology
ectodermal wall where the mesenchyme cells are densely located
(Figure 3a, yellow circle). Figure 3d shows the time-sequence of the
immunological response of four mesenchyme cells to the injected oildroplet (see also Supplementary Movie 3). During the first 10 min
immediately after injection, two mesenchyme cells (white arrowhead
and black arrow) came close to the oil-droplet and made contact with
it. In the next 10 min, a third mesenchyme cell (black arrowhead) got
in line with the first two mesenchyme cells. By 75 min, however, two
mesenchyme cells had left the oil-droplet (Figure 3d; black arrow and
arrowhead), and only one mesenchyme cell (white arrowhead)
remained in contact with the oil-droplet (Figure 3d; white arrowhead). A fourth mesenchyme cell (Figure 3d; white arrow) disappeared from this view without coming close to the oil-droplet, even
though the distance from this cell to the oil-droplet was similar to
that of the three mesenchyme cells that made contact with the oildroplet. As compared with control-MO larvae (Supplementary Movie
1), the mesenchyme cells in ApDOCK-MO larvae tended to retract
their lamellipodia once they were on the surface of the oil-droplet
(Supplementary Movie 3). In this way, the oil-droplet was not well
encapsulated by multiple mesenchyme cells in ApDOCK-MO larvae
(Figure 3e). This failure of encapsulation was observed in all 35
ApDOCK-MO larvae examined, whereas the mesenchyme cells of all
of the control-MO larvae (n ¼ 20) completely encapsulated the
injected oil-droplet.
We investigated whether the ApDOCK-mediated immunological
behaviors of mesenchyme cells occur at an earlier developmental stage
when mesenchyme cells vigorously migrate to the ectodermal and
mesendodermal walls in the blastocoel. When the oil-droplet was
injected into control-MO gastrula (30 h after fertilization) as shown in
Figure 3, no mesenchyme cells underwent encapsulation even after
they contacted the oil-droplet (Supplementary Movie 4). Rather, this
dynamic aspect was similar to those of the ApDOCK-MO bipinnaria
larva shown in Figure 3d and Supplementary Movie 3. The
mesenchyme cells did not cancel their developmental migration
under this experimental condition. These observations were made
from two experiments that each used 15 gastrula embryos.
Mesenchyme cells undergo normal cell-to-cell fusion on a
large oil-droplet and engulfment of micro-beads in ApDOCK
loss-of-function larvae
Next, we examined mesenchyme cell dynamics with regard to
multinucleated cell formation and phagocytosis in 4-day-old
ApDOCK loss-of-function larvae within 2 h of injecting an oil-droplet
or micro-beads. As we wished to observe each type of mesenchyme
cell dynamics when the larval mesenchyme cells come into contact
with the large oil-droplet and micro-beads (see the earlier section), we
performed two experiments in which each of these foreign substances
was injected in the vicinity of the ectodermal wall, as in Figure 3d.
Concerning cell-to-cell fusion, we attempted to locate the nucleus of
the mesenchyme cells as they were seen partially spreading over the
oil-droplet. By double fluorescence staining with MC5 mAb and
propidium iodide (PI), the mesenchyme cells were found to contain
two nuclei in their cytoplasm and thus showed normal cell-to-cell
fusion (Figure 4a). Similar results were obtained from 17 out of 20
ApDOCK-MO samples. Likewise, the mesenchyme cells revealed
active engulfment of the micro-beads in all ApDOCK-MO larvae
(Figure 4b). There was no marked difference in the engulfment
activity of mesenchyme cells around the micro-beads between
ApDOCK-MO and control-MO samples in either of two experiments
(n ¼ 15 larvae).
