Bruton’s Tyrosine Kinase (BTK) and Vav1 Contribute to
Dectin1-Dependent Phagocytosis of Candida albicans in
Karin Strijbis1, Fikadu G. Tafesse1, Gregory D. Fairn2, Martin D. Witte1, Stephanie K. Dougan1,
Nicki Watson1, Eric Spooner1, Alexandre Esteban1, Valmik K. Vyas1, Gerald R. Fink1, Sergio Grinstein2,3,
Hidde L. Ploegh1*
1Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America, 2Keenan Research Centre of the Li Ka Shing Knowledge Institute, St.
Michael’s Hospital, Department of Surgery, University of Toronto, Toronto, Ontario, Canada, 3Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada
Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent
infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting
proteins of Dectin-1 in macrophages, Bruton’s Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic
cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions
surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid
PI(3,4,5)P3and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal
membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the
subsequent recruitment of PKCe. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C.
albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans,
comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection
than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.
Citation: Strijbis K, Tafesse FG, Fairn GD, Witte MD, Dougan SK, et al. (2013) Bruton’s Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent
Phagocytosis of Candida albicans in Macrophages. PLoS Pathog 9(6): e1003446. doi:10.1371/journal.ppat.1003446
Editor: Robin Charles May, University of Birmingham, United Kingdom
Received January 5, 2013; Accepted May 7, 2013; Published June 27, 2013
Copyright: ? 2013 Strijbis 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: We acknowledge funding from The Netherlands Organization for Scientific Research (KS, MDW, FGT), the Clay Postdoctoral Fellowship (KS), the
National Institutes of Health (GM040266; GRF and F32 AI729353; VKV), Fulbright/Spanish Ministry of Education and Science Visiting Scholar Program (FU2006-
0983; AE) and the Margaret and Herman Sokol Fellowship in Biomedical Research (VKV). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Innate immune cells eliminate pathogens by phagocytosis, a
process of internalization followed by degradation of the pathogen.
Germline-encoded pattern recognition receptors (PRRs) recognize
pathogen-associated molecular patterns (PAMPs) on bacteria,
viruses, yeast and other microorganisms. Recognition of a PAMP
by its receptor initiates a coordinated sequence of events that
includes the recruitment of ancillary proteins and the formation of
various second messengers at -or close to- the site of initial contact
with the pathogen.
Macrophages and neutrophils are the first line of defense against
Candida albicans, a common cause of human fungal infections .
C. albicans is an opportunistic commensal yeast that is part of the
normal gut microbiota . Innate immune cells must therefore
tolerate commensal C. albicans, yet adequately deal with its
pathogenic counterpart. The major PRR involved in anti-fungal
immunity is Dectin-1, a C-type lectin present on neutrophils,
macrophages and dendritic cells that recognizes fungal b-glucan.
Dectin-1 contains an extracellular C-type lectin domain and an
intracellular ITAM-like domain essential for downstream signaling
. Upon activation of Dectin-1, phosphorylation of its ITAM-like
domain leads to the recruitment of spleen tyrosine kinase (Syk) .
The role of Syk in Dectin-1-mediated phagocytosis is cell type-
dependent: Syk is essential for phagocytosis in dendritic cells, but
not in macrophages [3,4]. Other proteins that interact with
Dectin-1 are PKCd , the tetraspanin CD37 , Galectin-3 
and TLR2 . Ectopic expression of Dectin-1 in fibroblasts or
kidney cells confers phagocytic capacity to these cells [3,7]. Dectin-
1 is thus a bona fide phagocytic receptor, but the detailed
mechanisms that underlie Dectin1-mediated phagocytosis are not
Actin drives phagocytosis: formation of the phagocytic cup
depends on the formation of F-actin, and closure of the
phagosome requires the reversal of actin polymerization . In
the course of FccR-mediated phagocytosis -the best-understood
model of phagocytosis-, several phosphoinositides (PI) are formed
in the phagosomal membrane, which serve as docking stations for
proteins with PI-specific interaction domains. Phosphatidylinositol
4,5-bisphosphate (PI(4,5)P2) is ubiquitously present in the plasma
membrane and is transiently enriched in phagocytic cups . As
the cup forms, phosphatidylinositol 3-kinase (PI3K) converts
PI(4,5)P2to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)
which in turn can be converted to PI(3,4)P2by the SH2-containing
PLOS Pathogens | www.plospathogens.org 1 June 2013 | Volume 9 | Issue 6 | e1003446
inositol 59-phosphatase (SHIP). Proteins with a Pleckstrin Homol-
ogy (PH) domain can bind to PI(4,5)P2, PI(3,4,5)P3or PI(3,4)P2,
allowing their recruitment to the maturing phagosome. In
addition, phospholipase C (PLCc) acts on PI(4,5)P2to yield the
second messengers IP3and diacylglycerol (DAG). IP3triggers an
increase in cytoplasmic Ca2+concentration, while DAG serves as a
docking site for proteins with a C1 domain. The quantity, timing
and localization of PI(3,4,5)P3, PI(3,4)P2and DAG formation vary,
depending on the phagocytic receptor and the identity of the
particle engaged. Formation of PI(3,4,5)P3/PI(3,4)P2and locali-
zation of actin to the phagocytic cup are also known to occur
during phagocytosis of C. albicans by macrophages [11,12] but
downstream pathways engaged by the PI during Dectin-1
phagocytosis remain to be studied in detail.
Here we describe two new interactors of Dectin-1: Bruton’s
Tyrosine Kinase (BTK) and the guanine nucleotide exchange
factor Vav1. We provide evidence that these proteins bind to
PI(3,4,5)P3-rich membranes and that BTK is involved in the
production of DAG during C. albicans phagocytosis. BTK and
Vav1-deficient macrophages show reduced rates of phagocytosis
and BTK and Vav1-deficient mice succumb more readily to C.
albicans systemic infections than wild type mice.
b-glucan exposure on Candida albicans yeasts and
To facilitate imaging of phagocytosis, we applied two new
imaging tools. First, we used a Candida strain that expresses a
variant of blue fluorescent protein (Candida-BFP). Second, to study
b-glucan exposure on C. albicans yeasts and hyphae, we site-
specifically fluorescently labeled the extracellular carbohydrate
recognition domain of Dectin-1 (Dectin1-CRD-Alexa647) using
the bacterial enzyme sortase . Candida-BFP was incubated in
DMEM with 10% serum for 15, 30, 90 and 180 min followed by
staining with Dectin1-CRD-Alexa647 (Figure 1A). At 15 and
30 min we observed moderately stained C. albicans yeasts with
increased staining of bud scars (white arrows), congruent with
Figure 1. BTK and Vav1 interact with Dectin-1 during C. albicans phagocytosis by macrophages. (A): Morphology and b-glucan exposure
of C. albicans expressing blue fluorescent protein (Candida-BFP) at indicated time points after incubation in DMEM with 10% IFS. Candida-BFP was
stained with fluorescent carbohydrate recognition domain of Dectin-1 (Dectin1-CRD-Alexa647) that binds b-glucan. Arrows indicate increases
staining at bud scars. (B): Immunoblotting experiment showing expression of different proteins in RAW-Dectin1 cells during co-incubation with live C.
albicans for the indicated time points. Phagocytosis of C. albicans occurs throughout the time course, but the morphology of the ingested particles
changes over time. (C): Co-incubation of RAW-Dectin1 with C. albicans followed by co-immunoprecipitation with anti-BTK or anti-Vav1 antibody and
immunoblotting with anti-HA to detect Dectin-1. BTK/Dectin-1 and Vav1/Dectin-1 complexes were detected at different time points during the co-
incubation. (D): Quantification of BTK/Dectin-1 and Vav1/Dectin-1 complexes showing strongest interactions at the 90- and 180-minute time points,
respectively. Means +/2 SD of three independent experiments are shown.
The opportunistic yeast Candida albicans is a commensal
organism of the human digestive tract, but also the most
common cause of human fungal infections. Phagocytosis,
the process by which innate immune cells engulf
pathogens, is vital to prevent C. albicans infections. The
major phagocytic receptor involved in anti-fungal immu-
nity is Dectin-1. We identify two new interacting proteins
of Dectin-1 in macrophages: Bruton’s Tyrosine Kinase (BTK)
and Vav1. In the course of phagocytosis, different
phosphoinositides (PIs) are formed in the phagosomal
membrane to allow the recruitment of proteins equipped
with specialized lipid-interaction domains. We show that
BTK and Vav1 colocalize with the lipid PI(3,4,5)P3at the
phagocytic cup, but not with diacylglycerol (DAG), which
marks more mature phagosomal membranes. Inhibition of
BTK affects the production of DAG and the recruitment of
DAG-interacting proteins. BTK and Vav1 are essential for C.
albicans immune responses, as BTK- or Vav1-deficient
macrophages show reduced uptake of C. albicans and BTK-
or Vav1-deficient deficient mice are more susceptible to
systemic C. albicans infection. This work identifies an
important role for BTK and Vav1 in immune responses
against C. albicans.
