Molecular Biology of the Cell

Vol. 19, 1241–1251, March 2008

RNA Interference in J774 Macrophages Reveals a Role

for Coronin 1 in Mycobacterial Trafficking but Not in
Actin-dependent Processes
Rajesh Jayachandran,* John Gatfield,* Jan Massner, Imke Albrecht,
Bettina Zanolari, and Jean Pieters

Biozentrum, University of Basel, 4056 Basel, Switzerland

Submitted July 9, 2007; Revised November 20, 2007; Accepted December 14, 2007
Monitoring Editor: Sandra Schmid

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of
macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family
of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial
phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes.
However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating
phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using
RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages
expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell
locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobac-
teria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a
specific role in modulating phagosome–lysosome transport upon mycobacterial infection and that it is dispensable for
most F-actin–mediated cytoskeletal rearrangements.

INTRODUCTION sists of five WD repeats and two additional sequence

stretches that are predicted to fold into a seven-bladed
The coronin protein family consists of a series of highly ␤-propeller region (Gatfield et al., 2005). WD repeat-contain-
conserved proteins that are found in all eukaryotic species ing proteins are involved in a variety of biological processes,
(Okumura et al., 1998). Coronin was originally isolated as an ranging from cytoskeletal organization to membrane traf-
actin/myosin-interacting protein from Dictyostelium discoi- ficking to signal transduction (Smith et al., 1999), and they
deum (de Hostos et al., 1991). In Dictyostelium, coronin is may represent a regulatory domain involved in protein–
involved in phagocytosis, macropinocytosis, cell locomo- protein interaction, signal transduction, or both.
tion, and cytokinesis (de Hostos et al., 1993; Gerisch et al., Mammalian coronin 1, also known as P57 or Tryptophan
1993). In yeast, there is a single coronin homologue (Crn1) Aspartate containing COat protein (TACO) is exclusively
that interacts in vitro with the actin-related protein 2/3 expressed in leukocytes (Suzuki et al., 1995; Ferrari et al.,
(Arp2/3) complex, and it has been proposed to regulate the 1999). Although in T cells coronin 1 has been suggested to
formation of actin filamentous networks (Humphries et al., regulate F-actin dynamics, in phagocytic cells, coronin 1
2002). In mammalian cells, up to seven coronin isoforms does not affect F-actin–mediated processes (Jayachandran et
have been identified of which coronins 2 and 3 are ubiqui- al., 2007). Instead, coronin 1 transiently accumulates at the
tously expressed, whereas the other coronins are expressed cytosolic side of nascent phagosomes during phagocytosis
in a tissue-specific manner: coronins 4 and 5 are expressed in (Grogan et al., 1997; David et al., 1998; Schuller et al., 2001;
the brain, coronin 6 (corSE) in epithelial tissues, and coronin Itoh et al., 2002; Foger et al., 2006), and it is actively retained
1 in hematopoietic cells (de Hostos, 1999; Parente et al., during and after internalization of pathogenic mycobacteria,
1999). Although several of the mammalian coronin isoforms thereby preventing lysosomal delivery (Pieters, 2001; Tail-
are known, for many their physiological roles have not been leux et al., 2003). In neutrophils, coronin 1 has been sug-
well defined in macrophages. gested to play a role in the control of NADPH oxidase
Coronins are characterized by the presence of an N-ter- activity by interacting with the soluble component p40phox of
minal WD repeat domain, separated by a unique region the NADPH oxidase (Grogan et al., 1997; Allen et al., 1999).
from a coiled coil C-terminal domain (de Hostos, 1999; Gat- However, despite many parallels between phagocyte coro-
field et al., 2005). In coronin 1, the N-terminal domain con- nin 1 and Dictyostelium coronin in localization and dynamics
during internalization processes no direct evidence for a role
of mammalian coronin 1 in actin-related processes is avail-
This article was published online ahead of print in MBC in Press
able.
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07– 07– 0640)
on December 27, 2007. To analyze a function of coronin 1 in cytoskeletal rear-
rangement during internalization and locomotion processes,
* These authors contributed equally to this work. coronin 1-deficient macrophages were generated by RNA
Address correspondence to: Jean Pieters ([email protected]). interference technology. In mammalian cells short interfer-