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Figure 3 Effect of ApDOCK-MO on immunological reactions of larval mesenchyme cells against a large oil-droplet. (a) The two blastocoelic regions used in
the injection experiments. Shown are paired Nomarski and confocal microscope images of normal 4-day-old bipinnaria larva that were immunofluorescently
stained with MC5 mAb. The fluorescent photo is a stacked image consisting of seven optical sections at 1-mm intervals. Red circle: the anterior blastocoelic
region where the mesenchyme cells are sparsely distributed. Yellow circle: the blastocoelic region near the ectodermal wall where multiple mesenchyme
cells are densely located. (b) Immunological reactions of mesenchyme cells in 4-day-old bipinnaria larvae obtained from eggs injected with 120 mM MO. An
oil-droplet was injected into the blastocoelic region as shown by red circle in (a). Shown are paired confocal microscope and Nomarski images of
mesenchyme cells and their nuclei that have been immunofluorescently stained with MC5 mAb (green) and PI (red), respectively. The images are shown by
means of the stacking and optical sectioning functions of the confocal microscope. (c) Dose-dependent effect of ApDOCK-MO and control-MO on the
immune response of larval mesenchyme cells. Boxplots show the number of the mesenchyme cells in contact with the oil-droplet 2 h after injection (n ¼ 35,
respectively). The number of the cells was calculated by the nucleus stained by PI. The concentrations of the injected MO are indicated. ***Po0.001;
**Po0.01; NS, not significant (t-test). (d) Immunological reactions of mesenchyme cells against a large oil-droplet injected into the blastocoel as shown by
yellow circle in (a). Sequential frames were captured from the time-lapse movies under a light microscope (see also Supplementary Movie 3). Times
indicate minutes after injection. Note that two mesenchyme cells separated from the oil-droplet after contacting it (black arrow and arrowhead). See text for
further explanation. (e) Merge of Nomarski and confocal microscope images shows insufficient encapsulation of the oil-droplet by mesenchyme cells in
ApDOCK-MO larvae. The image is from an ApDOCK-MO larva that has been immunofluorescently stained with MC5 mAb at 2 h after injection. Scale bars:
100 mM (a); 20 mM (b, d and e).
Mesenchyme cells in ApDOCK loss-of-function larvae display
similar defensive behaviors against the injected bacteria
To generate a more realistic immune response, we conducted the
injection experiment with bacteria that had been previously labeled
with rhodamine. Two hours after injection of bacteria into the central
region of the blastocoel of 4-day-old control-MO larva, multiple
mesenchyme cells formed an aggregate that encapsulated bacteria
within (Figure 5a: control-MO). On the other hand, in ApDOCK-MO
larva, approximately the same numbers of mesenchyme cells were
dispersed without forming an aggregate(s) and the individual cells
phagocytosed the bacteria (Figure 5a: ApDOCK-MO). Leftover
bacteria were widely observed in the area of injection in ApDOCKMO larva. These effects were observed in all of the 30 control- MO
and 23 ApDOCK-MO larvae examined.
Figure 5b shows an experiment in which the rhodamine-labeled
bacteria were injected in the immediate vicinity of the ectodermal wall
of ApDOCK-MO larva, wherein the mesenchyme cells were relatively
abundant, as shown in Figure 3d. In this case, most of the individual
mesenchyme cells phagocytosed bacteria, but they failed to form any
easily detected aggregates. Occasionally, however, there was an
indication of cell-to-cell fusion, as confirmed by more than two
mesenchyme cells that were closely connected to each other by the
bulk of their cell bodies (Figure 5b; arrow). Even in this situation, part
of bacteria remained unphagocytosed. Similar images were obtained
from all 7 ApDOCK-MO larvae.
The expression level of ApDOCK mRNA is significantly altered
with the progress of the immune reaction of mesenchyme cells in
culture
We profiled ApDOCK regulation at the mRNA level with regard to
the behavior of the mesenchyme cells during the immune response.
To this end, we used mesenchyme cells isolated on a culture dish from
4-day-old bipinnaria larvae (see Methods). After being allowed to
spread out for 4 h, the mesenchyme cells exhibited a network pattern
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Figure 4 Immunological reactions of mesenchyme cells against a large oildroplet and micro-beads in ApDOCK loss-of-function larvae. In 4-day-old
ApDOCK-MO bipinnaria larvae, an oil-droplet (a) and micro-beads (b) were
injected into the blastocoel where multiple mesenchyme cells are located,
and then fixed with PFA 2 h later. (a) Paired confocal microscope and
Nomarski images that were processed for staining with MC5 mAb and PI
are shown by means of the stacking and optical sectioning functions of the
confocal microscope, respectively. Note that two nuclei (red) are contained
in the mesenchyme cell spreading on the oil-droplet. (b) Paired confocal
microscope and Nomarski images. In the three pairs of images, three
mesenchyme cells are seen to engulf four, six and one micro-bead(s),
respectively. See text for further explanation. Scale bars: 20 mM.
in which each cell was associated with each other by cellular processes
(Figure 6a). They fidgeted at their position, while preserving the
structural network pattern under the naive situation (see
Supplementary Movie 5). Figure 6b shows the time course of the
immunological reaction of mesenchyme cells when they were
challenged with bacteria in culture. After 30 min, the mesenchyme
cells continued to rapidly move about the field, thereby distorting the
original network pattern. Throughout this period, the mesenchyme
cells continued to phagocytose bacteria (see Supplementary Movie 5,
arrows). The same results were obtained in each of the two
experiments.