BTK and Vav1 as New Interactors of Dectin-1
PLOS Pathogens | www.plospathogens.org 2 June 2013 | Volume 9 | Issue 6 | e1003446
increased b-glucan exposure at these sites . At 90 and
180 min, formation of hyphae was extensive with strong,
homogeneous b-glucan exposure. Under these growth conditions,
when compared to C. albicans yeast, C. albicans hyphae thus expose
higher levels of b-glucan, which is expected to affect signaling
Identification of Dectin1-interacting proteins
To identify proteins that interact with Dectin-1 during
phagocytosis of live C. albicans, we performed immuno-isolation
experiments using the RAW-Dectin1 macrophage cell line
(Esteban et al., 2011). The macrophages were co-incubated with
live C. albicans for one hour, a time point that marks early hyphal
formation and increased b-glucan exposure. Cells were lysed and
Dectin-1, together with its interacting partners, was immunopre-
cipitated using anti-HA antibodies. Proteins present in these
samples were analyzed and identified by SDS-PAGE, followed by
LC-MS-MS. From the list of proteins, we selected two that were
retrieved in complex with Dectin-1 in the samples with C. albicans,
but that were absent from the control sample. These proteins were
Bruton’s Tyrosine Kinase (BTK) and the guanine nucleotide
exchange factor Vav1.
To investigate the expression of BTK, Vav1 and other proteins
already known to be involved in Dectin1-mediated phagocytosis,
we incubated RAW-Dectin1 macrophages with live C. albicans.
Phagocytosis continues throughout the period of coincubation, but
C. albicans morphology changes from yeast form to hyphal form
over time. Total cell lysates from the different time points were
analyzed by immunoblot. Spleen tyrosine kinase (Syk), a known
interactor of Dectin-1, was present at constant levels at all-time
points (Figure 1B). Phosphorylation of Syk increased at 15 min
and then waned. BTK and Vav1 were likewise present at constant
levels. PLCc2, a key enzyme in phagocytosis, was present at
constant levels throughout the time course; its phosphorylation (at
Y1217) was most pronounced around 90 and 120 min.
Next we confirmed the interaction between Dectin-1 and BTK,
and between Dectin-1 and Vav1. Immunoprecipitation with
polyclonal anti-BTK antibody, followed by immunoblotting with
anti-HA, showed an increased interaction between BTK and
Dectin-1 starting at 15 min,
(Figure 1C). The interaction between Vav1 and Dectin-1 is
strongest at the later time points (Figure 1C). Quantification of
the Dectin-1/BTK and Dectin-1/Vav1 interactions using ImageJ
software showed that the level of the Dectin-1/BTK complex
peaks at 60–90 min, while the strongest Dectin-1/Vav1 interac-
tion was observed at 180 min (Figure 1D). We conclude that
BTK and Vav1 are expressed at constant levels, and that their
interactions with Dectin-1 are strongest at the later time points
when C. albicans has formed hyphae.
Confirmation of BTK and Vav1 localization to the
To study the subcellular localization of BTK, Vav1 and Syk, we
constructed stable RAW macrophage cell lines that express these
proteins as N- or C-terminal mCherry fusions in the RAW-
Dectin1 background. In unstimulated cells, BTK-mCherry and
mCherry-Vav1 were cytosolic, whereas mCherry-Syk localized to
both the cytosol and nucleus (Figure 2, top). Next the distribution
of the mCherry-tagged proteins was investigated after coincuba-
tion with Candida-BFP for 30, 90 or 180 min. At 30 min, when C.
albicans yeast-form cells are present, BTK-mCherry, mCherry-
Vav1 and mCherry-Syk showed a clear localization to the Candida-
BFP-containing phagocytic cup (Figure 2A, arrows). After 90 and
180 min, when C. albicans had formed extensive hyphae, BTK-
mCherry and mCherry-Vav1 showed enrichment in a cuff region
of the phagocytic cup when engulfing Candida-BFP hyphae
(Figure 2A, arrows). mCherry-Syk was more evenly distributed
along the phagosomal membrane, with additional foci of
enrichment outside the cuff region. There was no enrichment of
BTK-mCherry or mCherry-Vav1 in membranes of closed, more
mature, phagosomes. N- or C-terminal mCherry fusions with
Figure 2. Localization of BTK-mCherry and mCherry-Vav1 to
the Candida-containing phagocytic cup. (A): Confocal images
showing localization of BTK-mCherry, mCherry-Vav1 and mCherry-Syk
in RAW-Dectin1 macrophages incubated with Candida-BFP. Images
were taken without Candida-BFP or after 30 minutes, 90 minutes and
180 minutes of co-incubation with Candida-BFP to study phagocytosis
of yeast, hyphae, and very long hyphae, respectively. White arrows
indicate areas of mCherry recruitment to the Candida-containing
phagocytic cup, visible during ingestion of C. albicans yeast (30 min-
utes) and C. albicans hyphae (90 and 180 minutes). (B): XYZ images of C.
albicans phagocytosis by the BTK-mCherry, mCherry-Vav1 and mCherry-
Syk cell lines. Cuff regions of protein recruitment are circular bands
around C. albicans hyphae that are being ingested. (C): Quantitation of
BTK-mCherry, mCherry-Vav1 and mCherry-Syk recruitment to the
phagocytic cup after 90 minutes of coincubation with Candida-BFP.
(D): Model showing localization of BTK and Vav1 to the phagocytic cup
but not to mature phagosomes during phagocytosis of C. albicans yeast
and hyphae by macrophages. Representative micrographs and means
+/2 SD of 3 independent experiments are shown. For statistical
analysis, all data were analyzed by unpaired t test.
BTK and Vav1 as New Interactors of Dectin-1
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vimentin, expressed as controls in the same RAW macrophage cell
line, did not show recruitment to the phagocytic cup (data not
shown). 3D reconstructions of RAW macrophages in the process
of ingesting C. albicans hyphae showed that the cuff regions of
BTK/Vav1/Syk recruitment are cylindrical sleeves surrounding
the phagosomal membrane (Figure 2B). Recruitment of BTK-
mCherry, mCherry-Vav1 and mCherry-Syk to the phagocytic cup
was quantified by comparing the fluorescence intensity in the cup/
phagosomal membrane to that of the cytosol. Recruitment of
mCherry-Syk was strongest, followed by mCherry-Vav1 and
BTK-mCherry (Figure 2C).
Is recruitment of BTK and Vav1 to the phagocytic cup
dependent on Dectin-1? We incubated the RAW mCherry cell
lines with b-glucan-coated beads or with zymosan, the phagocy-
tosis of which is Dectin1-dependent . All three fusion proteins
localized to phagosomes containing b-glucan-coated beads or
zymosan. Recruitment of BTK, Vav1 and Syk therefore indeed
relies on Dectin-1 (Figure S1A and B). To address the difference
in geometry of yeast versus hyphal particles, we incubated the
RAW mCherry cell lines with UV-killed C. albicans yeasts or
hyphae. While the shape of the phagocytic cup differs, recruitment
of BTK, Vav1 and Syk occurs in all cases (Figure S1C and D).
Regardless of the geometry of the ingested particle, BTK and
Vav1 are recruited to the phagocytic cup but not to the mature
phagosome during Dectin1-mediated phagocytosis (Figure 2D).
Immunofluorescence was performed to investigate the localization
of Dectin1 during phagocytosis of C. albicans hyphae. Dectin1 was
enriched in the cuff region of the phagocytic cup to which BTK/
Vav1/Syk were recruited, but also showed areas of enrichment
outside the cuff region (Figure 3). Phagosomes that contain
completely internalized C. albicans yeasts or hyphae showed very
little Dectin1 staining.