© 2008 by The American Society for Cell Biology 1241

R. Jayachandran et al.

ing RNAs (siRNAs) can be expressed in a stable manner to genomic DNA. RNA integrity was then checked using the RNA 6000 Nano
persistently suppress gene expression (Brummelkamp et al., Assay kit (Agilent Technologies, Palo Alto, CA). RT reactions were set up
according to the manufacturer’s protocol with 2 ␮g of DNAse I-treated total
2002). This allowed the analysis of loss-of-function of coro- RNA, SuperScript III reverse transcriptase (Invitrogen), and random hex-
nin 1 in macrophages. As shown in this paper, no defects in anucleotide primers (Promega, Madison, WI). Polymerase chain reactions
actin-mediated processes could be observed in coronin 1 using coronin-specific primers and cDNA templates from the RT reactions
deficient macrophages. However, when macrophages de- consisted of 30 cycles of 96°C for 30 s, 56°C for 30 s, and 72°C for 45 s. As a
control, PCR reactions were performed concurrently with primers for mouse
pleted for coronin 1 through RNA interference (RNAi)-me- glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following prim-
diated gene silencing were infected with pathogenic myco- ers were used (Okumura et al., 1998): coronin-1 forward, 5⬘-AAACCACTT-
bacteria, the internalized bacilli were rapidly delivered to GGGACAGTGGCT-3⬘; coronin-1 reverse, 5⬘-CATCCGGGCCCAGCGT-
lysosomes and killed. Together, these data suggest that in CAGCA-3⬘; coronin-2 forward, 5⬘-GTGAGCGGTCA GGATGCTAATCCAA-
3⬘; coronin-2 reverse, 5⬘-TTCTCCCTGCTCCTTGACCAG-3⬘; coronin-3
J774 macrophages coronin 1 specifically modulates phago- forward, 5⬘-TTTGAGGGGAAGAACGCGGAC-3⬘; coronin-3 reverse, 5⬘-AGT-
some–lysosome fusion rather than being involved in the GTCTCCCTCTCTGCCCTC-3⬘; coronin-4 forward, 5⬘-CGACTAGGGAT
regulation of F-actin dynamics. TGTCCTCCA-3⬘; coronin-4 reverse, 5⬘-GGTCAGGTGAGGTTTCTCCA-3⬘; co-
ronin-5 forward, 5⬘-GATCCCCATCACCAAGAATG-3⬘; coronin-5 reverse, 5⬘-
GGCTGC CGTCTGTATTGAAG-3⬘; coronin-6 forward, 5⬘-GTGCTGGACAT-
MATERIALS AND METHODS TGACTGGTG-3⬘; coronin-6 reverse, 5⬘-TTGCTTGTGTCCATCTCCTG-3⬘;
coronin-7 forward, 5⬘-GAGCTGCCAGTGGAGGTACT-3⬘; and coronin-7 re-
DNA Constructs verse, 5⬘-GCAACTCATGAC AGCCAGTG-3⬘. The size of the PCR products is
as follows: coronin-1, 430 base pairs; coronin-2, 260 base pairs; coronin-3, 370
For siRNA-mediated depletion of coronin 1 in J774 cells, 64-nt oligonucleo- base pairs; coronin-4, 297 base pairs; coronin-5, 482 base pairs; coronin-6, 551
tides were designed containing a unique 19-nt sequence derived from the base pairs; coronin-7, 600 base pairs; and GAPDH, 450 base pairs.
target transcript. These were annealed and ligated into the pSUPER vector
(OligoEngine) by using BglII/HindIII sites. For selection of J774 clones stably
expressing siRNA they were further subcloned together with the H1-RNA
Immunostaining and Imaging
promoter as a BamHI/XhoI fragment in the StuI site of the pEGFP-C1 vector Cells were grown on 10 well Teflon-coated glass slides (Polysciences, War-
(Clontech, Mountain View, CA). The following oligonucleotides have been rington, PA). After fixation (10 min; 3% paraformaldehyde in phosphate-
used for siRNA directed against mouse coronin 1 (siRNAmCORO1nt198-216, buffered saline [PBS]; 37°C) and permeabilization in 0.1% saponin/2% bovine
target sequence in italics): 5⬘-GATCCCCGACTGGACGAGTAGACAAG serum albumin (BSA) in PBS, cells were incubated for 30 min with primary
TTCAAGA GACTTGTCTACTCGTCCAGTCTTTTTGGAAA-3⬘ (forward oli- antibodies (anti-coronin 1 antiserum, 1:4000; anti-tubulin, immunoglobulin G
gonucleotide) and 5⬘-AGCTTTTCCAAAAAGACTGGACGAGTAGAC AAG- [IgG]1 ascites clone E7, 1:5000). After washing (3 ⫻ 0.1% saponin/2% BSA in
TCTCTTGAACTT GTCTACTCGTCCAGTCGGG-3⬘ (reverse oligonucleotide). PBS), phalloidin-fluorescein isothiocyanate (FITC) (Invitrogen, Carlsbad, CA)
As a control, oligonucleotides containing a target sequence from human and secondary antibodies (goat-anti-mouse Alexa Fluor 633, goat-anti-rabbit
coronin 1 were designed (siRNAhCORO1nt594-612): 5⬘-GATCCCCGCGCGT- Alexa Fluor 568; Invitrogen) were applied for 30 min at 1:200 dilutions. The
GCGCATCAT CGAGTTCAAGAGACTCGATGATGCGCACGCGCTTTTTGG- slides were washed three times with 0.1% saponin/2% BSA in PBS and three
AAA-3⬘ (forward oligonucleotide) and 5⬘-AGCTTTTCCAAAAAGCGCGT- times with PBS and mounted using Fluoroguard antifade mounting medium
GCGCATCATCGAG TCTCTTGAACTCGATGATGCGCACGCGCGGG-3⬘ (reverse (Bio-Rad, Hercules, CA) and analyzed using an LSM510 Meta confocal laser-
oligonucleotide). Sequences were verified using the BigDye reagent scanning microscope (Carl Zeiss, Jena, Germany) and the corresponding
(PerkinElmer Life and Analytical Science, Boston, MA) on an ABI 310 Se- software. Video microscopy was performed as described previously (Gatfield
quencer (Applied Biosystems, Foster City, CA) and MacVector software (Ac- et al., 2000).
celrys, Munich, Germany).
Isolation of Membrane and Cytosol Fractions
Antibodies and Immunoblotting To examine subcellular actin distribution biochemically, cells were sedi-
The coding sequence of coronin 1 was fused to the glutathione transferase mented at 300 ⫻ g for 5 min and resuspended in 10 volumes of homogeni-
sequence in the pGEX-4T-1 expression vector, the fusion protein was ex- zation buffer (10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, and
pressed in Escherichia coli, purified, and used for immunization of rabbits to 0.25 M sucrose, pH 7.4). Homogenization of cells was performed by mechan-
obtain a polyclonal anti-coronin 1 antiserum; anti-peptide antiserum was ical disruption using a syringe and 22-gauge needle. The postnuclear super-
raised against residues 5-20 of coronin 1 as described in Gatfield et al. (2005). natant was prepared by centrifugation (240 ⫻ g; 15 min; 4°C) and subse-
Antisera against coronins 2, 3, 6, and 7 were generated using synthetic quently subjected to ultracentrifugation (100,000 ⫻ g; 30 min). Membranes
peptides spanning amino acid residues 428-439 (coronin 2), 419-430 (coronin (pellet) and cytosol (supernatant) were then analyzed for actin distribution by
3), and 910-922 (coronin 7) as described previously (Ferrari et al., 1999). SDS-polyacrylamide gel electrophoresis (PAGE) and subsequent immuno-
Analysis of protein expression was performed as described previously (Tulp blotting using anti-actin antibody. Protein equivalents were loaded on SDS-
et al., 1994; Ferrari et al., 1997). The monoclonal anti-tubulin antibody (clone PAGE.
E7) was obtained from the Developmental Studies Hybridoma Bank (Univer-
sity of Iowa, Iowa City, IA). For anti-actin immunoblotting, anti-actin ascites Isolation of a Cytoskeleton-containing Detergent-
(mouse monoclonal antibody [mAb] clone C4; Chemicon International, Te- insoluble Fraction
mecula, CA) was used. Detection of coronin 1 and actin was performed using
anti-coronin 1 peptide antiserum (1:1000) and anti-actin mAb (1:2000), respec- To isolate a cytoskeleton-containing detergent-insoluble fraction, cells were
tively, followed by goat-anti-rabbit or goat anti-mouse antisera coupled to sedimented at 300 ⫻ g for 5 min and resuspended in 10 volumes of ice-cold
horseradish peroxidase (Southern Biotechnology Associates, Birmingham, cytoskeletal isolation buffer (1% Triton X-100 in 80 mM PIPES, pH 6.8, 5 mM
AL) as described previously (Tulp et al., 1994). Blots were exposed to x-ray EGTA, and 1 mM MgCl2). The samples were then spun in a tabletop centri-
films (Hyperfilm ECL; GE Healthcare, Little Chalfont, Buckinghamshire, fuge at 3000 ⫻ g for 2 min at 4°C. Pellet and supernatant were analyzed for
United Kingdom) after enhanced chemiluminescence reaction (GE Health- actin distribution by SDS-PAGE and subsequent immunoblotting using anti-
care). actin antibody. Cell equivalents rather than protein equivalents were loaded
on SDS-PAGE.
Mammalian Cell Culture and Transfection
Analysis of Fluid Phase Uptake and Macropinocytosis by
The J774A.1 mouse macrophage-like cell line was grown in DMEM (Invitro-
gen, Paisley, United Kingdom) and 10% fetal calf serum (Invitrogen). Cells
Immunofluorescence
(4 ⫻ 106) were transfected by electroporation with 40 ␮g of circular DNA in Fluid phase uptake and macropinocytosis were analyzed by seeding macro-
a volume of 800 ␮l in 4-mm cuvettes (570 V, 50 ␮s; Eppendorf multiporator phages (20,000 cells per well in a 10 well Teflon slide) and allowing these cells
and buffer system; Eppendorf, Hamburg, Germany). After transfection, stable to adhere for 4 h in serum-free DMEM at 37°C and 5% CO2 to induce serum
cell lines expressing siRNA constructs were obtained by selection using 1.2 starvation. The cells were then stimulated with 100 nM phorbol 12-myristate
mg/ml G418 (Invitrogen). 13-acetate (PMA; for fluid phase uptake) or 100 ng/ml epidermal growth
factor (Cell Signaling Technology, Danvers, MA; to analyze macropinocyto-
Reverse Transcription-Polymerase Chain Reaction sis) for a period of 20 min at the end of which they were shifted to 4°C and
layered with a solution of ice-cold FITC-dextran (140,000 Da; 0.5 mg/ml). The
(RT-PCR) of Total Cellular RNA cells were reincubated at 37°C and 5% CO2 for 20 min to allow uptake of
Total RNA was isolated from 5 ⫻ 106 cells of the cell lines J774A.1 and RAW FITC-dextran, shifted to 4°C, fixed with 3% paraformaldehyde, and analyzed
264.7 by using TRIzol reagent (Invitrogen) in accordance with the manufac- using confocal microscopy (LSM510 Meta; Carl Zeiss). The number of FITC-
turer’s protocol. Then, 10 ␮g of total RNA was subjected to a 30-min treatment positive vacuoles per cell (n ⫽ 20 –25) and the percentage of cells having
at 37°C with DNAse I (Ambion, Austin, TX) to remove contaminating internalized FITC macropinosomes was quantitated (n ⫽ 100).

