Microscopy, 2016, 233242

doi: 10.1093/jmicro/dfv376
Advance Access Publication Date: 10 January 2016

Article

Colin contributes to phagocytosis of IgG-

opsonized particles but not non-opsonized
particles in RAW264 macrophages

Downloaded from http://jmicro.oxfordjournals.org/ at University of North Carolina at Chapel Hill on June 24, 2016
Yanmeng Lu1,2, Lei Cao1,3, Youhei Egami1, Katsuhisa Kawai1, and
Nobukazu Araki1,*
1
Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa
761-0793, Japan, 2Laboratory of Electron Microscopy, Southern Medical University, Guangzhou 510515,
China, and 3Department of Information Technology, School of Biomedical Engineering, Southern
Medical University, Guangzhou 510515, China
*To whom correspondence should be addressed. E-mail: naraki@med.kagawa-u.ac.jp
Received 27 October 2015; Accepted 30 November 2015

Abstract
Colin is an actin-binding protein that severs actin laments. It plays a key role in regulating
actin cytoskeletal remodeling, thereby contributing to diverse cellular functions. However,
the involvement of colin in phagocytosis remains to be elucidated. We examined the
spatiotemporal localization of colin during phagocytosis of IgG-opsonized erythrocytes,
IgG-opsonized latex beads and non-opsonized latex beads. Live-cell imaging showed that
GFP-colin accumulates in the sites of IgG-opsonized particle binding and in phagocytic
cups. Moreover, immunouorescence microscopy revealed that endogenous colin loca-
lizes to phagocytic cups engulng IgG-opsonized particles, but not non-opsonized latex
beads. Scanning electron microscopy demonstrated a notable difference in morphology
between phagocytic structures in IgG-dependent and IgG-independent phagocytosis. In
phagocytosis of IgG-opsonized particles, sheet-like pseudopodia extended along the
surface of IgG-opsonized particles to form phagocytic cups. In contrast, in opsonin-inde-
pendent phagocytosis, long nger-like lopodia captured non-opsonized latex beads.
Importantly, non-opsonized beads sank into the cells without extending phagocytic cups.
Our analysis of colin mutant expression demonstrates that phagocytosis of IgG-opsonized
particles is enhanced in cells expressing wild-type colin or active mutant colin-S3A,
whereas the uptake of non-opsonized latex beads is not. These data suggest that colin
promotes actin cytoskeletal remodeling to form phagocytic cups by accelerating actin turn-
over and thereby facilitating phagosome formation. In contrast, colin is not involved in
opsonin-independent phagocytosis of latex beads.
Key words: phagocytosis, colin, live-cell imaging, electron microscopy, latex beads, macrophages