Phosphoinositide metabolism during C. albicans
Different PIs are present in the phagosomal membrane when
the phagocytic cup forms, as well as during phagosomal
maturation. To visualize membrane PI composition, PI-binding
protein domains fused with fluorescent proteins have been used as
imaging tools (biosensors or probes) . We transfected the
RAW-Dectin1 cell line with biosensors for PI(4,5)P2(PH-PLCd-
GFP), PI(3,4,5)P3(PH-BTK-GFP), PI(3,4,5)P3/PI(3,4)P2(PH-Akt-
RFP) and DAG (C1-PKCd-GFP) to study their formation during
C. albicans phagocytosis.
PI(4,5)P2 was present in the plasma membrane at rest, but
multiple regions of enrichment were observed at 30 and 90 min at
sites where macrophages contacted Candida-BFP yeast or hyphae
(Figure S2). Membranes of sealed phagosomes no longer showed
PI(4,5)P2enrichment, consistent with the reported localization of
PI(4,5)P2during FccR-medicated phagocytosis . PI(3,4,5)P3as
visualized by the BTK-PH domain showed enrichment in some,
but not all, of the PI(4,5)P2-rich regions (Figure S2, arrows).
Next we investigated the localization of PI(3,4,5)P3/PI(3,4)P2
and DAG. High levels of both PIs were present in cups or
phagosomes containing C. albicans. PI(3,4,5)P3/PI(3,4)P2and DAG
colocalized in some early phagosomes, but phagosomes containing
only PI(3,4,5)P3/PI(3,4)P2 or only DAG were also present
(Figure 4A, white and green arrows). Recruitment of PH-Akt-
RFP (PI(3,4,5)P3/PI(3,4)P2) and C1-PKCd-GFP (DAG) or both to
Candida-containing cups and phagosomes was quantified at the 30-
min time point (Figure 4B). More than 20% of cups/phagosomes
were PI(3,4,5)P3/PI(3,4)P2-positive and a comparable percentage
was positive for both PI(3,4,5)P3/PI(3,4)P2and DAG. At this stage
4% of cups/phagosomes were positive for DAG only. PI(3,4,5)P3/
PI(3,4)P2were more prominent in phagocytic cups, while DAG
was more abundant in sealed phagosomes. These results indicate
that during Dectin1-mediated phagocytosis of b-glucan-exposing
C. albicans yeast or hyphae, PI(3,4,5)P3/PI(3,4)P2are formed early
during initiation of phagocytosis and that DAG-rich membranes/
phagosomes appear at a more advanced stage (Figure 4C).
The presence of different PIs allows the sequential docking of a
specialized set of effector proteins to the membrane during
phagosomal maturation. DAG is a potent second messenger,
activating members of the Protein Kinase C (PKC) family. We
transfected the RAW-Dectin1 cell line with GFP-tagged PKCa,
PKCb1, PKCd, PKCe or PKCf to determine their localization
during C. albicans phagocytosis. They all localized to the C. albicans-
containing phagocytic cup (Figure 4D), albeit to varying degrees.
The recruitment of PKCd and PKCe was most pronounced, while
the recruitment of PKCa, PKCb and PKCf was moderate
Colocalization of BTK and Vav1 with PI(3,4,5)P but not
BTK and Vav1 both contain a Pleckstrin Homology (PH)
domain that can bind to PI(3,4,5)P3. Vav1 also contains a
putative DAG-binding C1 domain, incapable of binding DAG
owing to the presence of hydrophilic and non-charged residues in
key binding positions . The Syk polypeptide does not contain
predicted PH or C1 domains and presumably localizes to the
phagocytic cup through interaction of its tandem SH2 domains
with the phosphorylated ITAM-like motif of Dectin-1. We
investigated the possible colocalization of BTK, Vav1 and Syk
with PI(3,4,5)P3and/or DAG during phagocytosis of C. albicans.
The BTK-mCherry, mCherry-Vav1 and mCherry-Syk RAW cell
lines were transfected with the biosensor construct PH-BTK-GFP
to visualize formation of PI(3,4,5)P3 and then incubated with
Candida-BFP. The BTK-mCherry and mCherry-Vav1 fusion
proteins colocalized with PI(3,4,5)P3 at the phagocytic cup,
mCherry-Syk was more diffuse, confirming PI(3,4,5)P3-indepen-
dent recruitment of Syk (Figure 5C, arrows). Next, we examined
colocalization of BTK, Vav1 and Syk with DAG using the C1-
PLCd-GFP biosensor. BTK-mCherry showed some colocaliza-
5A,B),while recruitment of
Figure 3. Localization of Dectin-1 during phagocytosis of C.
albicans hyphae. Confocal images showing localization of Dectin-1
during phagocytosis of C. albicans hyphae by the BTK-mCherry,
mCherry-Vav1 and mCherry-Syk RAW macrophage cell lines. Dectin1-
HA was visualized by anti-HA immunofluorescence.
BTK and Vav1 as New Interactors of Dectin-1
PLOS Pathogens | www.plospathogens.org4 June 2013 | Volume 9 | Issue 6 | e1003446
tion with DAG at 30 min. After 90 min, BTK-mCherry and
DAG clearly localized to different regions of the phagocytic cup
(Figure 5D, arrows). mCherry-Vav1 and mCherry-Syk showed
a similar distribution, and neither colocalized with DAG
(Figure 5E,F). BTK and Vav1 thus bind to PI(3,4,5)P3and
not to DAG during phagocytosis of C. albicans, consistent with the
presence of PH domains in both proteins and a non-DAG
binding C1 domain in Vav1. The colocalization of BTK and
Vav1 with PI(3,4,5)P3suggests a role for these proteins at an early
stage of phagocytosis, as PI(3,4,5)P3marks cups and immature
phagosomes (see above).
BTK and Vav1 localize to areas of F-actin formation at the
Rearrangement of the actin cytoskeleton drives phagocytosis,
enabling engulfment of the fungal particle by the macrophage. We
investigated cytoskeletal changes during Candida phagocytosis by
electron microscopy using fixation with tannic acid, a method that
preserves actin structures . RAW-Dectin1 macrophages were
incubated with C. albicans for 30 min. Areas of decreased staining
intensity were observed surrounding the C. albicans-containing
phagocytic cups, indicative of actin polymerization (Figure 6A).
Figure 4. Localization of phospholipids and PKC family proteins during C. albicans phagocytosis. (A): PH-Akt-RFP and C1-PKCd-GFP
biosensors showing localization of PI(3,4,5)P3/PI(3,4)P2and DAG, respectively, without challenge or after 30 or 90 minutes of co-incubation with
Candida-BFP. White arrows indicate areas of PI(3,4,5)P3/PI(3,4)P2and DAG co-localization, while red and green arrows indicate areas of speciation. (B):
Quantitation of PI(3,4,5)P3/PI(3,4)P2- and DAG-positive phagosomes after 30 minutes of coincubation with Candida-BFP. (C): Model showing
localization of PI(3,4,5)P3/PI(3,4)P2and DAG during engagement and internalization of C. albicans yeast and hyphae by macrophages. (D): Localization
of GFP-tagged PKCa, PKCb, PKCd, PKCe and PKCf after 30 or 90 minutes of Candida-BFP phagocytosis. (E): Quantitation of PKCa-GFP, PKCb-GFP,
PKCd-GFP, PKCe-GFP and PKCf-GFP recruitment to the phagocytic cup after 90 minutes of coincubation with Candida-BFP. Representative
micrographs and means +/2 SD of 3 independent experiments are shown. For statistical analysis, all data were analyzed by unpaired t test.
BTK and Vav1 as New Interactors of Dectin-1
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These cuffs had a smooth appearance, were of uniform thickness
across the sections examined and were distinct from the cytosol,
which was more granular in appearance. We also examined actin
polymerization with the biosensor LifeAct, which reports on the
distributionoffilamentous(F-) actin,incombination with biosensors
that bind to PI(3,4,5)P3and DAG. F-actin formation was detectable
at the phagocytic cup of Candida-BFP yeast and hyphae at 30 and
90 min. There is a clear separation of DAG-rich regions of the
phagosome from the F-actin rich membranes (Figure 6B). This
suggests regional membrane specializations, with different func-
tionalities and different peripheral proteins associated with them. F-
actin showed perfect colocalization with PI(3,4,5)P3at the Candida-
BFP containing phagocytic cup (Figure 6C). Also, BTK-mCherry,
mCherry-Vav1 and mCherry-Syk colocalize with F-actin after 30
and 90 min of C. albicans phagocytosis (Figure 6D). We conclude
that PI(3,4,5)P3-rich areas are formed in the course of Dectin1-
mediated phagocytosis of C. albicans and that BTK and Vav1 are
recruited to these areas, with ensuing formation of F-actin.
Inhibition of BTK affects phagocytosis and production of
Having established interactions of BTK and Vav1 with Dectin-
1 in the course of phagocytosis and their recruitment to
PI(3,4,5)P3-rich membranes, we next investigated the functional
importance of these proteins in phagocytosis of C. albicans. We
synthesized the highly selective irreversible BTK inhibitor PCI-
32765 . The IC50of this BTK inhibitor for BTK, Tec kinase
and Syk is 0.5, 78 and .10,000 nM, respectively, corresponding
to a BTK selectivity of 156 fold (Tec) and .10,000 fold (Syk) .