1242 Molecular Biology of the Cell

 RNAi Knockdown of Coronin 1

Figure 1. Localization of coronin 1 and F-

actin in macrophages. Macrophages (J774)
were seeded on coverslips, and they were left
untreated (A–D) or activated with 100 nM
PMA (E–L). After fixation in 3% paraformal-
dehyde and permeabilization with 0.1% sapo-
nin/2% BSA in PBS, cells were stained with
anti-coronin 1 antiserum followed by Texas
Red-conjugated secondary antibodies (B, F,
and J) or phalloidin-FITC (C, G, and K). Coro-
nin 1 and F-actin colocalized at sites of pseu-
dopod formation (D), at the leading edge (H),
and at membrane ruffles (L). Bar, 10 ␮m.

Analysis of Lamellipodia 1:200 diluted rabbit anti-sheep RBC immunoglobulin M (IgM) (CEDARLANE
Laboratories, Burlington, ON, Canada) in 1 ml of PBS at room temperature.
Macrophages (20,000 cells in 10% fetal bovine serum [FBS] supplemented
RBCs were washed twice in PBS and resuspended in 50 ␮l of PBS. Fifty
DMEM) were seeded per well in a 10-well Teflon slide and allowed to adhere
microliters of C5-deficient human serum (Sigma Chemie, Deisenhofen, Ger-
at 37°C and 5% CO2 for 12 h at the end of which they were stimulated with
10 ng/ml lipopolysaccharide for 1 h, fixed in methanol, and stained for actin
by using mouse anti-actin primary antibody and Alexa Fluor 568-tagged
anti-mouse secondary. Cells were analyzed using confocal microscopy
(LSM510 Meta; Carl Zeiss). The width of the lamellipodia was analyzed using
the software provided (n ⬃ 25).