Next, we examined the expression profile of ApDOCK mRNA in
the bacteria application experiment (Figure 6c). At 30 min, ApDOCK
mRNA expression had increased 1.7-fold as compared with naive
mesenchyme cells at 0 min. After 60 min, the expression of ApDOCK
mRNA began to decrease to the original level when the bacteria
challenge was started.
Immunology and Cell Biology
Figure 5 Immunological reactions of mesenchyme cells against bacteria in
ApDOCK loss-of-function larvae. Four-day-old control- or ApDOCK-MO
bipinnaria larvae were injected with rhodamine-labeled bacteria (red) into
the arbitrary region of the blastocoel in the anterior portion of the larva, and
immunohistochemically stained by MC5 mAb (green) at 2 h after injection.
(a) Typical images of mesenchyme cells after the bacteria had been injected
at the central region of the blastocoel, as performed in Figure 3. Note that
the mesenchyme cells form an aggregate in the control-MO, but not in the
ApDOCK-MO samples. The green and red merged image shows that the
bacteria were phagocytosed by mesenchyme cells. (b) Another image of
ApDOCK-MO mesenchyme cells. Here, injection of bacteria was carried out
in the vicinity of the ectodermal wall wherein the mesenchyme cells were
closely distributed with one other. Arrow indicates that cell-to-cell fusion
occured between two mesenchyme cells. Confocal microscope images were
obtained by the stacking and optical sectioning functions for the
immunofluorescent and Nomarsky photographs, respectively. Scale bars:
20 mM.
ApDOCK loss-of-function results in not only imperfect
lamellipodium formation but also deficient membrane ruffles at
the leading edge of larval mesenchyme cells in the culture
Under the same culture conditions used in Figure 6, we examined the
effect of ApDOCK loss-of-function on the detailed morphology and
cell dynamics of mesenchyme cells. The mesenchyme cells from
control-MO larvae highly extended the lamellipodium over a large
area (Figure 7a), while dynamically changing their shape by expansion
and contraction of the lamellipodium with the membrane ruffles
(Supplementary Movie 6). In contrast, mesenchyme cells from
ApDOCK-MO sample were apparently deprived of part of the
lamellipodium (Figure 7a). In addition, their shape change was
relatively slow as compared with that of cells from control-MO larvae
(Supplementary Movie 7). In a comparison of the area occupied by
individual single mesenchyme cells (see Methods), the area occupied
by mesenchyme cells from control-MO larvae was an B1.4-fold
greater than that from ApDOCK-MO larvae (Po0.001, t-test, three
experiments) (Figure 7b).
Assembly of the fibrous form of actin (F-actin) has been known to
be closely involved in the formation of membrane ruffles.17 We
performed double fluorescence analysis with rhodamine-phalloidin
(F-actin marker) and MC5 mAb (mesenchyme cell surface marker) to
observe the distribution of F-actin in larval mesenchyme cells under
the same culture conditions as in Figure 7a. In mesenchyme cells from
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Figure 6 Time dependency of ApDOCK mRNA expression in cultured mesenchyme cells at 30, 60, 120 min after challenge with bacteria. (a) Phasecontrast microscopy of cultured mesenchyme cells prepared from 4-day-old bipinnaria larvae. The mesenchyme cells crawled out from the extracellular
matrix over a period of 4 h. The cells enclosed by the rectangle are also shown in (b) and Supplementary Movie 6. (b) Phase-contrast micrographs of the
mesenchyme cells after the bacteria were applied in culture. Each photograph was captured from Supplementary Movie 5. (c) Real-time PCR of ApDOCK
mRNA in the bacteria application experiment. Data are shown as the level of ApDOCK mRNA expression relative to that in naive mesenchyme cells (0 min).