The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved.
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234
Introduction
Phagocytosis is a specialized form of endocytosis involving
the ingestion of large particles (>0.5 m in diameter) and is
an essential component of innate immunity and tissue
remodeling. Professional phagocytes, such as macrophages
and neutrophils, are equipped for ingesting foreign particles,
invading microorganisms and apoptotic cells, contributing
to the host defense against infections and clearing senescent
or damaged cells. Invading microorganisms, often coated
by soluble host opsonins such as complement C3 or immu-
noglobulins, are recognized by specic cell surface recep-
tors, such as complement receptors (CRs) and Fc receptors
(FcRs) [1]. Phagocytosis through FcRs, which is the best
characterized, is accompanied by actin polymerization and
reorganization, which induce the formation of phagocytic
cups around IgG-opsonized particles [25]. The extension
of pseudopodia ( phagocytic cups) is driven by the forma-
tion of branched F-actin networks generated by Arp2/3 [6].
During FcR-mediated phagocytosis, the recruitment and
activation of the Arp2/3 complex is mainly dependent on
GTP-bound (activated) forms of Rho GTPases, Rac1 and
Cdc42 [7]. Moreover, coronin1, which is complexed with
Arp2/3 to cause its dissociation from the branch junction of
F-actin [8], has been shown to accumulate in phagocytic
cups [9]. These molecules coordinately regulate Arp2/
3-mediated phagocytic cup formation and subsequent cup
closure ( phagosome formation). After internalizing parti-
cles, the newly formed phagosomes mature via a series of
interactions with other endocytic compartments before
fusing with lysosomes for particle degradation [10,11].
Colin is a member of the actin-depolymerizing factor
(ADF)/colin family that severs actin laments to promote the
rapid turnover of lamentous actin [12,13]. The ADF/colin
protein family consists of ADF, colin 1 and colin 2. Colin 1
(usually referred to as colin) is the most abundant and ubiqui-
tous member of the family in vertebrate non-muscle tissues and
is the only one required for viability [14]. The actin lament-
severing activity of colin is reversibly regulated by phosphoryl-
ation and dephosphorylation at its serine 3 (Ser-3) [15,16].
LIM kinase (LIMK), which is regulated by Rho GTPases, cata-
lyzes phosphorylation of colin on Ser-3 and thereby inhibits
the actin-severing activity of colin [17]. In contrast, phos-
phorylated colin is re-activated through dephosphorylation
by specic phosphatases such as Slingshot [18]. In general,
non-phosphorylated colin is localized in locomotory such as
lamellipodia, whereas phosphorylated colin is more uni-
formly distributed throughout the cytoplasm [19,20]. The
localization and activity of colin conne actin polymerization
and depolymerization through its severing activity, thereby
dening the site of membrane protrusion formation. Besides its
roles in directional cell protrusion, colin has been shown to be
Microscopy, 2016, Vol. 65, No. 3
involved in the internalization of several types of bacteria
through phagocytic pathways [2124]. During CR3-mediated
phagocytosis, colin controls phagosome formation in macro-
phages [21]. However, the involvement of colin in other types
of phagocytosis, such as FcR-mediated phagocytosis, remains
to be examined. Thus, it is of interest to investigate whether or
not colin regulates FcR-mediated phagocytosis.
In this study, we focused on the role of colin in IgG-
dependent and IgG-independent phagocytosis. Our micros-
copy and quantitative analysis of the phagocytosis of particles
demonstrated that colin facilitates ingestion of IgG-opsonized
particles, but not non-opsonized particles. Based on our data,
Downloaded from http://jmicro.oxfordjournals.org/ at University of North Carolina at Chapel Hill on June 24, 2016
it is suggested that, depending on the local requirement for
F-actin remodeling, colin contributes differently to phago-
some formation by facilitating the rapid turnover of actin
lament to form phagocytic cups.
Materials and methods
Cell culture
Mouse macrophage RAW264 cells (Riken Cell Bank,
Tsukuba, Japan) were cultured in DMEM (Nakalai, Kyoto,
Japan) supplemented with 10% heat-inactivated fetal bovine
serum (FBS), and 100 U/ml of penicillin and 0.1 mg/ml
streptomycin (Nakalai, Kyoto, Japan). The cells were main-
tained at 37C in a humidied 5% CO2 incubator. Before the
experiments, the culture medium was replaced with Ringers
buffer (RB) consisting of 155 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 2 mM Na2HPO4, 10 mM D-glucose,
10 mM HEPESNaOH (pH 7.2) and 0.5 mg/ml bovine
serum albumin (BSA) [25,26].
DNA constructs and transfection
To construct pEGFP-colin-wild type (wt), the cDNA frag-
ment comprising the entire coding region for mouse colin was
generated by PCR amplication of mouse cDNA. Primers used
were 50 -TCTCGAGCTATGGCCTCTGGTGTGGCTGTC-30
and 50 -TGGATCCTCACAAAGGCTTGCCCTCCAG-30 . The
PCR product was subcloned into pGEM-T Easy Vector
(Promega, Madison, WI), digested using XhoI/BamHI res-
triction enzymes and cloned into the XhoI/BamHI restric-
tion sites of pEGFP-C1 (Clontech, Palo Alto, CA). To
generate pEGFP-colin-S3A (a nonphosphorylatable
mutant) and pEGFP-colin-S3D (a phosphomimetic mu-
tant), the entire coding region of colin was amplied using