Quantitative Determination of Phagocytosis by Flow

Cytometry
J774 macrophages were grown to 80% confluence in 24-well plates, and then
they were shifted to 4°C followed by the addition of yellow-green fluorescent
polystyrene beads (FluoSpheres beads, 1 ␮m in diameter, 1:10,000 dilution of
an aqueous 2% suspension; Invitrogen). Next, cells were shifted to 37°C or
kept on ice (cold control, to account for particles that are not internalized but
merely surface bound) for 30 min, followed by washing in PBS/5% fetal calf
serum. Cells were harvested by scraping. Bead uptake was determined by
flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA) gating on the
live population as assessed by the forward-scatter and side-scatter profiles.
Fluorescence-activated cell sorting (FACS) data were analyzed using the
FlowJo (Tree Star, Ashland, OR) software package.

Fc Receptor-mediated Phagocytosis of Red Blood Cells

(RBCs)
Sheep red blood cells (300 ␮l of a 10% suspension; Cappel Laboratories/ICN,
Durham, NC) were washed twice with PBS, and then they were opsonized by
incubation with 1:1000 diluted rabbit-anti-sheep RBC IgG (Cappel Laborato-
ries/ICN) in 1 ml of PBS at room temperature. RBCs were washed three times
in PBS and resuspended in DMEM. Opsonized RBCs in ice-cold DMEM were
allowed to bind for 15 min to J774 macrophages grown on 10-well Teflon-
coated glass slides (Polysciences). We added 800,000 RBCs per 10,000 J774
macrophages.
Unbound RBCs were washed away with ice-cold DMEM. Prewarmed
DMEM was added to the slides to initiate phagocytosis of bound RBCs. After
15-min incubation at 37°C and 5% CO2, slides were exposed to ice-cold
distilled water for 15 s to lyse RBCs that were not internalized, washed three Figure 2. Depletion of coronin 1 by RNA interference. Macrophages
times in PBS, and fixed (3% paraformaldehyde in PBS for 10 min at 37°C). To (J774) transiently transfected with pEGFP-C1::siRNAmCORO1nt198-216
visualize internalized RBCs, slides were stained with rabbit-anti-sheep RBC (A) or pSUPER::siRNAmCORO1nt198-216 (B and C) were seeded
IgG (diluted 1:2000), phalloidin-FITC, and goat anti-rabbit IgG Alexa Fluor
on coverslips, fixed, and permeabilized on day 4. Cells were stained
568 (Invitrogen).
The average number of internalized RBC per J774 macrophage was deter- for coronin 1 (Texas Red-labeled secondary antibody) and tubulin
mined using a fluorescence microscope (LSM510 Meta; Carl Zeiss). Data are (Alexa Fluor 633 secondary antibody) (A) or coronin 1 (Texas Red-
means ⫾ SD of three experiments, with 90 –130 cells counted in each case. labeled secondary antibody) and phalloidin-FITC (B). In C, wild-
type J774 or the indicated stable clones grown on 10-well Teflon-
Complement Receptor-mediated Phagocytosis of RBCs coated glass slides were activated with 100 nM PMA for 20 min and
Three hundred microliters of a 10% suspension of sheep red blood cells subsequently stained with phalloidin-FITC to visualize the actin
(Cappel Laboratories/ICN) were washed twice with PBS and incubated with cytoskeleton. Bar, 10 ␮m.

Vol. 19, March 2008 1243

R. Jayachandran et al.

Figure 3. Analysis of J774 cell lines depleted for

coronin 1. (A) Equal amounts of wild-type J774
macrophages or the indicated stably transfected
cells were lysed in Laemmli sample buffer, and
coronin 1 expression was analyzed by immuno-
blotting after SDS-PAGE by using anti-coronin 1
and anti-actin antibodies. Clones 3–10 and 10 –11
express pEGFP-C1::siRNAm CORO1nt198-216;
clone 21 expresses the control construct pEGFP-
C1::siRNAhCORO1nt 594-612. (B) Wild-type J774
macrophages or the indicated stable clones were
homogenized, and the postnuclear supernatant
was subjected to ultracentrifugation (100,000 ⫻ g
for 30 min). Actin distribution between mem-
branes (pellet, p) and cytosol (supernatant, s)
was analyzed by SDS-PAGE and subsequent im-
munoblotting by using anti-actin antibody. Pro-
tein equivalents were loaded on SDS-PAGE. (C)
Analysis of actin distribution between a Triton
X-100 –insoluble fraction (p) and cytosol (s) by
immunoblotting as described in Materials and
Methods.