In B cells, PCI-32765 irreversibly inhibited autophosphorylation of
BTK (IC50: 11 nM), phosphorylation of BTK’s physiological
substrate PLCc (IC50: 29 nM), and phosphorylation of down-
stream kinase ERK (IC50: 13 nM) . RAW-Dectin1 macro-
phages were pre-incubated with different concentrations of BTK
inhibitor (starting from 50 nM), followed by coincubation with
Candida-BFP for 1 hour and staining with fluorescently labeled
Concanavalin A to distinguish accessible (extracellular) particles
from internalized Candida-BPF particles. Preincubation of RAW-
Dectin1 macrophages with 50 nM PCI-32765 reduced uptake of
C. albicans by 30% and increasing inhibitor concentrations
progressively blocked phagocytosis (Figure 7A). These results
indicate an important role for BTK during C. albicans phagocy-
PLCc, which converts PI(4,5)P2into DAG, is a key enzyme
during phagocytosis by innate immune cells and can be regulated
by BTK [18,19]. We hypothesized that during phagocytosis of C.
Figure 5. BTK-mCherry and mCherry-Vav1 colocalize with PI(3,4,5)P3but not with DAG. (A): Colocalization of BTK-mCherry and PH-BTK-
GFP that binds to PI(3,4,5)P3at 30 and 90 minutes of coincubation with Candida-BFP showing phagocytosis of yeast and hyphae, respectively. (B):
Colocalization of mCherry-Vav1 and PH-BTK-GFP at 30 and 90 minutes of coincubation with Candida-BFP. (C): Localization of mCherry-Syk and PH-
BTK-GFP at 30 and 90 minutes of coincubation with Candida-BFP. (D): Localization of BTK-mCherry and C1-PKCd-GFP at 30 and 90 minutes of
coincubation with Candida-BFP. (E): Localization of mCherry-Vav1 and C1-PKCd-GFP at 30 and 90 minutes of coincubation with Candida-BFP. (F):
Localization of mCherry-Syk and C1-PKCd-GFP at 30 and 90 minutes of coincubation with Candida-BFP. White arrows indicate areas of co-localization,
while red and green arrows indicate areas of speciation. Experiments were performed at least three times, representative micrographs are shown.
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albicans, early localization of BTK to the phagocytic cup activates
PLCc to increase synthesis of DAG and so enables recruitment of
PKC members to the phagosomal membrane. To address this
possibility, RAW-Dectin1 macrophages were incubated with BTK
inhibitor or the PLC inhibitor U73112, followed by coincubation
with C. albicans for 1 hour. Total DAG levels in these macrophages
were measured by lipid extraction, DAG kinase assays and
quantification of the product, phosphatidic acid, by thin layer
chromatography. Addition of the PLC inhibitor U73112 reduced
total DAG levels by 50% while the BTK inhibitor did not
significantly affect total DAG levels (Figure 7B and C). However,
in samples without inhibitor we observed a small increase in DAG
levels in the presence of C. albicans, possibly due to increased
production of DAG at the phagocytic cup during phagocytosis. To
examine the effect of the BTK inhibitor on local production of
DAG at the phagocytic cup, we performed confocal microscopy
with the C1-PLCd-GFP biosensor and the PKCe-GFP construct
in the absence and presence of the BTK inhibitor. Addition of the
inhibitor strongly reduced recruitment of the C1-PLCd-GFP
biosensor and the PKCe-GFP construct to the phagocytic cup
(Figure 7D and E). These results underscore the importance of
investigating local changes in lipid composition as opposed to
changes in total levels. BTK is thus involved in the production of
DAG and the subsequent recruitment of PKC family proteins at
the phagocytic cup (Figure 7F). The interactions of BTK and
Vav1 with Dectin-1, their recruitment to PI(3,4,5)P3-rich mem-
branes, the possible contribution of Vav1/BTK to actin
rearrangements and the role of BTK in the production of DAG
are summarized in Figure 7G.
btk2/2 and vav12/2 macrophages display reduced
To further investigate the contributions of BTK and Vav1 to
phagocytosis we determined the phagocytic capacity of peritoneal
macrophages and bone marrow-derived macrophages (BMDMs)
from wild type mice and from dectin-1, btk and vav1 knockout mice.
Phagocytic indices were determined after incubation of peritoneal
macrophages with zymosan for 30 min or with C. albicans for
30 min or 1 hour. While Dectin1-deficient peritoneal macrophag-
es did not phagocytose zymosan, the btk and vav1 deficient cells
showed an intermediate phenotype (Figure 8A). Uptake of C.
albicans by btk and vav1 knockout peritoneal macrophages at the
30-min time point was also significantly reduced compared to wild
type cells (p,0.05). In addition, the btk2/2 and vav12/2
BMDMs displayed a reduction in phagocytosis similar to that seen
for the dectin12/2 BMDMs (p,0.05) (Figure 8A). BTK and
Vav1 are thus important contributors to Dectin1-mediated uptake
of C. albicans by BMDMs.
btk2/2 and vav12/2 mice show increased sensitivity to
C. albicans infections
In vivo immune responses of wild type and dectin12/2, btk2/2
and vav12/2 mice to C. albicans were tested in a systemic
candidiasis model. Tail vein injection of the four groups of mice
with C. albicans showed that dectin12/2 mice were most
susceptible to C. albicans infections, while the majority of the wild
type animals survived the systemic infection. Btk2/2 and vav12/
2 animals displayed an intermediate phenotype, both during
systemic infection with 56104colony forming units (CFU)
(Figure 8B) and with 16105colony forming units (CFU) of C.
albicans (data not shown). We found no statistical difference in C.
albicans loads in kidneys harvested from mice about to succumb to
infection (Figure 8C). In addition, histological analysis of the
kidneys showed similar fungal loads (Figure 8D) and comparable
immune cell invasion of the tissues in the different groups
(Figure 8E). Elevated chemokine and cytokine levels in the
kidney represent early responses to C. albicans infection and
correlate with virulence . We determined secretion of the
proinflammatory cytokines TNFa and IL-6. Wild type, dectin12/
Figure 6. BTK, Vav1 and Syk colocalize with F-actin and
PI(3,4,5)P3. (A): Electron microscopy of C. albicans phagocytosis by
RAW-Dectin1 macrophages. Cells were fixed to visualize polymerized
actin. (B): Localization of LifeAct-RFP detecting F-actin and C1-PKCd-GFP
that binds to DAG after 30 and 90 minutes of coincubation with
Candida-BFP. (C): Colocalization of LifeAct-RFP detecting F-actin and
PH-BTK-GFP that binds to PI(3,4,5)P3 after 30 and 90 minutes of
coincubation with Candida-BFP. (D): Localization of BTK-mCherry,
mCherry-Vav1 and Syk-mCherry with LifeAct-GFP after 90 minutes of
coincubation with Candida-BFP. White arrows indicate areas of co-
localization, while red and green arrows indicate areas of speciation.
Experiments were performed multiple times, representative micro-
graphs are shown.
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2, btk2/2 and vav12/2 peritoneal macrophages secreted both
TNFa and IL-6 (Figure 8F,G) in response to exposure to C.
albicans. Btk2/2 and vav12/2 macrophages generally secreted
more TNFa and IL-6 than wild type and dectin12/2 macro-
phages (the difference between wild type and btk2/2 TNFa
secretion reached statistical significance). Next we investigated
TNFa and IL-6 levels in mouse kidney during systemic C. albicans
infection. Kidneys were harvested 11 days after tail vein injection
with 56104CFU. Although levels of TNFa and IL-6 were slightly
higher in the dectin12/2, btk2/2 and vav12/2 mice than in wild
type, these differences did not reach statistical significance
(Figure 8H,I). We conclude that disease progression in response
to C. albicans systemic infection is accelerated in dectin12/2, btk2/
2 and vav12/2 animals compared to wild type animals.