Vertical bars represent the mean±s.e. (n ¼ 3). Scale bars: 20 mM.
control-MO larvae, the fluorescent signal of rhodamine-phalloidin
was concentrated at the leading edge of lamellipodia, where the
fluorescent signal of MC5 mAb was also condensed (Figure 7c;
arrowheads of upper panel). This co-localization image of F-actin and
MC5 antigen was obtained for 3 of 28 mesenchyme cells in two
experiments. In contrast, in a mesenchyme cell from ApDOCK-MO
larvae, the MC5 mAb exhibited no condensed pattern of fluorescent
signals (Figure 7c; lower panel) (32 cells examined in two experiments). The fluorescent signal of phalloidin could be also detected
throughout the cytoplasm of the mesenchyme cells. On the other
hand, between the control-MO and ApDOCK-MO larvae, there was
no obvious difference in the intensity of the fluorescent signals of
rhodamine-phalloidin at the filopodia of the mesenchyme cells
(Figure 7c; arrows). Except for these regions, the intensity of
fluorescent signals of rhodamine-phalloidin was weaker in the
cytoplasm of mesenchyme cells from ApDOCK-MO larvae than in
that of mesenchyme cells from control-MO larvae.
DISCUSSION
In the present study, we identified a cDNA clone encoding a protein
that belongs to the DOCK180 superfamily in the starfish Asterina
pectinifera, and have termed it ApDOCK. We prepared ApDOCKMO-injected 4-day-old bipinnaria larvae and their experimental
counterparts (control-MO samples), and principally examined the
effect of loss-of-function of ApDOCK on several types of mesenchyme
cell dynamics in the larval defense system (see the earlier section). A
combined experiment with oil-droplet injection and dose-dependent
studies of ApDOCK-MO confirmed the specificity of ApDOCK-MO
as a reliable tool to examine mesenchyme cell dynamics in both a
series of injection experiments (Figures 3–5) and analyses of the
detailed morphology of mesenchyme cells under culture conditions
(Figure 7). Furthermore, the real-time PCR approach using a culture
system of mesenchyme cells underpins the actual involvement of
ApDOCK in the innate immune system of the starfish larvae
(Figure 6). Below, our findings on ApDOCK protein are discussed
with regard to its structural features, immunological roles and the
possible regulatory mechanism of mesenchyme cell dynamics during
the defense processes.
Phylogenetic tree analysis disclosed that ApDOCK is an ancestral
protein of the deuterostome DOCK-A subfamily (Figure 1d). It has
been well documented that proteins of the DOCK-A subfamily share
an intramolecular structure comprising Src Homology 3 domain,
DHR-1 and DHR-2 domains, which are involved in recruitment of
the proteins to the plasma membrane3 and directive organization of
the actin cytoskeleton.18 In this way, in many cell types, proteins of
the DOCK-A subfamily has essential and also common roles in cell
dynamics, such as cell migration, phagocytosis, cell-to-cell fusion and
shape determination (see the earlier section). It should be noted,
however, that DOCK2 protein in the DOCK-A subfamily is restricted
to expression in hematopoietic cell lines.4,11,14 Intriguingly, this
defined expression of DOCK2 indicates a functional identity with
ApDOCK protein, because both proteins has an essential role(s) in
immunological situations (Figures 2–6). The Crk-binding consensus
motif (Figure 1a: PPxLPxK) raises concern about a structural
difference between the DOCK2 and ApDOCK proteins. This consensus motif is absent from DOCK2, but present in ApDOCK protein.
Crk is a well-known adaptor protein that mediates the recruitment of
cytoplasmic proteins to tyrosine kinase19,20 and that interacts with
proteins of the DOCK-A subfamily.9,21,22 It might be possible that,
although the role of ApDOCK protein in the immune response is
conserved in evolution, the Crk-binding consensus motif of ApDOCK
protein has been replaced by another sequence in DOCK2.
Our results indicate that ApDOCK protein regulates mesenchyme
cell dynamics exclusively in the larval defense system. The injection
experiments, in which large oil-droplet and micro-beads were injected
at arbitrary blastocoelic regions, facilitated direct visualization of
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ApDOCK protein in starfish larval mesenchyme cells
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Figure 7 Appearance of lamellipodium of cultured mesenchyme cells from ApDOCK loss-of-function larvae. (a) Phase-contrasted image of mesenchyme
cells prepared from 4-day-old control-MO and ApDOCK-MO bipinnaria larvae (4 h after inoculation). Note that the lamellipodium are markedly
underdeveloped in mesenchyme cells from ApDOCK-MO larvae, thereby appearing as a refractile morphology. See text for further explanation.