many) were added, and the RBC suspension was incubated for 20 min at Life Sciences), and they were allowed to adhere for 2 h at 37°C and 5% CO2.
37°C. RBCs were washed again three times in PBS, and then they were Then, 100 ␮l of Mycobacterium bovis bacillus Calmette-Guérin (BCG) washed
resuspended in DMEM. J774 macrophages grown on 10-well Teflon-coated three times in fresh DMEM resuspended to a final OD of 0.02 was replaced
glass slides (Polysciences) were serum starved for 2 h in DMEM at 37°C and gently over the adhered cells, and it was allowed to infect the macrophages
5% CO2, and they were activated with 100 nM PMA in DMEM for 20 min. for 1 h at 37°C and 5% CO2. Free bacteria were removed by three washes and
Subsequently, 800,000 RBCs were added per 10,000 J774 macrophages, and treatment with 200 ␮g/ml amikacin for 1 h. To initiate the chase, 200 ␮l of
phagocytosis was allowed to occur for 1 h at 37°C and 5% CO2. Slides were fresh medium was added per well and chased for the times indicated. At the
exposed to ice-cold distilled water for 15 s to lyse RBCs that were not end of chase, the medium was removed, and the macrophages were lysed by
internalized, they were washed three times in PBS, and then they were fixed addition of 100 ␮l of incorporation media (7H9 medium with 10% DS sup-
(3% paraformaldehyde in PBS for 10 min at 37°C). To visualize internalized plement, 0.15% saponin, and 10 ␮Ci/ml tritiated uracil) to release the intra-
RBCs, slides were stained as described above. cellular mycobacteria, and then the macrophages were further incubated for
The average number of internalized RBC per J774 macrophage was deter- 24 h at 37°C and 5% CO2. Mycobacteria were lysed by addition of 20 ␮l of 1
mined using a fluorescence microscope (LSM510 Meta; Carl Zeiss). Data are N NaOH and incubation at 50°C for 30 min. Proteins from the lysate were
means ⫾ SD of three experiments, with 30 –50 cells counted in each case. precipitated with 80 ␮l of 50% trichloroacetic acid, and the supernatant was
harvested using a FilterMate harvester (PerkinElmer Life and Analytical
Determination of Superoxide Production Sciences) with Unifilter-96, GF/C filter. The incorporated counts were mea-
sured using a TopCount microplate scintillation counter (PerkinElmer Life
J774 macrophages (1 ⫻ 106) resuspended in 1 ml of 10% DMEM were either
and Analytical Sciences) according to the manufacturer’s protocol.
treated with DMSO or 100 nM PMA for 30 min at the end of which they were
treated with reactive oxygen species indicator 2,7-dichlorodihydrofluorescein
diacetate (Invitrogen) at 1.5 ␮M concentration for 15 min. The cells were RESULTS
washed twice in 10 ml of PBS containing 2% FBS and resuspended in 1 ml of
the same and analyzed using FACSCaliber (BD Biosciences) with excitation at Coronin 1 Localization in Resting and Activated J774
488 nm and emission measured at FL-1.
Macrophages
Cell Migration Assay In Dictyostelium, coronin colocalizes with actin at crown-
Macrophages were harvested, washed twice with DMEM/10 mM HEPES, pH shaped structures at the leading edge of the cell, and in
7.3, and seeded at a density of 4 ⫻ 105 cells/well into the upper part of the phagocytic cups and macropinosomes (de Hostos et al., 1991;
Transwell chamber (8-␮m pore size, 6.5 mm in diameter; Corning Life Sci- Maniak et al., 1995). To analyze the localization of coronin 1
ences, Acton, MA) containing 0.6 ml of DMEM/10 mM HEPES, pH 7.3, with
or without chemoattractant (either 200 ng/ml macrophage chemoattractant
in J774 macrophages, cells were analyzed by immunofluo-
protein 1 or 10% zymosan-activated human serum) in the lower well. As a rescence microscopy. Coronin 1 strongly colocalized with
control, the cells were treated with 4 ␮M latrunculin B. After 4 h of incubation F-actin in pseudopods of resting J774 macrophages (Figure
at 37°C, the Transwell filters were fixed in 3% paraformaldehyde and wiped 1D), lamellipodia/leading edges (Figure 1H), and mem-
on the top with a cotton swab to remove nonmigrated cells. Filter membranes
were stained with propidium iodide solution (2 ␮g/ml PBS), excised, and
brane ruffles (Figure 1L) that extend from the upper part of
mounted onto glass slides using Fluoroguard antifade mounting medium PMA-activated cells. Thus, coronin 1 is enriched in struc-
(Bio-Rad). Migrated cells were counted in five to eight optical fields per filter tures where the F-actin cytoskeleton is being remodeled
by using a confocal microscope (LSM510 Meta; Carl Zeiss) at 63⫻ magnifi- similarly to the localization of coronin in Dictyostelium.
cation.
Establishing RNA Interference Technology in J774
Mycobacterial Survival
Macrophages
Mycobacterial survival was essentially performed as described previously
(Walburger et al., 2004). In brief, 5 ⫻ 104 J774 cells in 200 ␮l of DMEM To address a function for coronin 1 in actin-related processes in
supplemented with 10% FBS was seeded per well in a 96-well plate (Corning J774 macrophages, coronin 1 expression was ablated using

1244 Molecular Biology of the Cell

 RNAi Knockdown of Coronin 1

Figure 4. Fluid phase uptake, macropinocytosis and lamellipodia formation in coronin 1-depleted macrophages. (A) Immunofluorescence
analysis of transiently siRNA-transfected J774 cells (day 4) incubated with the fluid phase marker FITC-dextran (2 mg/ml; 70,000 Da) for 20
min. Cells were stained with anti-coronin 1 antibody (Texas Red-labeled secondary antibody). (B) Flow cytometric analysis of bead uptake
by stable siRNA-expressing J774 cells. Wild-type (WT), coronin 1-depleted (clones 3–10 and 10 –11), or control J774 clones (clone 21) were
incubated for 30 min with yellow-green fluorescent polystyrene beads as described in Materials and Methods. Bead uptake was quantified by
determining the mean fluorescent intensity (MFI) of the living population (n ⫽ 10,000). MFI of the cold controls is subtracted from the MFI
of the 37°C samples, and MFI for J774 WT cells is set to 100%. MFI for the indicated clones is expressed in percentage relative to WT. Data
are means ⫾ SD from three independent experiments. (C) Macropinosome formation in stably transfected J774 clones incubated with the fluid
phase marker FITC-dextran (0.5 mg/ml; 140,000 Da) for 20 min in the presence of epidermal growth factor. (D) Quantitation of the number
of macropinosomes per cell as analyzed by confocal microscopy upon stimulation with EGF (n ⫽ 25 ⫾ SD). (E) Analysis and (F) quantitation
of the lamellipodia width in various J774 clones (n ⫽ 25 ⫹/⫺ SD).