Figure 7. BTK is involved in DAG production at the phagocytic cup. (A): RAW-Dectin1 macrophages were pre-incubated with the indicated
concentrations of BTK inhibitor PCI-32765 followed by coincubation with Candida-BFP (MOI 10) for 1 hour. Graphs represent number of internalized
Candida-BFP per macrophage as determined by microscopy. (B): DAG measurements in RAW-Dectin1 macrophages preicubated with the indicated
inhibitors and in the presence or absense of C. albicans. Thin layer chromatography was performed to visualize the phosphatidic acid (PA) product of
the DAG kinase assay. (C): Quantification of PA signal from three independent DAG kinase experiments. (D): Confocal images of C1-PKCd-GFP (DAG)
and PKCe-GFP distribution in RAW-Dectin1 macrophages pre-incubated with 0.5 mM BTK inhibitor PCI-32765. (E): Quantification of C1-PKCd-GFP and
PKCe-GFP recruitment to phagosomes in absence and presence of 0.5 mM BTK inhibitor. All graphs display means +/2 SD of three independent
experiments. (F): Schematic showing localization of PI(3,4,5)P3, BTK, Vav1, DAG and PKC family proteins during engagement and internalization of C.
albicans yeast and hyphae by macrophages. (G): Model summarizing this studies findings. BTK, Vav1 and Syk interact with Dectin-1 during
phagocytosis of C. albicans (left). Phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) can be converted to phosphatidylinositol 3,4,5-triphosphate
(PI(3,4,5)P3) by PI3K or to diacylglycerol (DAG) by phospholipase C c (PLCc). Specialized PI(3,4,5)P3- and DAG-rich phagosomal membranes can be
distinguished during C. albicans phagocytosis. Bruton’s Tyrosine Kinase (BTK) and Vav1 localize to PI(3,4,5)P3-rich membrane regions and colocalize
with F-actin. Vav1 might play an active role in actin rearrangements at the phagocytic cup through activation of small GTPases Rac1, Cdc42 and/or
Rho1. BTK is involved in the production of DAG at the phagocytic cup, possibly through the activation of PLCc. Protein Kinase C (PKC) family proteins
localize to DAG-rich membranes. For statistical analysis, all data were analyzed by unpaired t test.
BTK and Vav1 as New Interactors of Dectin-1
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BTK and Vav1 are best known for their role in adaptive
immunity: BCR signaling in B cells (BTK) and B and T cell
development as well as activation of mature lymphocytes (Vav).
Vav family members orchestrate cytoskeletal rearrangements, with
Vav1 being expressed in the hematopoietic system in particular
. BTK and Vav also participate in innate immune reactions.
Figure 8. Phenotypic analysis of BTK- and Vav1-deficient macrophages and mice. (A): Peritoneal macrophages or bone marrow-derived
macrophages (BMDM) from wild type, dectin12/2, btk2/2 or vav12/2 mice were incubated with zymosan-Alexa647 or live Candida-BFP for
30 minutes or 1 hour at an MOI of 10 and the number of internalized Candida-BFP was determined by microscopy. Graphs represent means and
standard deviations of experiments with three different mice. (B): The contribution of BTK and Vav1 to overall immune responses to C. albicans was
determined using the model for systemic candidiasis. Tail vein injection of wild type, dectin12/2, btk2/2 or vav12/2 mice were performed with
0.56104colony forming units (CFU) of C. albicans and disease was monitored over time. (C): C. albicans CFU in kidneys of indicated mice at final stage
of disease, means +/2 SD are indicated. (D): GMS staining of kidney histology slides of wild type, dectin12/2, btk2/2 or vav12/2 mice, at final stage
of disease showing extensive fungal invasion of tissues. (E): H&E staining of kidney histology slides of wild type, dectin12/2, btk2/2 or vav12/2
mice, at final stage of disease showing extensive immune cell invasion of tissues. Representative images are shown. TNFa (F) and IL-6 (G) levels in
supernatant of mouse peritoneal macrophages 12 hours after incubation without or with C. albicans. Graphs represent means and standard
deviations of experiments with three different mice. TNFa (H) and IL-6 (I) levels in kidney lysates of mice infected with 56104CFU C. albicans at 11
days after infection. Each dot represents one mouse; means and standard deviations are indicated. Values did not differ significantly. For statistical
analysis, all data were analyzed by unpaired t test.
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BTK contributes to Fc-mediated phagocytosis  and was
previously implicated in Dectin1-dependent pathways [23,24],
while the Vav protein family participates in Dectin-1/Mac-1
signaling in neutrophils . We identified BTK and Vav1 as
novel interaction partners of the b-glucan receptor Dectin-1 and
confirmed their importance during C. albicans phagocytosis and
immune responses during systemic infection with C. albicans.
Dectin-1/BTK and Dectin-1/Vav1 complexes form during
phagocytosis of live C. albicans, particularly during ingestion of C.
albicans hyphae (Figure 1 and 2). BTK and Vav1 are found at
membranes enriched for PI(3,4,5)P3and colocalize with markers
for F-actin (Figures 5 and 6) where BTK is involved in
production of DAG at the phagocytic cup (Figure 7). Macro-
phages deficient in BTK or Vav1 display reduced phagocytosis
and BTK- or Vav1-deficient mice succumb more readily to
systemic C. albicans infections than do wild type animals (Figure 8).
BTK and Vav1 can now be added to the list of Dectin1-
interacting proteins, which includes Syk , PKCd , the
tetraspanin CD37 , Galectin-3  and TLR2 . The multiple
interactions of Dectin-1 with its partners reflect the complexity of
Dectin-1 signaling and the (sub)complexes in which it participates.
Under non-phagocytic conditions, the receptor remains at the
plasma membrane, but engagement by a b-glucan ligand initiates
phagocytosis and signaling from the nascent phagosome. With
multiple Dectin1-interacting proteins identified, the interesting
possibility of tripartite or multicomponent signalosomes arises.
Dectin-1 multicomponent signalosomes exist, as complexes were
found that encompassed PKCd, Syk and Dectin-1 . We tested
the possibility of a Dectin1/BTK/Vav1 tripartite complex, but
could not detect the three proteins in a single complex (data not
shown). However, Vav1 could be a target of BTK phosphoryla-
tion, as the SH3 domain of BTK interacts with Vav1 in B cells
. The signaling events that occur after engagement of Dectin-1
and the relationships between BTK, Vav1 and the known Dectin-
1 mediator Syk remain to be clarified. Although interaction and
signaling data cannot be extrapolated to different cell types,
relevant information can be extracted from the literature. Syk and
Vav interact in yeast-two-hybrid experiments and in B and T cells,
where Syk directly phosphorylates Vav .
BTK and Vav1 localize to the Candida-containing phagocytic
cup, both when C. albicans yeasts and hyphae are internalized
(Figure 2). During phagocytosis of hyphae, BTK-mCherry and
mCherry-Vav1 show strong recruitment to a ‘‘cuff’’ region where
engulfment of the hyphae is ongoing. C. albicans hyphae grown
under these conditions expose high levels of b-glucan (Figure 1).
The strong RAW-Dectin1 engagement in the cuff regions and the
ensuing recruitment of BTK and Vav1 could therefore be a result
of increased exposure of b-glucan on the C. albicans hyphae under
these conditions. The exposure of b-glucan on C. albicans yeast and
hyphae is a matter of ongoing debate.
Our data show that hyphae generated by growth in DMEM
media with serum at 37uC for 90–180 min display high levels of b-
glucan (Figure 1A), which is also the case during disseminated
infection . However, it was also reported that Dectin1 does not
bind to C. albicans hyphae generated by overnight growth at 37uC
in serum-free RPMI media , producing hyphae that appear
morphologically distinct. Different growth conditions may well
produce differences in b-glucan exposure and yield distinct hyphal
Phosphoinositides (PIs) formed in the phagosomal membrane
serve as docking stations for proteins with the appropriate binding
domains. During phagocytosis of C. albicans, PI(4,5)P2is enriched
at sites of contact, followed by production of PI(3,4,5)P3at the
phagocytic cup and disappearance of PI(4,5)P2 and DAG-
enrichment as the phagosomes seal (Figures S2 and Figure 3).
BTK and Vav1 colocalize with PI(3,4,5)P3(Figure 5), which
suggests a role for these proteins at an early stage of phagocytosis.
F-actin also colocalizes with BTK and Vav1 in the PI(3,4,5)P3-rich
areas, which fits the known role of Vav1 in actin cytoskeleton
rearrangement and suggests a possible contribution of BTK to F-
actin formation (Figure 6). The observation that Vav1 colocalizes
with PI(3,4,5)P3but not with DAG supports the notion that the C1
domain of Vav1 is not a functional DAG-binding domain .
During CR3- and FccR-mediated phagocytosis, actin tail forma-
tion follows local production of PI(3,4,5)P3 . BTK also
localizes to actin-rich cups during FccR-mediated phagocytosis
. Our data and those in the literature emphasize similarities
between Dectin1- and FccR-mediated phagocytosis.