primers with pEGFP-colin-wt as a template. Primers used
were as follows: colin-S3A: 50 -TCTCGAGCTATGGCCG
CTGGTGTGGCTGTCTCTG-30 and 50 -TGGATCCTCAC
AAAGGCTTGCCCTCCAG-30 ; colin-S3D: 50 -TCTCGAG
CTATGGCCGATGGTGTGGCTGTCTCTG-30 and 50 -TGG
Microscopy, 2016, Vol. 65, No. 3
ATCCTCACAAAGGCTTGCCCTCCAG-30 . The fragments
were nally cloned into the XhoI/BamHI restriction sites of
pEGFP-C1. pTagRFP-actin was purchased from Evrogen
(Moscow, Russia). All constructs were veried by sequencing
prior to use. Plasmid transfection was performed using the
Neon Transfection System (Invitrogen, Gaithersburg, MD)
following the manufacturers instructions. Briey, 100 l of a
RAW264 cell suspension (1 107 cells/ml) in buffer R was
mixed with 3 g of the indicated plasmids and electroporated
once at 1680 V for 20 ms. The cells were then seeded onto
25-mm circular coverslips in 35-mm culture dishes and main-
tained in the culture medium. At 2024 h after transfection,
the cells were subjected to live-cell imaging.
Phagocytosis
Sheep erythrocytes were opsonized with rabbit anti-sheep
IgG (1 : 100; Organo Teknika-Cappel) and suspended in PBS
as described previously [2]. A total of 1 106 carboxylate-
modied latex beads (Polybead carboxylate 4.5 m micro-
sphere, Polysciences, Inc., Warrington, PA) were washed
in PBS and incubated in 10 mg/ml human IgG (hIgG) for
1 h at 37C. After washing three times, IgG-opsonized
beads were resuspended in PBS. For the quantitative assay
of phagocytosis, IgG-opsonized erythrocytes (IgG-Es) or
non-opsonized carboxylate-modied latex beads were
added to RAW264 macrophages. After 30 min incubation
with phagocytic targets at 37C, the cells on the coverslips
were xed with 4% paraformaldehyde. The number of
internalized particles was counted in 50 cells randomly
chosen under a phase-contrast and uorescence microscope.
The phagocytic index, that is, the mean number of particles
taken up per cell, was calculated. The index obtained for the
transfected cells was divided by that for the untransfected
(control) cells and expressed as a percentage of the control
cells.
Live-cell imaging
RAW264 cells cultured on a 25-mm circular coverslip were
assembled in a RB-lled chamber on the thermo-controlled
stage (Tokai Hit INU-ONI, Shizuoka, Japan) of an inverted
epiuorescence microscope (Nikon TE300). Phase-contrast
images of live cells were sequentially taken through a CCD
camera DOC-CAM HR-U3-60S6M (Molecular Devices,
Downingtown, PA) using shutters and lter wheels con-
trolled by the MetaMorph imaging system (Molecular
Devices, Downingtown, PA). Time-lapse images of phase-
contrast and uorescence microscopy were taken at 10 s
intervals and assembled into QuickTime movies by the
MetaMorph imaging system. At least three examples were
observed in each experiment.
235
Immunostaining and confocal laser microscopy
RAW264 cells cultured on a 13-mm circular coverslip were
co-incubated with hIgG-opsonized and non-opsonized latex
beads for 8 min at 37C. After xation with 4% paraformal-
dehyde, the cells were rinsed with PBS three times for 5 min,
followed by 0.25% NH4Cl in PBS for 10 min, and treated
with a blocking solution consisting of 1% BSA and 1% goat
serum in permeabilizing buffer (0.1% Triton X-100 in PBS)
for 10 min. For the detection of endogenous colin, we used
rabbit polyclonal anti-colin afnity-puried antibody (gen-
erous gift from Drs Ichiro Yahara and Kenji Moriyama at
The Tokyo Metropolitan Institute of Medical Science) at 1:5
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dilution in the blocking solution. As a secondary antibody,
Alexa Fluor 594 anti-rabbit IgG (Molecular Probes) at 1:500
dilution was used. For the detection of hIgG-opsonized parti-
cles, we used Alexa Fluor 488 goat anti-human IgG (F(ab0 )2
fragment specic) at 1:2000 dilution (Jackson ImmunoRe-
search Laboratories, West Grove, PA). The specimen cover-
slips were mounted on glass slides using PermaFlour
(Thermo Scientic) and observed by a confocal microscope
(LSM700, Zeiss) as previously described [26].
Electron microscopy
RAW264 cells were cultured on coverslips and incubated with
IgG-opsonized beads or non-opsonized beads at 37C. The cells
were xed with 2% glutaraldehyde in 0.1 M phosphate buffer
(pH 7.4) containing 5% sucrose for 60 min at room tempera-
ture. The cells on coverslips were rinsed in a buffer, post-xed
with 1% osmium tetroxide, treated with 1% tannic acid and
1% osmium tetroxide. The specimens were dehydrated through
a graded ethanol series and rapidly transferred into a liquid
carbon dioxide critical point-drier (Hitachi HCP-2). Critical-
point-dried specimens were coated with an osmium plasma
coater (OPC40; NLE Lab, Nagoya, Japan) and observed by a
eld emission scanning electron microscope (Hitachi S900) at
8 kV. Electron micrographs were pseudocolored using generic
image processing software (Adobe Photoshop).
Statistics
The data are as means standard error (SE) of ve inde-
pendent experiments. Statistical signicance was determined
by unpaired Students t-test. Differences between the ana-
lyzed data were considered signicant at P < 0.05.
Results
Colin is transiently recruited to phagocytic cups
during FcR-mediated phagocytosis
To examine whether or not colin is involved in FcR-
mediated phagocytosis, we observed the localization of colin
236 Microscopy, 2016, Vol. 65, No. 3