(b) Quantitative analysis of the expanding area of a single mesenchyme cell (4 h after inoculation). The mesenchyme cells of ApDOCK-MO larvae occupy
434.4±29.2 mM2 on average by extension of their lamellipodia (n ¼ 70), while the experimental counter mesenchyme cells (control-MO larvae, n ¼ 72)
occupy 625.3±39.5 mM2 (means±s.e.). Dots represent the outliers, ***represents Po0.001 in t-test. (c) Effects of ApDOCK loss-of-function on F-actin
distribution in mesenchyme cells in in vitro. Shown are confocal microscope and Nomarski (differential interference contrast microscope; DIC) images of an
optical section that was immunohistochemically double-stained with MC5 mAb (MC5) and rhodamine-phalloidin (F-actin). Mesenchyme cells obtained from
control-MO and ApDOCK-MO larvae were fixed with PFA 4 h after inoculation. Note that some F-actin was concentrated at the membrane ruffles at the edge
of the lamellipodia of mesenchyme cells from control-MO larvae (arrowheads). n: nuclei. Scale bars: 50 mM (a); 20 mM (c).
distinguishable overlapping mesenchyme cell dynamics; this regulation is essential for migratory congregation and persistence of
encapsulation. In addition to these inorganic foreign bodies, the
injection of bacteria also confirmed that the mesenchyme cell is
regulated by ApDOCK, and its regulation defines the response
regarding congregated migration but not phagocytosis (Figure 5a).
In ApDOCK-MO samples, demonstration of the failure of mesenchyme cells to encapsulate the bacteria (Figure 5b), even though some
mesenchyme cells were closely located to one other, is considerably
similar to that seen in Figure 3d. The regulation of mesenchyme cells
dynamics by ApDOCK protein was furthermore demonstrated under
culture conditions with applied bacteria (Figure 6). As shown in
Figure 6, expression of ApDOCK was altered at the mRNA level,
including both up- and downregulation, in a time-dependent manner
Immunology and Cell Biology
in response to the application of bacteria. Interestingly, the mesenchyme cell seems to increase its migration activity in concert with
timing of upregulation in ApDOCK mRNA, regardless of whether
phagocytosis occurs or not (Supplementary Movie 5). For this culture
system, details are currently unknown regarding other cell dynamics,
encapsulation and cell-to-cell fusion, or regarding the relationship
between the migration activity of mesenchyme cells and downregulation of ApDOCK at the mRNA level.
Actin organization under control of DOCK2 provides the crucial
mechanism for chemotactic migration of several types of immune
cells.12,14,23 This would be also the case when the larval mesenchyme
cells undergo migratory congregation, which is ostensibly a similar
process to chemotactic migration, although no chemokine molecules
have been identified as yet in the larval defense system of
ApDOCK protein in starfish larval mesenchyme cells
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A. pectinifera. In addition, actin organization under control of
ApDOCK protein is also essential for persistence of encapsulation
on the foreign body (Figures 3d and e), because in culture conditions
larval mesenchyme cells from ApDOCK loss-of-function larvae failed
to organize sufficient F-actin at the leading edge of lamellipodium
(Figure 7c), which could only exhibit a vigorless ruffling movement
(Supplementary Movie 7). Furthermore, attention should be paid to
lamellipodium formation, which is significantly inhibited in mesenchyme cells from ApDOCK loss-of-function larvae (Figures 7a and b).
We therefore consider that ApDOCK protein regulates lamellipodium
formation via actin organization, which contributes to generation of a
physical force that permits the persistence of encapsulation. This
possibility would explain why larval mesenchyme cells from ApDOCK
loss-of-function larvae can only undergo encapsulation transiently
(Figure 3d and e, and Supplementary Movie 3). Similarly, larval
mesenchyme cells are certain to require a physical force generated by
actin organization under control of ApDOCK to undergo congregated
migration (Figure 3b).