The enzyme PLCc converts PI(4,5)P2to DAG at the phagocytic
cup and the PLC inhibitor U73112 blocked C. albicans phagocy-
tosis by the RAW-Dectin1 macrophages (data not shown). PLCc is
also essential for FccR-mediated phagocytosis . DAG-rich
membranes recruit proteins of the PKC family that have a DAG-
interacting C1 domain [31,32]. All PKC isoforms examined
localized to the Candida-containing phagocytic cup, with the Ca2+-
independent family members PKCd and PKCe displaying the
strongest recruitment (Figure 3). Conventional PKCs require
increased intracellular Ca2+levels for activation and binding to
DAG  but DAG-independent recruitment of the different
PKC isoforms, for example via protein-protein interactions,
remains possible as well. PKCd and PKCe are involved in early
steps of phagocytosis: PKCd interacts with Dectin-1 and Syk and is
required for phagocytosis of zymosan  whereas PKCe enhances
FccR-mediated phagocytosis . Conventional PKCs also
contribute to other processes, such as the generation of the
respiratory burst, but are dispensable for FccR-mediated inter-
nalization . Further characterization of the contributions of
individual PKC members to FccR- and Dectin1-mediated
phagocytosis is necessary.
BTK and Vav1 are important for phagocytosis of C. albicans, as
btk2/2 and vav12/2 peritoneal macrophages and BMDMs
displayed reduced phagocytosis (Figure 8). BTK- and Vav1-
deficient mice are also more susceptible to systemic infections with
C. albicans. Dectin1-, BTK- and Vav1-deficient mice succumb
earlier to infections than do wild type mice, but fungal burdens,
immune cell invasion and cytokine levels in the kidney are
comparable (Figure 8). While the phenotype of the BTK- and
Vav1-deficient mice might be due to reduced phagocytosis by
macrophages, the mice used here are complete knockouts. We
therefore must remain vigilant to the possibility that defects in
macrophage or innate immune cells may not be solely responsible
for the results reported here. Btk2/2 mice have B cell defects
[36,37] while vav12/2 mice have reduced numbers of T and B
cells [38,39]. Although B and T cells are not thought to play a
major role in immune responses during systemic C. albicans
infections, a (minor) contribution cannot be excluded. In addition
to their role in phagocytosis, BTK and Vav1 might contribute to
other innate processes related to C. albicans immune responses,
such as production of reactive oxygen species or cytokines. The
complexities of these interconnections clearly require further
BTK and PLCc are functionally connected, as knockdown of
BTK resulted in reduced PLCc phosphorylation in response to
stimulation of the TREM-1/DAP12 pathway in a lymphoma cell
line . In our RAW-Dectin1 cell line, pharmacological
inhibition of BTK resulted in decreased phagocytosis, reduced
DAG levels and compromised recruitment of PKCe to the
phagocytic cup (Figure 7). We therefore hypothesize that in this
BTK and Vav1 as New Interactors of Dectin-1
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setting BTK is responsible for activation of the DAG-producing
enzyme PLCc. In FccR-mediated phagocytosis, PLCc phosphor-
ylation and recruitment to the phagocytic cup are Syk-dependent
[10,40]. It remains to be established if Syk or BTK or both are
responsible for PLCc phosphorylation and/or activation during
Dectin1-mediated phagocytosis. BTK was previously shown to be
involved in Dectin1-dependent arachidonate release by macro-
phages in response to incubation with zymosan or particulate b-
glucan [23,24]. Phosphorylation of BTK on tyrosine 223 and
phosphorylations of PLCc2 were induced by incubation with
zymosan or particulate b-glucan. However, incubation with the
BTK inhibitor LFM-A13 did not reduce phosphorylation of
PLCc2 during incubation with zymosan .
The role for Vav proteins remains incompletely understood.
Our data add to previous observations that Vav proteins are
instrumental in phagocytosis, and participate in a cell type-
dependent manner. vav1/vav2/vav3 triple-knockout macrophages
ingest IgG-opsonized erythrocytes normally  but vav12/2
and vav32/2 neutrophils are deficient in FccR-mediated
phagocytosis . With regards to fungal particles, in microglia
phosphorylation of Vav1 is induced by particulate b-glucan and
this phosphorylation is affected by a Src family kinase inhibitor as
well as a Syk inhibitor. Vav1 knockdown in a microglial cell line
resulted in reduced uptake of b-glucan particles . vav1/vav3
double knockout neutrophils show reduced binding to zymosan,
and vav1/vav3 mice have increased susceptibility to C. albicans
infection  accompanied by reduced PLCc phosphorylation.
While in our hands vav1 peritoneal macrophages have defects in
both C. albicans and zymosan uptake (Figure 8A), Li et al.
reported that thioglycollate-induced peritoneal macrophages do
not display reduced phagocytosis of zymosan . Whether these
differences are due to macrophage activation status remains to be
established. In addition, the precise role of Vav1 and a possible
link to PLCc during phagocytosis by macrophages remains
Differences in cell wall composition between C. albicans and a
non-pathogenic yeast like Saccharomyces cerevisiae might influence
recruitment of downstream factors. A systematic assessment of the
involvement of BTK, PLCc, DAG and PKC family members
during Dectin1-mediated phagocytosis of C. albicans, S. cerevisiae
and other fungi should help clarify their roles.
Materials and Methods
Animals used in this study were housed at the Whitehead
Institute for Biomedical Research, which is certified by the United
States Office of Laboratory Animal Welfare (OLAW) under the
guidance of the Public Health Service (PHS) Policy on Humane
Care and Use of Laboratory Animals. Whitehead Institute’s
Animal Welfare Assurance was approved 11/3/2009 (IACUC,
A3125-01) All studies were carried out in accordance with
procedures approved by the Massachusetts Institute of Technology
Committee on Animal Care (Ploegh lab, CAC# 1011-123-14).
Cells and culture conditions
C. albicans strain SC5314 was cultured in YPD + Uri (2%
bactopeptone, 1% yeast extract, 2% glucose and 80 mg/ml
uridine) at 30uC. To generate a blue fluorescent protein (BFP)-
expressing C. albicans strain, the GFP sequence of the pENO1-
yEGFP3-NAT plasmid  was replaced with the TagBFP
sequence (Evrogen) with codon usage adapted for C. albicans. C.
albicans SC5314 was transformed with the pENO1-TagBFP-NAT
plasmid and selected with 200 mg/ml nourseothricin (Werner
Bioagents, Jena, Germany) resulting in the Candida-BFP strain.
The RAW-Dectin1-LPETG-36HA cell line (RAW-Dectin1) 
was used for most phagocytosis experiments. Cells were grown in
DMEM medium with 10% inactivated Fetal Bovine Serum (IFS)
at 37uC and 5% CO2. For the production of retrovirus,
HEK293T cells (ATCC) were transfected using TransIT trans-
fection reagent (Mirus) and virus-containing supernatant was
harvested after 24 hours.
Plasmids, cloning, protein expression and construction of
stable cell lines
The imaging constructs used in this study are listed in Table 1.
A Dectin1-CRD-LPETG-His bacterial expression vector was
cloned and expressed as described for Dectin1-CRD .
Table 1. Imaging constructs used in this study.