during phagocytosis of IgG erythrocytes (IgG-Es) in live
RAW264 macrophages coexpressing EGFP-colin-wild type
(GFP-colin-wt) and TagRFP-actin by phase-contrast and
uorescence microscopy. Prior to the onset of phagocytosis,
GFP-colin-wt was diffusely localized in the cytosol, as previ-
ously shown in other cell types [27]. After the binding of
IgG-Es to the cells, GFP-colin-wt was rapidly recruited to
the sites where IgG-Es were binding to the cells and to
extending pseudopodia (phagocytic cups) along the surface
of IgG-Es (Fig. 1 and Supplementary data online, Movie 1).
In the process of phagosome formation, GFP-colin-wt was
colocalized with TagRFP-actin (Fig. 1, t = 0, 2.5 and 5 min).
observed by live-cell imaging. Time-lapse images in Fig. 2a
and Supplementary data online, Movie 2a show that colin
GFP-colin-S3A are recruited to the phagocytic cups disso-
ciated from internalized phagosomes, similar to wild-type
colin. However, GFP-colin-S3D was not recruited to the
sites of IgG-E binding, although IgG-Es internalization was
observed (Fig. 2b and Supplementary data online, Movie 2b).
These ndings indicate that phosphorylation of colin is indis-
pensable for its recruitment to phagocytic cups during
FcR-mediated phagocytosis.
To ascertain the specic involvement of colin in
FcR-mediated phagocytosis, we compared the localization