On the other hand, it should be noted that some types of
mesenchyme cell dynamics, other than migratory congregation and
persistence of encapsulation, does not appear to be regulated by
ApDOCK protein. Remarkably, the mesenchyme cells from ApDOCK
loss-of-function larvae underwent cell-to-cell fusion on the large oildroplet and uptake of micro-beads. Although mesenchyme cells
numbers are considerably few to evaluate multinucleated giant cell
formation, cell-to-cell fusion was evidenced by the near location of
two nuclei (Figure 4a). Regarding the uptake of micro-beads,
although quantitative analysis was not conducted, the numbers of
micro-beads (Figure 4b) were comparable to those seen in normal
4-day-old larvae.1 Previous studies have shown that the vertebrate
macrophage shares a lot of proteins for cell-to-cell fusion and
phagocytosis,24 and also both types of cell dynamics are regulated
by DOCK180 protein not but DOCK2 protein.21,25 Thus, in the larval
mesenchyme cell, DOCK180-related proteins other than ApDOCK
protein might regulate multinucleated giant cell formation and
phagocytosis via cell-to-cell fusion and uptake dynamics.
Our study is not only a functional demonstration of a new member
of the DOCK180 superfamily protein but also the first report of the
immunological function of a DOCK180 orthologue in a marine
invertebrate. In addition, ApDOCK protein is the first protein to be
identified as a regulator of actin organization involved in cell
behaviors in echinoderms. In order to understand the evolution of
mechanisms that regulate the behaviors of immune cells, further
studies are needed to explore the target(s) of ApDOCK protein, such
as the Rho GTPase family member Rac, which are well known to
be positioned downstream of DOCK180 superfamily protein.3 At
present, a member of the Rho GTPase family is yet to be identified in
starfish species. The present study raises two significant issues that
should be addressed in future studies. The first issue pertains to the
other roles of ApDOCK protein when the mesenchyme cell operates
in the larval defense system. Except for chemotactic migration of
hematopoietic cells (see the earlier section), DOCK2 protein has been
shown to regulate the production of immune modulators, such as
interleukin-2 in T cells,25 type 1 Interferon in dendritic cells26 and the
inflammasome adaptor ASC (apoptosis-associated speck-like protein
containing a caspase recruitment domain) in dendritic cells and
lymphocytes.27 The production of these immune modulators
has been also reported in the macrophage.28,29 Likewise, the larval
mesenchyme cell might utilize orthologues of these immune
modulators through ApDOCK regulation. The second issue
concerns the developmental expression of ApDOCK protein. Our
finding that the embryonic mesenchyme cell is indifferent to the oildroplet, similar to mesenchyme cells in ApDOCK loss-of-function
larva (Supplementary Movie 4) indicates the possibility that ApDOCK
is not expressed until the embryo reaches the bipinnaria stage. A
specific antibody against ApDOCK protein would enable us to
elucidate the developmental process of the larval defense system in
A. pectinifera.
METHODS
Discharged follicle-free eggs
Immature eggs were obtained from the starfish Asterina pectinifera by cutting
ovaries into small pieces in ASW (Artificial Seawater; MarineArt SF-1, Tomita
Pharmaceutical, Tokushima, Japan). Then, they were gently suspended for five
to ten times in Ca2 þ -free seawater (Jamarin, Osaka, Japan) to remove the
follicle cells, and then placed in ASW. The composition of ASW has been
previously described.2
cDNA cloning, sequencing and sequence analyses
Degenerate PCR cloning was used to explore the partial cDNA of an
orthologous gene of the DOCK180 superfamily from the mesenchyme cell
cDNA library that has been reported previously.2 degenerate primers,
DOCK1F (50 - GCGGGAGAAGATCTTCTTCgtntgycarat-30 ) and DOCK4R
(50 - GGTCGGCGATCAGGccdatdatraa-30 ), were designed by the CODEHOP
program (http://dbmi-icode-01.dbmi.pitt.edu/i-codehop-context) using the
following vertebrate and invertebrate DOCK180 superfamily members:
Homo sapiens, BAA09454; Mus musculus, NP_001028592; Rattus norvegicus,
XP_219424; Canis lupus familiaris, XP_544064; Equus caballus, XP_001489814;
Strongylocentrouts purpuratus, XP_785550. The PCR program was performed
using AmpliTaq Gold (Applied Biosystems, Tokyo, Japan) with the following
cycling conditions: 5 min at 94 1C; 35 cycles of 94 1C for 30 s, 49 1C for 60 s,
74 1C for 105 s; and a final extension step at 74 1C for 5 min. The amplification
product was sequenced and termed the ApDOCK clone. To obtain the fulllength ApDOCK cDNA, total RNA was isolated from cultured mesenchyme
cells as described previously.2 Then, 50 - and 30 -rapid amplification of cDNA
ends (RACE) were performed using a SMART RACE cDNA Amplification
Kit (Clontech, Shiga, Japan) according to the manual with the RACE
primers DOCK-50 race (50 -GGGCAGCACGGGTAGCCTTTTCCGCAGC-30 )
and DOCK-30 race (50 -CTTCAGGCCCACCCAGATCCCTTGGCCC-30 ). All of
the DNA sequencing was performed on a Fluorescence DNA sequencer (ABI
PRISM 3100, Applied Biosystems) using BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems).