PH-PLCd-RFP Phosphatidylinositol 4,5-bisphosphatePI(4,5)P2
PH-Akt-RFP Phosphatidylinositol 3,4,5-trisphosphate and
LifeAct-RFPFilamentous actin F-actin
LifeAct-GFPFilamentous actin F-actin 
PKCa-GFPProtein Kinase C isoform a
PKCb-GFP Protein Kinase C isoform b
PKCd-GFPProtein Kinase C isoform d
PKCe-GFPProtein Kinase C isoform e
PKCf-GFPProtein Kinase C isoform f
BTK-mCherry Bruton’s Tyrosine KinaseBTK This study
mCherry-Vav1 Guanine nucleotide exchange factor Vav1 Vav1 This study
mCherry-SykSpleen Tyrosine Kinase SykThis study
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PLOS Pathogens | www.plospathogens.org 11June 2013 | Volume 9 | Issue 6 | e1003446
Mammalian expression vectors were constructed for N- or C-
terminal mCherry fusions in vectors based on the retroviral
plasmid pMSCVpuro (Clontech) (pMSCVpuro-mCherry-N and
pMSCVpuro-mCherry-C). The BTK, Vav1 and Syk open reading
frames were cloned from mouse spleen cDNA into the tagging
vectors, resulting in N- or C-terminal fusion of all three genes
(mCherry-BTK, BTK-mCherry, mCherry-Vav1, Vav1-mCherry,
mCherry-Syk and Syk-mCherry). The vectors were used to create
stable cell lines in the RAW-Dectin1 background by retroviral
transduction and selection with puromycine. The resulting six cell
lines were tested for expression of the mCherry-fused proteins by
mCherry-Vav1 and mCherry-Syk cell lines were selected for
further experiments. For expression of the biosensor constructs in
the RAW macrophage cell lines, cells were transfected using
Immunoprecipitation, mass spectrometry, blotting and
To identify proteins that interact with Dectin-1 during
phagocytosis, RAW-Dectin1 macrophages were incubated with
live C. albicans at MOI 5 for 1 hour or left unchallenged. Cells were
harvested by scraping into ice-cold PBS and lysed in NP40 buffer
(25 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2with 0.5%
NP40 and protease inhibitors). Epitope-tagged Dectin-1 was
immunoprecipitated from the total lysates with anti-HA beads
(Roche). Eluates were run on a SDS-PAGE gradient gel and silver
stained to visualize proteins. Each lane was cut into regions
according to molecular weight, which were then reduced,
alkylated and subjected to trypsin digestion. The resulting peptides
were extracted, concentrated in vacuo, and analyzed by reverse-
phase chromatography and tandem mass spectrometry. The
resulting CID spectra were searched against a species-specific
database generated from NCBI’s non-redundant database using
SEQUEST. For the generation of anti-BTK and anti-Vav1
antisera, BTK and Vav1 were cloned from mouse spleen cDNA
into bacterial expression vector pET28a with an N-terminal His
tag (pET28a-BTK and pET28a-Vav1). Vectors pET28a-BTK
and pET28a-Vav1 were used to transform Rosetta cells and
transformants were induced with IPTG for protein expression.
His-Vav1 was isolated from the soluble fraction and His-BTK was
isolated from the insoluble fraction in 8 M urea. Both proteins
were purified using NiNTA beads and BTK was refolded by
stepwise dialysis to eliminate urea, followed by FPLC purification
of the peak containing the monovalent BTK. Purified Vav1 and
BTK were injected in rabbits and serum was harvested and used
for immunoblot and immunoprecipitation experiments (anti-BTK
and anti-Vav1). Other antibodies used were: anti-Syk (Cell
Signaling), anti-phospho-Syk (Tyr525/526; Cell Signaling), anti-
PLCc2 (Cell Signaling), anti-phospho-PLCc2 (Tyr1217; Cell
Signaling), anti-p97 , anti-HA-HRP (Roche). For immuno-
blotting, protein extracts were separated on 8% or 12% SDS-
polyacrylamide gels and transferred to a nitrocellulose membrane
using a semi-dry system. For BTK and Vav1 immunoprecipitation
experiments, cells were lysed in NP40 buffer followed by
immunoprecipitation with 2 ml of anti-BTK or anti-Vav1 antisera
and 30 ml of Protein-A beads (Repligen). Beads were washed and
eluates were analyzed using SDS-PAGE gels.
Spinning disk confocal and electron microscopy
Confocal images were collected in the W.M. Keck Facility for
Biological Imaging using a PerkinElmer Live Cell Imaging
spinning disk confocal system and Volocity software. The
PerkinElmer Live Cell Imaging spinning disk confocal system
was mounted on a Zeiss Axiovert 200M with a 10061.4NA Plan-
Apochromat objective. Excitation light was generated by gas and
solid state lasers (Argon laser for 488 nm, Krypton laser for
568 nm, solid state laser for 405 nm and 647 nm) and passed
through an AOTF for wavelength selection and laser power
control. A quadruple bandpass filter separated the excitation and
emission light inside the CSU-22 confocal scanhead (Yokogawa)
and a filter wheel (Prior Scientific) provided selection of emission
filters (TagBFP & RFP: dual-band 445/60 and 615/70 nm; GFP:
527/55 nm). Volocity image acquisition software was used to
capture images from a Hamamatsu Orca-ER cooled-CCD camera
and to control all the equipment. For 3D reconstructions of
phagocytic cells, Z planes were acquired at 0.15 mM distance and
Volocity image acquisition software was used to create the XYZ
views. Electron microscopy sections were examined using a FEI
Tecnai Spirit at 80 KV. Routine morphology was performed by
trimming and fixing the tissue in 2.5% gluteraldehyde, 3%
paraformaldehyde with 5% sucrose in 0.1 M sodium cacodylate
buffer (pH 7.4) and 0.2% tannic acid. Samples were post fixed in
1% osmium in veronal-acetate buffer. The tissue was stained in
block overnight with 0.5% uranyl acetate in veronal-acetate buffer
(pH 6.0), then dehydrated and embedded in em812 resin. Sections
were cut on a Leica Ultracut UCT microtome with a Diatome
diamond knife at a thickness setting of 50 nm, stained with uranyl
acetate, and lead citrate. The sections were examined using a FEI
Tecnai Spirit at 80 KV.
For general confocal microscopy, Candida-BFP was added to
RAW-Dectin1 macrophages at an MOI of 10, fixed with 4% PFA
in PBS and mounted on slides in 50% glycerol. b-1,3-glucan
conjugated beads were a kind gift of Jatin Vyas and prepared as
described . Zymosan A (Sigma) was labeled with Alexa647
carboxylic acid, succinimidyl ester (Invitrogen) by incubation in
0.1 M Na2CO3at room temperature. Candida-BFP was UV-killed
by exposure to 100.000 mJ/cm2in a UV-crosslinker for four
rounds. Recruitment of fluorescent proteins to the phagocytic cup
or phagosome was quantified using ImageJ software according to
the method of Flannagan and Grinstein . Phagocytic indices of
RAW-Dectin1 cells, BMDMs or peritoneal macrophages were
determined by incubation with Candida-BFP or zymosan-Alexa647
at MOI 10 for 30 minutes or 1 hour. Cells were fixed in 4% PFA
and stained with Concanavalin A-FITC (Sigma) to distinguish
unengulfed yeasts. Inhibitors were added to the media at the
indicated concentrations for 1 hour prior to incubation with
Candida-BFP. Images were aquired by confocal microscopy and the
number of intracellular Candida-BFP per macrophage was
determined by counting 75-200 cells per experiment. Dectin1-
CRD-LPETG was incubated with Staphylococcus aureus sortase A
enzyme and GGG-Alexa647 probe resulting in Dectin1-CRD-
LPETGGG-Alexa647 that was used for staining of Candida-BFP
yeasts and hyphae. For immunofluorescence, cells were grown on
coverslips and fixed in 4% PFA in PBS, washed with PBS and
incubated in 50 mM NH4CL in PBS for 10 min. Next, cells were
incubated in Binding Buffer (0.1% Saponin, 0.2% BSA in PBS) for
30 min followed by incubation in Binding Buffer with anti-HA-
Alexa488 (Invitrogen) antibody for 60 min, several washes with
PBS and mounting for spinning disk confocal microscopy.