RNA interference technology. Analysis of the stability of coro- nin 1 (pEGFP-C1::siRNAhCORO1nt594-612), no coronin
nin 1 mRNA translation products by pulse labeling of J774 cells 1-depleted cells were observed (data not shown). Down-
revealed that coronin 1 is stable over several days (data not regulation of coronin 1 affected neither the overall cellular
shown). To analyze down-regulation of coronin 1, cells were morphology nor the tubulin cytoskeleton (Figure 2A). To
transfected with the siRNA constructs expressing the murine analyze whether depletion of coronin 1 affects cytoskeletal
coronin 1-specific sequences together with the enhanced green structures such as the cortical actin cytoskeleton at the single
fluorescent protein (pEGFP-C1::siRNAmCORO1nt198-216; see cell level, J774 macrophages transiently transfected with
Materials and Methods), and they were analyzed after 4 d for the pSUPER::siRNAmCORO1nt198-216 were fixed, permeabil-
presence of coronin 1. As shown in Figure 2A, complete down- ized, and stained for F-actin. Neither the overall morphol-
regulation of coronin 1 translation was achieved at the single ogy of the cells, nor the F-actin cortical structures showed an
cell level (in ⬃5% of total cells). In J774 cells that had been
transfected with siRNA constructs specific for human coro-

Table 2. Migration speed of J774 WT and siRNA clones

Table 1. EGF induced macropinosomes in J774 cells Migration speed

J774 (␮m/30 min) ⫾ SEM
% of cells positive
J774 for macropinosomes WT 14.304 ⫾ 3.058
10–11 12.95 ⫾ 1.033
WT 65.4 3–10 13.236 ⫾ 1.887
10–11 69.7 21 13.804 ⫾ 1.283
3–10 67.8
21 65 n ⫽ 8 –12 cells/cell type.

Vol. 19, March 2008 1245

R. Jayachandran et al.

altered morphology upon coronin 1 depletion (Figure 2B). nized, and the postnuclear supernatant was separated by
Additionally, J774 macrophages that were depleted for co- sedimentation into a membrane and a cytosolic fraction.
ronin 1 (stable clones 3–10 and 10 –11; see below) were SDS-PAGE followed by immunoblotting using anti-actin an-
equally capable to spread on glass slides and form pseudopods tibody showed identical patterns of distribution for coronin
compared with nondepleted cells (Figure 2C). Together, these 1-negative (clones 3–10 and 10 –11) and coronin 1-positive
data indicate that coronin 1 is not required for cortical F-actin (clones 21 and J774) cell lines (Figure 3B), demonstrating that
colocalization or pseudopod formation upon cell spreading of the absence of coronin 1 does not affect steady state actin
J774 macrophages. association with the membrane. To further investigate po-
tential changes in the actin polymerization state due to
Generation of Stable Coronin 1-deficient Macrophage Cell coronin 1 depletion, the cytoskeleton was isolated by cell
Lines lysis in 1% Triton X-100 and low-speed centrifugation
To establish cell lines that are permanently depleted for coro- (3000 ⫻ g for 2 min). Pellet and supernatant were analyzed
nin 1, J774 macrophages were transfected with expression vec- by immunoblotting by using anti-actin antibody. Actin was
tors expressing the enhanced green fluorescent protein (EGFP) found to be equally distributed between the insoluble and
and the murine or human siRNA sequences (see Materials and the soluble fraction for coronin 1-negative (clones 3–10 and
Methods). Two coronin 1 negative clones were selected (clones 10 –11) and coronin 1-positive (clone 21 and J774) cell lines
3–10 and 10 –11; pEGFP-C1::siRNAmCORO1nt198-216) and a (Figure 3C).
control clone (clone 21; pEGFP-C1::siRNAhCORO1nt594-612).
To analyze the degree of down-regulation, equal cell numbers Fluid Phase Uptake, Macropinocytosis, and Lamellipodia
were lysed in Laemmli sample buffer, and the coronin 1 levels Formation in the Absence of Coronin 1
were analyzed by immunoblotting. Although clone 21 ex- Fluid phase uptake including macropinocytosis ensures the inter-
pressed coronin 1 levels similar to wild-type J774 cells, in clone nalization of nonparticulate material in an actin-dependent pro-
3–10 and 10 –11, no coronin 1 could be detected (Figure 3A). As cess (Maniak et al., 1995; Steinman and Swanson, 1995; Araki et al.,
a control, the levels of actin were analyzed, which were identical 1996; Amyere et al., 2000; Johannes and Lamaze, 2002). To analyze
for coronin 1-depleted and control cells, indicating that depletion a role for coronin 1 in fluid phase uptake and macropinocytosis,
of coronin 1 does not have an effect on the total levels of actin. coronin 1-depleted or control J774 macrophages that were tran-
To analyze the association of actin with membranes in siently transfected with pSUPER::siRNAmCORO1nt198-216
coronin 1-deficient J774 macrophages, cells were homoge- and pSUPER::siRNAhCORO1nt594-612, respectively, were in-

Figure 5. Phagocytosis of RBCs. Sheep RBCs

were either IgG opsonized (A and B) or com-
plement treated (C and D), and they were
allowed to be phagocytosed by J774 macro-
phages as described in Materials and Methods.
RBCs were stained with rabbit-anti-sheep RBC
IgG and goat-anti-rabbit IgG Alexa Flour 568.
The actin cytoskeleton was visualized using
phalloidin-FITC. (A) Representative image of
IgG-opsonized RBCs internalized by J774 WT
macrophages. RBCs are in red, and phalloidin-
stained J774 cells are in green. (B) Results from
determining the average number of internal-
ized, IgG-opsonized RBCs per macrophage.
Ninety to 130 cells were counted, and data are
means ⫾ SD from three independent experi-
ments. (C) Representative image of comple-
ment-treated RBCs internalized by J774 WT
macrophages. RBCs are in red, and phalloidin-
stained J774 cells are in green. (D) Quantita-
tion of the average number of internalized,
complement-treated RBCs per macrophage.
Thirty to 50 cells were counted, and data are
means ⫾ SD from three independent experi-
ments. Bar, 10 ␮m. Arrowhead, internalized
RBCs.