The ApDOCK cDNA and protein sequences were analyzed by the BLAST
algorithm (http://www.ncbi.nlm.nih.gov/BLAST), InterProScan (http://www.
ebi.ac.uk/Tools/InterProScan) and MOTIF (http://motif.genome.jp). The phylogenetic tree analysis was performed as described previously.15 Amino-acid
sequences of human, Drosophila melanogaster, Caenorhabditis elegans,
S. purpuratus, Dictyosterium discoideum and A. pectinifera family proteins
were aligned with ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
To generate the unrooted phylogenetic tree, neighboring-joining analysis was
applied to pairwise sequence distances calculated with the PHYLIP package
using the Kimura two-parameter method.15 The final output was generated
with FigTree v1.1.2 (http://tree.bio.ed.ac.uk/software/figtree/).
Microinjection
Antisense MO (ApDOCK-MO) and control-MO were obtained from Gene
Tools LLC (Philomath, OR, USA). The sequence of ApDOCK-MO was 50 ACCCAACCAGTCATGTTTCACAGTC-30 , which is complementary to the
sequence containing the translation start site of ApDOCK mRNA (underlined).
The control-MO differed from the ApDOCK-MO in the sequence of five base
pairs (50 -ACTCAACTAGTCACGTCTCACAATC-30 , mis-paired bases underlined). Each MO was dissolved in sterile water at 1 mM to generate a stock
solution, which was diluted to the appropriate concentration before injection.
The discharged follicle-free eggs were injected with ApDOCK-MO or controlMO. They were allowed to develop until the 4-day-old bipinnaria stage in
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ApDOCK protein in starfish larval mesenchyme cells
R Furukawa et al
964
ASW at 20 1C.16 The detailed injection procedure has been described
previously.30
Silicon oil (Shin-Etsu Chemical co, Ltd, Tokyo, Japan), fluorescently labeled
polystyrene latex beads (diameter 2 mm: Fluoresbrite carboxy YG microsphere;
Polysciences Inc., Warrington, PA, USA) and Escherichia coli (DH5a strain)
were used for injection experiments in ApDOCK-MO and control-MO
samples (4-day-old bipinnaria larvae) as described previously.1 E. coli were
labeled by rhodamine B isothiocyanate (Sigma-Aldrich, Tokyo, Japan) using
the following procedure: the bacteria were incubated for 1 h at room
temperature with ASW containing 0.6 mM rhodamine B isothiocyanate,
washed three times with ASW, and adjusted to a concentration corresponding
to OD600 ¼ 0.5 in ASW.