Subsequently, both proteins dissociated from newly formed
phagosomes after closing cups into phagosomes.
It has been shown that the actin-binding and severing activ-
ity of colin is inactivated by phosphorylation of the serine
residue at position 3 (Ser-3) by protein kinases and reversely
activated by phosphatases [16]. Interestingly, colin has been
reported to be transiently dephosphorylated and translocated
to the plasma membrane [27]. Therefore, we attempted to
determine whether or not the phosphorylation state of colin
affects its recruitment to phagocytic cups during FcR-
mediated phagocytosis. RAW264 cells were transfected with
GFP-colin-S3A (a constitutively active mutant that lacks the
physiological phosphorylation site) or GFP-colin-S3D (an
inactive mutant that mimics phosphorylated colin) and were
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of colin during IgG-dependent and opsonin-independent
phagocytosis. RAW264 macrophages expressing GFP-
colin-wt were allowed to ingest IgG-opsonized latex beads
or non-opsonized carboxylate-modied latex beads and
were observed by phase-contrast and uorescence micros-
copy. As shown in Fig 3a and Supplementary data online,
Movie 3a, colin readily accumulated in the binding sites of
IgG-opsonized beads, similarly to IgG-Es, and in the extend-
ing phagocytic cups. In contrast, GFP-colin-wt was not
recruited to the binding sites of non-opsonized beads
(Fig. 3b and Supplementary data online, Movie 3b). It is
also notable that the internalization of non-opsonized beads
into the cells was slower than that of IgG-opsonized beads
(Supplementary data online, Movie 3b).