1246 Molecular Biology of the Cell

 RNAi Knockdown of Coronin 1

The formation of cellular protrusions such as lamellipodia

is spatially controlled by actin polymerization (Pantaloni et
al., 2001). To determine whether coronin 1 plays a role in the
formation of lamellipodia, coronin 1-expressing and coronin
1-depleted cells were allowed to spread on glass slides, and
the dimensions of the lamellipodia formed were quantitated
after staining with anti-actin antibodies. No differences in
the appearance of lamellipodia were observed between co-
ronin 1-positive and -negative macrophages (Figure 4, E and
F; see also Supplemental Movies 1– 4), suggesting that coro-
nin 1 is dispensable for this actin-based process.
To analyze the kinetics of motility in the presence and
absence of coronin 1, migration speed was quantitated from
the accompanying video sequences (Supplemental Movies
1– 4). As shown in Table 2, no differences were apparent
between the different cell lines.

Phagocytosis in the Presence and Absence of Coronin 1

To analyze the contribution of coronin 1 to phagocytosis,
macrophage cell lines stably depleted for coronin 1 were
incubated with nonopsonized fluorescent polystyrene beads
for 30 min, and the degree of phagocytosis was determined
using FACS analysis (Figure 4B). Whereas nontransfected
J774 cells had a slightly higher capacity to phagocytose, the
coronin 1-depleted clones 3–10 and 10 –11 showed a similar
capacity for phagocytosis compared with the J774 cell line
stably expressing siRNA for human coronin 1 (clone 21). In
addition, the kinetics of phagocytosis of polystyrene beads
was assessed by time-lapse imaging (Supplemental Videos
5– 8). No differences were apparent between coronin 1-ex-
pressing and -depleted macrophages. Together, these data
demonstrate that coronin 1 is not involved in phagocytosis
of nonopsonized polystyrene beads.
The initial event in the phagocytosis of particles is recog-
nition of the particle by specific receptors on the plasma
membrane of the phagocytes (Aderem and Underhill, 1999).
To analyze a possible role for coronin 1 in receptor-mediated
phagocytosis, J774 wild-type and coronin 1-depleted cells
(clones 3–10 and 10 –11) were incubated with sheep RBCs.
Subsequently, the average number of internalized RBCs per
Figure 6. Chemotaxis in coronin 1-depleted macrophages. Coro- cell was determined by immunofluorescence microscopy.
nin 1-depleted or control J774 clones were placed in the upper well Although IgG-opsonized RBCs served as cargo for Fc recep-
of a Transwell assay plate (8-␮m pore size, 6.5 mm in diameter), and tor-mediated phagocytosis, treatment of RBCs with rabbit
they were subjected to a gradient of 10% zymosan-activated human anti-sheep RBC IgM and C5-deficient human serum allowed
serum or 200 ng/ml MCP-1 in culture media placed in the lower
us to study complement receptor (CR)-mediated uptake.
well for 4 h (A) or for 0, 1, 2, and 4 h (B and C). In A, the control
conditions contained medium only (untreated) in the lower well or Neither Fc receptor (Figure 5B) nor CR-mediated uptake
cells treated with 4 ␮M latrunculin B (Lat B). Cells that had tra- (Figure 5D) was found to be affected by RNAi-mediated
versed the filter were stained with propidium iodide and quanti- depletion of coronin 1. Therefore, we conclude that coronin
tated microscopically. Experiments were done in duplicate, and 1 is not required for receptor-mediated uptake of sheep
three to five fields were analyzed per filter. RBCs.

Macrophage Cell Locomotion and Chemotaxis in the

Absence of Coronin 1
cubated with the fluid phase marker FITC-dextran for 20 Coronin is critically involved in chemotaxis in Dictyostelium
min after activation with PMA, and the number of FITC- (de Hostos et al., 1993). To analyze a role for coronin 1 in
dextran–positive vesicles per cell and the percentage of chemotaxis in J774 macrophages, coronin 1-deficient J774
cells with macropinosomes were analyzed. As seen in macrophages were placed in the upper reservoir of a Trans-
Figure 4A, no differences were observed in fluid phase well chamber, and they were subjected to a chemotactic
uptake between coronin 1-negative and -positive cells. To gradient from the lower chamber containing either 10%
analyze macropinocytosis in the presence and absence of zymosan-activated human serum or 200 ng/ml monocyte
coronin 1, coronin 1-expressing or -depleted cells were chemoattractant protein-1 (MCP-1) in cell culture medium.
stimulated with the epidermal growth factor (EGF) before Alternatively, the lower chamber contained medium alone
incubation with FITC dextran. As shown in Figure 4, C (no gradient) or cells treated with actin-depolymerizing
and D, and Table 1, no qualitative and quantitative dif- agent latrunculin B serving as internal controls. Chemotaxis
ferences were observed in the appearance and number of was assessed by analyzing the number of cells that had
macropinosomes in either coronin 1-expressing and coro- traversed the filter by staining with propidium solution
nin 1-depleted macrophages. either after 4 h (Figure 6A) or by analysis after 1, 2, and 4 h