Preparation of larval mesenchyme cells in a culture dish and
bacteria challenge
Mesenchyme cells from ApDOCK-MO or control-MO bipinnaria larvae were
transferred to the culture dishes. The basic principle of this method has been
previously reported.31 Briefly, ten bipinnaria larvae of each MO type were
treated with 1 ml of the dissociate medium (DM; 1.2 M glycine in distilled
water containing 1% ASW and 6% newborn calf serum). After 5 min, the
medium was replaced with 1 ml of fresh DM, and the larvae were then gently
suspended in a glass mouth pipet (tube diameter, B150 mm; Drummond
Scientific Company, Broomall, PA, USA) to remove the ectodermal epithelial
cells. These peeled larvae were dropped onto a glass coverslip in a plastic
culture dish Falcon 1007 (diameter, 3.5 cm; BD, Tokyo, Japan) containing 3 ml
of ASW supplemented with 4% newborn calf serum. Samples were incubated
at 20 1C for 4 h to allow the mesenchyme cells to crawl out of the extracellular
matrix, and then the culture medium was refreshed. For bacteria challenge, the
concentration of E. coli was adjusted to an OD600 of 0.5 in ASW, and 500 ml of
E. coli-ASW was added to the cultured mesenchyme cells.
Fluorescent microscopy and measurement of the extension areas of
cultured mesenchyme cells
To determine the localization, morphological shape and immune response of
mesenchyme cells from 4-day-old ApDOCK-MO and control-MO bipinnaria
larvae, indirect immunofluorescent microscopy was performed using MC5
mAb, as described previously.1 MC5 mAb has been previously reported to
react with a membranous type of metalloproteinase of the astacin family.30 Its
antigen is intensely synthesized in embryonic and larval mesenchyme cells,16
and therefore it used as a ‘mesenchyme cell marker’. In some samples, the
nuclei were fluorescently viewed with PI (0.1 mg ml 1, Wako, Osaka, Japan).1
To detect the intracellular localization of the F-actin, cultured mesenchyme
cells from the ApDOCK-MO and control-MO larvae were processed on a glass
coverslip for indirect immunofluorescence with MC5 mAb and then treated
with rhodamine-phalloidin (1/1000 dilution, Invitrogen, Tokyo, Japan). All
specimens were examined with a laser confocal microscope (Fluoview,
Olympus, Tokyo, Japan).
To measure the extension area of cultured mesenchyme cells prepared from
each MO sample, the cells were processed for double fluorescent staining with
PI and MC5 mAb as described above. Next, the extension area was measured
for each single cell of cultured mesenchyme cells by Image J software (National
Institutes of Health, Bethesda, MD, USA).
Real-time PCR
A cDNA mix corresponding to cultured mesenchyme cells at each time point
of the bacteria challenge experiment was synthesized by using a ReverTra Ace
qPCR RT kit (TOYOBO, Osaka, Japan) as a template. A 127-bp fragment of
the ApDOCK gene (nucleotides: 415–541) was amplified by a pair of primers,
DOCK-qPCR-F (50 -AGAATGGGGAGTGCTGTGGA-30 ) and DOCK-qPCR-R
(50 -TACAGGGAGGGTGCCTGAGA-30 ). As a reference gene, a 114-bp fragment of the A. pectinifera 18S rRNA gene was amplified by 18S-3-f and 18S-3-r
primers as described previously.2 A five-fold serial dilution of the cDNA mix
was used to generate a standard curve. Real-Time reverse transcriptase-PCR
was performed by using a StepOne Real-Time PCR System (Applied
Biosystems) with the KAPA SYBR FAST qPCR master mix (KAPA
BIOSYSTEMS, Woburn, MA, USA). Each sample was analyzed in triplicate
Immunology and Cell Biology
wells, and the PCR products were resolved by electrophoresis to confirm that
the expected PCR products were obtained.
Time-lapse video microscopy
For in vivo time-lapse imaging, silicon oil-injected ApDOCK-MO or controlMO larvae or embryos were dropped between the spacer of two double-stick
tapes (NM-10, Nichiban, Tokyo, Japan) that were attached to a glass bottom
dish (35 mm dish, D110400, Matsunami, Osaka, Japan). They were then
immobilized with a nylon mesh (pore size; 200 mm), and further covered with
a glass coverslip lain on the spacers. Lastly, the glass bottom dish was filled
with ASW. Time-lapse observation of mesenchyme cell behavior was
performed by a light microscope (IX71, Olympus) with DP controller
software (Olympus). The images were captured every 5 min for 2 h. To
investigate the lamellipodium formations and phagocytosis, time-lapse images
of larval mesenchyme cells in culture were captured every 3 min in the culture
condition described above.
ACKNOWLEDGEMENTS
We thank members of the Asamushi Marine Biological Station of Tohoku
University and Dr Osamu Kawase for supplying the starfish. We also thank
members of Tateyama Marine Laboratory, Marine Coastal Research Center,
Ochanomizu University for keeping the adult starfish. We are particularly
grateful to Dr Motonori Hoshi for critical discussion. This work was supported
by Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education
and Research (to HK) and Keio Gijyuku Academic Development Funds (to RF).
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Supplementary Information that accompanies this paper is available on the Immunology and Cell Biology website (http://www.nature.com/icb)
Immunology and Cell Biology