Fig. 1. Live-cell imaging of GFP-colin-wt and TagRFP-actin in RAW264 macrophages during

phagocytosis of IgG-Es. RAW264 cells cotransfected with GFP-colin-wt and TagRFP-actin were
allowed to contact with IgG-Es and observed by phase-contrast and uorescence microscopy.
Time-lapse images were acquired using the MetaMorph imaging system. Binding of IgG-Es to
the cell surface is set as time 0. Top panels are selected phase-contrast images at the indicated
times. Note that GFP-colin-wt is transiently recruited to both the binding sites of IgG-Es and
forming phagocytic cups and colocalized with TagRFP-actin (arrowheads). Representative
images are shown. Scale bars: 10 m.
Microscopy, 2016, Vol. 65, No. 3 237

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Fig. 2. Colin dephosphorylation of Ser-3 is indispensable for its recruitment to phagocytic cups.
Time-lapse images of RAW264 cells expressing GFP-colin-S3A (a) or GFP-colin-S3D (b) were
acquired. (a) It was found that an active, phosphorylation defective mutant colin-S3A localizes to
phagocytic cups engulng IgG-Es and concurrently dissociates from the internalized phagosomes. (b)
An inactive, phospho-mimic mutant colin-S3D was not recruited to the sites of phagocytosis of IgG-Es.
Representative images are shown. Scale bars: 10 m.

Next, we dissected the localization of endogenous colin
in RAW264 cells after co-incubation with uorescently
labeled IgG-opsonized beads and unlabeled non-opsonized
beads. Confocal immunouorescence microscopy showed
that colin is enriched at the phagocytic cups surrounding
IgG-opsonized latex beads (Fig. 4); however, no increase in
colin immunoreactivity was detected at sites of phagocyt-
osis of non-opsonized latex beads (Fig. 4). These data indi-
cate that opsonization of particles by IgG is required for the
recruitment of colin to the phagocytic cups.
Marked differences in morphology between
phagocytic structures on the cell surface during
opsonin-dependent and -independent
phagocytosis
In this study, we found a remarkable difference in colin
dynamics during opsonin-dependent and -independent
phagocytosis. To compare ultrastructural differences in the
morphology of phagocytic structures between IgG-dependent
and -independent phagocytosis, we performed scanning elec-
tron microscopy (SEM). RAW264 macrophages were incu-
bated for 8 or 30 min with IgG-opsonized or non-opsonized
latex beads. Then, the cells were xed and processed for
SEM. Eight minutes after the addition of IgG-opsonized
targets to the cells, some particles were observed on the dor-
sal cell surface (Fig. 5a). At high magnication, we observed
that phagocytic cups develop well along the surface of IgG-
opsonized latex beads (Fig. 5b). In contrast, in RAW264
macrophages incubated with non-opsonized beads, long
nger-like lopodia grasped non-opsonized phagocytic tar-
gets (Fig. 5d). Intriguingly, the captured beads sank into the
cells without extending phagocytic cups (Fig. 5e). After
30 min incubation with phagocytic targets, IgG-opsonized
beads were scarcely observed on the cell surface (Fig. 5c)
because almost all of the particles had been internalized into
the cell body. However, non-opsonized beads were frequently
seen on the dorsal surface of the cells (Fig. 5f), indicating that
non-opsonized latex beads are internalized more slowly than
IgG-opsonized particles.
The expression of colin-wt and colin-S3A
promotes FcR-mediated, but not
opsonin-independent, phagocytosis
To explore the functional contribution of colin to phago-
some formation, we examined the effect of colin mutant
expression on the internalization of IgG-opsonized and
238 Microscopy, 2016, Vol. 65, No. 3