Synthesis of BTK inhibitor PCI-32765
(1) was prepared from 4-phenoxybenzoic acid and malonitrile as
described (International Patent Publication No. WO 01/019829
and . Alkylation of pyrazole (1) with 3-methanesulfonyl N-Boc
BTK and Vav1 as New Interactors of Dectin-1
PLOS Pathogens | www.plospathogens.org12 June 2013 | Volume 9 | Issue 6 | e1003446
hydroxypiperidine (2) followed by removal of the Boc-protecting
group and acylation with acryloyl chloride gave the racemate of
PCI-32756 in three steps (Figure S3). All chemicals were of
commercial sources and were used as received. DriSolv anhydrous
CH2Cl2, DriSolv anhydrous MeOH, DriSolv anhydrous DMF
were purchased from EMD Chemicals. Redistilled, anhydrous
N,N9- diisopropylethylamine (DiPEA), trifluoroacetic acid (TFA),
triisopropylsilane (TIS) N-methylpyrrolidone (NMP) was obtained
from Sigma-Aldrich. LC-ESI-MS analysis was performed using a
Micromass LCT mass spectrometer (Micromass MS Technolo-
gies, USA) and a Paradigm MG4 HPLC system equipped with a
HTC PAL autosampler (Michrom BioResources, USA) and a
Waters Symmetry 5 mm C8 column (2.1650 mm, MeCN:H2O
(0.1% formic acid) gradient mobile phase, 150 mL/min). HPLC
purifications were achieved using an Agilent 1100 Series HPLC
system equipped with a Waters Delta Pak 15 mm, 100 A˚C18
column (7.86300 mm) using A: H2O, B: MeCN and C: 1%
aqueous trifluoroacetic acid as mobile phase (3 mL/min). (R/S)-1-
Boc-3-Hydroxypiperidine (1.05 g, 5 mmol) was dissolved in
CH2Cl2 (25 mL) and subsequently triethylamine (1.39 mL,
10 mmol) and methanesulfonyl chloride (0.394 mL, 5.1 mmol)
were added. After stirring overnight, the reaction was concentrat-
ed under reduced pressure, redissolved in ethyl acetate, washed
with water and brine, dried over MgSO4and concentrated in
vacuo. The crude mesylate was dissolved in anhydrous DMF
(20 mL). Pyrazole 1 (1.02 g, 3.33 mmol) and potassium carbonate
(0.92 g, 6.66 mmol) were added and the reaction was stirred until
TLC analysis showed complete conversion. The reaction was
diluted with water and extracted with CH2Cl2. The organic layer
was dried over MgSO4, concentrated in vacuo. Purification over
silica gel chromatography(CH2Cl2RMeOH/CH2Cl2) gave
intermediate 3. Intermediate 3 was dissolved in dioxane (20 mL)
and freshly prepared hydrochloric acid (35 mmol) was added. The
solution was stirred for 1 h, concentrated in vauo. The crude amine
(1 g, 2.58 mmol) was redissolved in CH2Cl2(10 mL). To this was
added Et3N (1.8 mL, 12.9 mmol) and acryloyl chloride (0.22 mL,
2.71 mmol). After 5 h, the reaction was quenched by the addition
of water. The solution was extracted and the organic layer was
dried over MgSO4and concentrated in vacuo. The crude product
was purified by reverse phase HPLC (28R43%B in 20 min,
3 mL/min) affording the title compound (46.3 mg, 0.105 mmol)
as a white solid. LC/MS: Rt8.52 min; linear gradient 5R80% B
in 10 min; ESI/MS: m/z=441.2 [M+H]+.1H NMR (400 MHz,
CD3OD) d ppm 8.39 (s, 1H), 7.69-7.66 (m, 2H), 7.44-7.39 (m,
2H), 7.21-7.08 (m, 5H), 6.82 (dd, J=16.8, 10.8 Hz, 0.6H), 6.66
(dd, J=16.8, 10.8 Hz, 0.4H), 6.17 (d, J=16.8 Hz, 0.6H), 6.15 (d,
J=16.8 Hz, 0.4H) 5.77 (d, J=10.8 Hz, 0.6H), 5.65 (d,
J=10.8 Hz, 0.4H), 4.95-4.93 (m, 1H), 4.56 (d, J=12.4 Hz,
0.6H), 4.24 (d, J=11.2 Hz, 1H), 4.06 (d, J=14.0 Hz, 0.6H), 3.89
(dd, J=12.8, 8.8 Hz, 0.4H), 3.58 (dd, J=12.4, 9.6 Hz, 0.6H),
3.42-3.36 (m, 1H), 2.45-2.34 (m, 1H), 2.28-2.23 (m, 1H), 2.15-2.05
(m, 1H), 1.80-1.68 (m, 1H). 2.72 (t, J=7.6 Hz, 2H), 2.15 (dt,
J=7.2, 2.8 Hz, 2H), 1.96 (d, J=2.8 Hz, 1H), 1.82 (quin.,
J=7.6 Hz, 2H), 1.52 (quin., J=7.2 Hz, 2H).
Quantitative analysis of cellular diacylglycerol content
RAW-Dectin1 macrophages were coincubated with Candida
albicans in 6-well dishes for 1 hour, washed with phosphate-
buffered saline and subjected to lipid extraction . The
chloroform/methanol phase was dried under N2and DAG kinase
assays were performed as described . See supplementary
materials for further details. The dried lipids were dissolved in
40 ml of solubilizing buffer (7.5% octyl-ß-D-glucoside and 5 mM
cardiolipin in 1 mM diethylenetriaminepenta acetic acid (DETA-
PAC, pH 7.0) by vigorously vortexing for 20 sec and incubating at
RT for 10 min. Then, 100 ml of 26 reaction buffer (100 mM
imidazole HC1, pH 6.6, 100 mM NaCl, 25 mM MgCl2, and
2 mM EGTA), 4 ml of 100 mM freshly prepared DTT and 20 ml
of E. coli DAG kinase (Sigma-Aldrich)) were added while keeping
the samples on ice. The reaction was initiated by addition of 3 mCi
[c33P]-ATP prepared by dilution in 20 ml of 1 mM DETAPAC,
pH 6.6. After vortexing briefly, the reaction was incubated at
25uC for 30 min. Lipids were extracted as described above and the
reaction products were analyzed by TLC, which was developed in
acetone followed by CHCl3/MeOH/acetic acid (65/15/5 [vol/
vol/vol]) solution. Radiolabelled lipids were detected by exposure
to imaging screens (BAS-MS; FujiFilm), scanned on a BAS-2500
(FujiFilm)), and quantified with Quantity One software.
Animal care, primary cells and mouse model for systemic
Animals were housed at the Whitehead Institute for Biomedical
Research and maintained according to protocols approved by the
Massachusetts Institute of Technology Committee on Animal
Care. C57BL/6 mice were purchased from Jackson Labs,
dectin12/2 , btk2/2  and vav12/2 mice  were
kind gifts from Stu Levitz, Whasif Khan and Victor Tybulewicz,
respectively. Bone marrow-derived macrophages (BMDMs) were
differentiated from mouse bone marrow by growth in DMEM
(high glucose; Gibco) with 10% HI-FBS (Hyclone) and 5% M-
CSF-containing culture supernatant from L929 cells. Experiments
were performed after 7 days of differentiation. Peritoneal
macrophages were harvested by peritoneal lavage with upto
10 ml PBS. Cells were seeded for experiments in DMEM (high
glucose; Gibco) with 10% HI-FBS (Hyclone) and used for
experiments the next day. For cytokine analysis, macrophages
were incubated with C. albicans and supernatants were harvested
after 16 hours. Cytokine concentrations were determined by
Discovery assay cytokine array by Eve Technologies. For the
mouse model of systemic candidiasis, 56104or 16105CFU of C.
albicans SC5314-derived strain Candida-BFP was administered
intraveneously to age-matched C57BL/6 wild type, dectin-1, btk
and vav1 2/2 mice in a final volume of 200 ml in PBS. Mice were
weighed and monitored daily and euthanized when .20% of
initial body weight was lost. Kidneys were harvested for
enumeration of fungal burden, histology or cytokine analysis. To
determine fungal burden, kidneys were homogenized and lysates
were plated on YPD plates with antibiotics. For histology, tissues
were fixed in 4% formalin in PBS, embedded and sectioned in
paraffin and slides were stained with Hematoxylin and Eosin
(H&E) or Gomori Methenamine Silver (GMS). For cytokine
analysis, kidneys were homogenized in 1 ml PBS followed by
cytokine analysis using a Th1/Th2/Th17 mouse cytometric bead
array (BD Biosciences) and LSR Flow Cytometer according to the
For statistical analysis unpaired t tests were used with 95%
confidence interval. All graphs show means and standard
deviations of three independent experiments.
Supplemental information includes three figures.
mCherry to the phagocytic cup. Confocal images showing
Localization of BTK-mCherry and Vav1-
BTK and Vav1 as New Interactors of Dectin-1
PLOS Pathogens | www.plospathogens.org 13June 2013 | Volume 9 | Issue 6 | e1003446
localization of BTK-mCherry, mCherry-Vav1 and mCherry-Syk
in RAW-Dectin1 macrophages at the indicated time points during
co-incubation with b-glucan-coated beads (A), zymosan-Alexa647
(B), UV-killed Candida-BFP yeast (C) and UV-killed Candida-BFP
hyphae (D). Experiments were performed multiple times, repre-
sentive micrographs are shown.
during C. albicans phagocytosis. PH-PKCd-RFP and PH-
BTK-GFP biosensors showing localization of PI(4,5)P and
PI(3,4,5)P, respectively, without challenge or after 30 or
90 minutes of coincubation with Candida-BFP. White arrows
indicate areas of PI(4,5)P2and PI(3,4,5)P3co-localization.
Localization of PI(4,5)P2 and PI(3,4,5)P3
Synthesis of PCI-32765.
We want to thank Wendy Salmon for advice on spinning disk imaging and
we are grateful to Ana Maria Avalos, Bastien Pare ´ and Ekaterina
Spivakovsky for experimental input.
Conceived and designed the experiments: KS GDF AE GRF SG HLP.
Performed the experiments: KS FGT MDW SKD NW ES. Analyzed the
data: KS MDW FGT HLP. Contributed reagents/materials/analysis tools:
VKV. Wrote the paper: KS HLP.
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