Vol. 19, March 2008 1247

R. Jayachandran et al.

macrophage cell lines were assayed for constitutive and

Table 3. NADPH oxidase activity in coronin 1-depleted macro- stimulated superoxide production by incubation with the
phages chromogenic substrate 2,7-dichlorodihydrofluorescein diac-
etate in the presence or absence of PMA. Superoxide pro-
Mean fluorescence value
duction in resting and PMA-stimulated cells was not influ-
J774 Basal PMA (200 nM) enced by the presence or absence of coronin 1, excluding a
role for coronin 1 in the regulation of NADPH oxidase
WT 274 1894 activity (Table 3).
10–11 264 1640
3–10 255 1778 Mycobacterial Trafficking and Survival in Coronin
21 316 1715 1-negative J774 Macrophages
Although coronin 1 was dispensable for the above-men-
tioned F-actin– dependent processes in J774 cells, upon
mycobacterial uptake coronin 1 is retained around phago-
(Figure 6, B and C). As shown in Figure 6, A–C, although the somes, thereby blocking phagosome lysosome fusion
siRNA expression slightly reduced the chemotactic activity (Ferrari et al., 1999). To analyze whether mycobacterial
of macrophages independently of coronin 1, coronin 1-de- trafficking is affected in J774 cells lacking coronin 1 ex-
pleted and the control siRNA macrophages (clone 21) dis- pression, cells were infected with M. bovis BCG for 1 h,
played similar chemotactic activity, suggesting that coronin followed by a 3-h chase. As shown in Figure 7, A and B,
1-deficient J774 macrophages were not adversely affected in although in J774 cells expressing the control siRNA con-
cell locomotion as induced by chemotactic stimuli. struct the mycobacteria remained outside lysosomes,
in coronin 1-negative macrophages mycobacteria were
NADPH Oxidase Activity in the Absence of Coronin 1 in largely transported to lysosomes, to a similar degree as M.
J774 Macrophages bovis BCG⌬pkng, a mycobacterial mutant that lacks the
Coronin 1 has been suggested to function as an inhibitor of capacity to withstand lysosomal delivery (Walburger et
the superoxide-generating NADPH oxidase by sequestra- al., 2004; Nguyen et al., 2005; Scherr et al., 2007). As
tion of the soluble enzyme subunit p40phox (Grogan et al., expected, on the basis ofthe intracellular localization of
1997). To analyze the capacity of coronin 1 to regulate mycobacteria in the coronin 1-negative cell lines, the pro-
NADPH oxidase activity, coronin 1-deficient and control liferation of mycobacteria was severely affected. Whereas

Figure 7. Trafficking and survival in wild-type and coronin 1-depleted macrophages. (A) Macrophages were infected with M. bovis BCG,
chased for 3 h, and then fixed with methanol and stained for lysosome-associated membrane protein (LAMP)1 (568) or mycobacteria (488).
Cells were analyzed by confocal microscopy. (B and C) Quantitation of the lysosomal delivery of wild-type or PknG-deficient M. bovis BCG
in the different macrophage clones indicated as determined by confocal microscopy (n ⬃ 150). The error bars represent the SD values from
three independent experiments. (D) Survival of mycobacteria in the macrophage clones indicated as determined by incorporation of tritiated
uracil. The error bars represent SD values from triplicate values, and the data shown are representative of at least three independent
experiments.

1248 Molecular Biology of the Cell

 RNAi Knockdown of Coronin 1

Figure 8. Expression of coronin 1-7 in cell

lines as determined by RT-PCR. Total RNA
was extracted from J774 and RAW264.7 mac-
rophages, reversely transcribed, and analyzed
for the presence of coronin 1-7 transcripts by
PCR. Expression of coronin 1-7 in J774 (A) and
RAW264.7 (B) cells. RT, reverse transcriptase.
For immunoblotting, equal cell numbers were
loaded per well from the cell lines indicated in
C after denaturation in Laemmli sample
buffer. Blots were probed for the coronin iso-
forms indicated using anti-tubulin antibodies
as a control.

in control J774 cells, mycobacteria readily survived and the basis of the homology with Dictyostelium coronin and the
proliferated, in the absence of coronin 1 expression my- capacity of in E. coli-expressed coronins to interact with actin
cobacteria failed to proliferate, similar to mycobacteria in vitro. To analyze a role for coronin 1, also known as P57
lacking the essential intracellular survival factor PknG or TACO in actin-related processes in macrophages, we
(Figure 7D). Together, these data indicate that coronin 1 have generated cell lines depleted for coronin 1 by RNA
specifically modulates the intracellular transport of patho- interference. Coronin 1-depleted cells displayed a normal
genic mycobacteria. morphology, and they were indistinguishable from wild-
type cells with respect to the actin and tubulin cytoskeletal
Analysis of the Expression of Coronin Family Members structures, lamellipodia formation, spreading, migration,
The lack of any obvious phenotype of coronin 1-negative phagocytosis, and macropinocytosis. Furthermore, chemo-
J774 macrophages could be explained by the expression of taxis and the activity of the NADPH oxidase was normal.
several coronin homologues within J774 macrophages. However, when macrophages depleted for coronin 1 by
Therefore, the expression of the seven coronin homologues RNAi interference were infected with pathogenic mycobacte-
that have been so far described to be present in mammalian ria, all bacilli were transferred to lysosomes and killed, in
cells (Rybakin and Clemen, 2005) was assessed by RT-PCR. contrast to the phagosomal residence and survival of mycobac-
As shown in Figure 8, coronins 1, 2, 3, 6, and 7 were found teria phagocytosed by control macrophages. We conclude that
to be expressed in the macrophage cell lines J774 and in macrophage cell lines coronin 1 is fully dispensable for
RAW264.7, whereas coronins 4 and 5 were not expressed in F-actin based processes but specifically mediates the intracel-
these cell types. Consistent with these results, no coronin 4 lular survival of mycobacteria by blocking phagosome–lyso-
or 5 was detected by immunoblotting by using the lysates of some fusion.
coronin 1-expressing or -depleted cells. In addition, we were The data presented here are in sharp contrast to recent
unable to detect a specific signal using antisera raised work claiming a role for coronin 1 in phagocytosis on the
against coronin 6 (data not shown). However, as shown in basis of TAT-mediated transduction of the coronin 1 WD
Figure 8C, the amounts of coronins 2, 3, and 7 were unal- domain (Yan et al., 2005). However, in our hands, expression
tered upon coronin 1 depletion, suggestion that coronin 1 of exclusively the coronin 1 WD domain, either alone or as a
depletion did not result in the modulation of the expression fusion protein, resulted in aggregation of the resulting prod-
of these other coronin isoforms. uct due to misfolding in the absence of the C-terminal region
(Gatfield et al., 2005; data not shown). Therefore, the ob-
DISCUSSION served reduction by Yan et al. (2005) might result from
expressing or transducing misfolded proteins inside J774
The WD repeat-containing family of mammalian coronins macrophages, which may compromise cellular functions
has been implicated in various actin-related processes, on such as spreading, membrane ruffling, and phagocytosis.

Vol. 19, March 2008 1249

R. Jayachandran et al.

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