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Fig. 3. Live-cell imaging of GFP-colin-wt in RAW264 macrophages during phagocytosis of
IgG-opsonized and non-opsonized latex beads. (a) RAW264 cells expressing GFP-colin-wt were
observed by phase-contrast and uorescence microscopy. The binding of IgG-opsonized latex beads to
the cell surface was set as time 0. Phase-contrast images are shown in the top panels. GFP-colin-wt
was recruited to the binding sites of IgG-opsonized beads and to the phagocytic cups (arrows). Scale
bars: 10 m. (b) Time-lapse images of GFP-colin-wt during phagocytosis of non-opsonized latex beads
were acquired in the same way as for (a). Note that GFP-colin-wt is not recruited to the sites of
phagocytosis of non-opsonized latex beads. Scale bars: 10 m.

Fig. 4. Confocal microscopy of endogenous colin in RAW264 macrophages during phagocytosis of

IgG-opsonized and non-opsonized latex beads. RAW264 cells were co-incubated with IgG-opsonized
latex beads (uorescent) and non-opsonized latex beads (nonuorescent) for 8 min before xation,
immunostained with anti-colin antibody and observed by confocal laser microscopy. Endogenous
colin accumulated at phagocytic cups engulng IgG-opsonized beads (arrows). In contrast, colin was
scarcely recruited to the binding and phagocytic sites of non-opsonized latex beads (arrowheads).
Scale bars: 10 m.

non-opsonized targets. We transiently transfected RAW264
macrophages with GFP-colin-wt, GFP-colin-S3A (active
mutant) or GFP-colin-S3D (inactive mutant). Then, quan-
titative assay of phagocytosis of IgG-opsonized and non-
opsonized particles was performed using RAW264 cells
transfected with each colin construct. As shown in Fig. 6,
the expression of either colin-wt or constitutively active
mutant colin-S3A facilitated the uptake of IgG-opsonized
particles. In contrast, the expression of inactive mutant
colin-S3D had no effect on the internalization of opsonized
particles. Importantly, the efciency of opsonin-independent
phagocytosis was unaffected by the expression of colin-wt,
Microscopy, 2016, Vol. 65, No. 3 239

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Fig. 5. SEM showing differences in morphology between phagocytic structures on the cell
surface during IgG-dependent and -independent phagocytosis. RAW264 macrophages were
incubated with IgG-opsonized beads (ac) or non-opsonized latex beads (df ) for 8 or 30 min
and were xed and processed for SEM. (a and b) In macrophages 8 min after the addition of
IgG-opsonized particles, some opsonized beads were seen on the dorsal cell surface, and others
were internalized in the cell body. Under this condition, phagocytic cups along the surface of
IgG-opsonized particles were observed at higher magnication (b). (d and e) In RAW264 cells
engulng non-opsonized latex beads, long nger-like lopodia grasped non-opsonized beads.
Interestingly, the phagocytic targets directly sank into the cells without forming phagocytic cups
(e). After 30 min, almost all of IgG-opsonized beads were internalized in the cells (c), whereas
some non-opsonized beads were observed on the cell surface (d). Phagocytic targets were
pseudocolored by Adobe Photoshop. Scale bars: 5 m.

colin-S3A or colin-S3D. The results indicate that colin
promotes FcR-mediated phagocytosis, but is not involved
in opsonin-independent phagocytosis.
Discussion
In this study, we revealed that colin is transiently recruited
to phagocytic cups during IgG-dependent phagocytosis, but
not opsonin-independent phagocytosis. This nding indi-
cates that the binding of IgG Fc region to FcR is required
for the recruitment of colin to phagocytic cups. It is known
that colin is transiently dephosphorylated at its Ser-3 residue
and binds to actin laments [1620]. However, colin-S3D,
a phosphorylation-mimic mutant in which Ser-3 was
replaced with Asp, fails to bind to and sever actin laments.
Therefore, colin-S3D localizes more uniformly in cyto-
plasm, while colin-S3A non-phosphorylated mutant, in
which Ser-3 was replaced with Ala, appropriately localizes
to actin-enriched regions beneath the plasma membrane
[19,20,27]. Consistently, we observed that GFP-colin-S3D
does not localize to sites of IgG-particle binding, suggesting
that colin dephosphorylation at Ser-3 is indispensable for
its recruitment to phagocytic cups during FcR-mediated
phagocytosis by macrophages. It is of interest to elucidate
how IgG-FcR signaling recruits and activates colin in
future studies.
Our live-cell imaging and quantitative assay demon-
strated that colin contributes to IgG-dependent phagosome
formation rather than opsonin-independent phagocytosis.
The overexpression of wild-type colin promotes the uptake
of IgG-opsonized particles by FcR-mediated phagocytosis,
although phagocytosis of non-opsonized latex beads was
not signicantly affected. Moreover, RAW264 cells expres-
sing non-phosphorylation-mimic colin-S3A appear to
form phagocytic cups more efciently than those expressing
wild-type colin. It is now generally accepted that non-
phosphorylated active colin severs actin laments at the
pointed end and increases actin monomers. Furthermore,
active colin speeds up actin polymerization via its actin-
severing activity providing free barbed ends for further poly-
merization and nucleation by the Arp2/3 complex.
May et al. (2000) reported that the Arp2/3 complex
plays a crucial role in FcR-mediated phagocytosis. Pseu-
dopod extension along the surface of IgG-opsonized
phagocytic targets is driven by the formation of branched
F-actin networks generated by Arp2/3 [6]. In the process of
240
Fig. 6. Expression of colin-wt or active mutant colin-S3A promotes
ingestion of IgG-opsonized particles. To quantify phagosome formation,
RAW264 cells transiently expressing each construct indicated were
incubated with IgG-opsonized particles (lled bars) or non-opsonized
latex beads (open bars) for 20 min at 37C and xed. The efciency of
phagocytosis was calculated based on 50 transfected and 50
untransfected cells. The results are expressed as a percentage of control
(untransfected) cells. The data are expressed as the means SE of ve
independent experiments. Students t-test was used for statistical
analysis. *P < 0.05.
FcR-mediated phagocytosis, the GTP-bound form of
Cdc42, a member of the Rho GTPase family, binds and
activates WASP, which induces actin polymerization via
activation of the Arp2/3 complex. Importantly, the activa-
tion and inactivation of Cdc42 have been shown to be
essential for phagocytic cup formation and subsequent cup
closure [28], implying that the spatiotemporal regulation
of Arp2/3 activity is required for phagosome formation.
Actin lament severing leads to the generation of barbed
ends, which are preferred sites for Arp2/3-mediated nucle-
ation and for release of G-actin [16,27]. In this study, we
observed that colin is transiently recruited to phagocytic
cups where actin remodeling actively occurs. Thus, it is
assumed that colin may provide nucleation sites via its
severing activity and may promote rapid turnover of actin
in the process of lamellipodium extension to form phago-
cytic cups.
Unexpectedly, we found that the expression of an inactive
phospho-mimic mutant colin-S3D does not signicantly
inhibit FcR-mediated phagosome formation as compared
with the control. Therefore, it is likely that colin is not an
essential component but a facilitator of FcR-mediated phago-
some formation. Alternatively, other actin-depolymerizing/
severing proteins such as destrin or gelsolin may function
redundantly in FcR-mediated phagocytosis [29,30].
Microscopy, 2016, Vol. 65, No. 3
In our quantitative assay, unlike FcR-mediated phagocyt-
osis, opsonin-independent phagocytosis of latex beads was
not affected by overexpression of any colin mutants. We also
showed, using both live-cell imaging and immunouorescence
microscopy, that colin does not localize to the sites of pha-
gocytosis of non-opsonized latex beads. These ndings
suggest that colin is not involved in phagocytosis of non-
opsonized particles. Therefore, we consider that the process of
opsonin-independent phagocytosis is mechanically different
from that of FcR-mediated phagocytosis. A notable nding
in our study is that there are marked differences in morph-
ology between phagocytic structures on the cell surface during
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IgG-dependent and opsonin-independent phagocytosis. In the
process of FcR-mediated phagocytosis, lamellipodia extend
along the surface of IgG-opsonized particles to form tight
phagocytic cups, whereas long and thin lopodia extend to
grasp non-opsonized latex beads. Intriguingly, non-opsonized
latex beads sank into the cells without extension of phago-
cytic cups. Moreover, we found that non-opsonized beads are
internalized more slowly than IgG-opsonized particles. This
observation is in line with the previous nding that non-
opsonized latex beads are insufciently captured by macro-
phages compared with IgG-opsonized particles [31]. Because
phagocytosis of non-opsonized beads did not show the fully
grown phagocytic cups, it is postulated that the uptake of
non-opsonized latex beads might be less actin-dependent than
FcR-mediated phagocytosis. This is consistent with the fact
that colin was not recruited to the sites of phagocytosis of
non-opsonized latex beads. Even though the extension of thin
nger-like protrusions to capture non-opsonized particles
would require actin laments, it is unlikely that a rapid actin
turnover is essential.
Concluding remarks
In conclusion, our study demonstrates that colin is tempor-
arily associated with phagocytic cups and facilitates the
uptake of IgG-opsonized particles but not non-opsonized
latex beads. Although colin function may be dispensable
for slow phagocytosis, it is an important facilitator for rapid
phagosome formation during FcR-mediated phagocytosis.
Our study leads to the view that colin-mediated actin
depolymerization and polymerization coupled with Arp2/3
complex activity may facilitate the phagocytic cup exten-
sion along the surface of IgG-opsonized particles. As for
opsonin-independent phagocytosis, striking differences in
morphology and colin independency from IgG-dependent
phagocytosis were noteworthy. Carboxylate-modied latex
beads have often been used as a surrogate to study phago-
cytosis of apoptotic cells because their surface electrostatic
property mimics the negative charge on dying cells. How-
ever, it is unclear whether the mechanisms of phagocytosis
Microscopy, 2016, Vol. 65, No. 3
of carboxylate-modied latex beads and apoptotic cells are
the same. Further studies would be needed to explore the
roles of colin and other actin-binding proteins in phagocyt-
osis of different kinds of particles.
Acknowledgements
We are very grateful to Dr Ying Jie Piao for his continuous encourage-
ment. We also thank Dr Katsuya Miyake, Mr Kazuhiro Yokoi, Mr
Toshitaka Nakagawa and Ms Yukiko Iwabu for their advice and
technical support.
Supplementary data
Supplementary data are available at http://jmicro.oxfordjournals.
org/.
Funding
This study was supported by the Japan Society for the Promotion of
Science (JSPS) KAKENHI No. 25860142 (to Y.E.), and also sup-
ported in part by JSPS KAKENHI No. 26860136 (to K.K.), No.
26670094 (to N.A.) and the research fund of Kagawa University.
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