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Vol. 19, Issue 3, 1241-1251, March 2008
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Biozentrum, University of Basel, 4056 Basel, Switzerland
Submitted July 9, 2007;
Revised November 20, 2007;
Accepted December 14, 2007
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Coronin was originally isolated as an actin/myosin-interacting protein from Dictyostelium discoideum (de Hostos et al., 1991). In Dictyostelium, coronin is involved in phagocytosis, macropinocytosis, cell locomotion, and cytokinesis (de Hostos et al., 1993; Gerisch et al., 1993). In yeast, there is a single coronin homologue (Crn1) that interacts in vitro with the actin-related protein 2/3 (Arp2/3) complex, and it has been proposed to regulate the formation of actin filamentous networks (Humphries et al., 2002). In mammalian cells, up to seven coronin isoforms have been identified of which coronins 2 and 3 are ubiquitously expressed, whereas the other coronins are expressed in a tissue-specific manner: coronins 4 and 5 are expressed in the brain, coronin 6 (corSE) in epithelial tissues, and coronin 1 in hematopoietic cells (de Hostos, 1999; Parente et al., 1999). Although several of the mammalian coronin isoforms are known, for many their physiological roles have not been well defined in macrophages.
Coronins are characterized by the presence of an N-terminal WD repeat domain, separated by a unique region from a coiled coil C-terminal domain (de Hostos, 1999; Gatfield et al., 2005). In coronin 1, the N-terminal domain consists of five WD repeats and two additional sequence stretches that are predicted to fold into a seven-bladed β-propeller region (Gatfield et al., 2005). WD repeat-containing proteins are involved in a variety of biological processes, ranging from cytoskeletal organization to membrane trafficking to signal transduction (Smith et al., 1999), and they may represent a regulatory domain involved in protein–protein interaction, signal transduction, or both.
Mammalian coronin 1, also known as P57 or Tryptophan Aspartate containing COat protein (TACO) is exclusively expressed in leukocytes (Suzuki et al., 1995; Ferrari et al., 1999). Although in T cells coronin 1 has been suggested to regulate F-actin dynamics, in phagocytic cells, coronin 1 does not affect F-actin–mediated processes (Jayachandran et al., 2007). Instead, coronin 1 transiently accumulates at the cytosolic side of nascent phagosomes during phagocytosis (Grogan et al., 1997; David et al., 1998; Schuller et al., 2001; Itoh et al., 2002; Foger et al., 2006), and it is actively retained during and after internalization of pathogenic mycobacteria, thereby preventing lysosomal delivery (Pieters, 2001; Tailleux et al., 2003). In neutrophils, coronin 1 has been suggested to play a role in the control of NADPH oxidase activity by interacting with the soluble component p40phox of the NADPH oxidase (Grogan et al., 1997; Allen et al., 1999). However, despite many parallels between phagocyte coronin 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 available.
To analyze a function of coronin 1 in cytoskeletal rearrangement during internalization and locomotion processes, coronin 1-deficient macrophages were generated by RNA interference technology. In mammalian cells short interfering RNAs (siRNAs) can be expressed in a stable manner to persistently suppress gene expression (Brummelkamp et al., 2002). This allowed the analysis of loss-of-function of coronin 1 in macrophages. As shown in this paper, no defects in actin-mediated processes could be observed in coronin 1 deficient macrophages. However, when macrophages depleted for coronin 1 through RNA interference (RNAi)-mediated gene silencing were infected with pathogenic mycobacteria, the internalized bacilli were rapidly delivered to lysosomes and killed. Together, these data suggest that in J774 macrophages coronin 1 specifically modulates phagosome–lysosome fusion rather than being involved in the regulation of F-actin dynamics.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies and Immunoblotting
The coding sequence of coronin 1 was fused to the glutathione transferase sequence in the pGEX-4T-1 expression vector, the fusion protein was expressed in Escherichia coli, purified, and used for immunization of rabbits to obtain a polyclonal anti-coronin 1 antiserum; anti-peptide antiserum was raised against residues 5-20 of coronin 1 as described in Gatfield et al. (2005). Antisera against coronins 2, 3, 6, and 7 were generated using synthetic peptides spanning amino acid residues 428-439 (coronin 2), 419-430 (coronin 3), and 910-922 (coronin 7) as described previously (Ferrari et al., 1999). Analysis of protein expression was performed as described previously (Tulp et al., 1994; Ferrari et al., 1997). The monoclonal anti-tubulin antibody (clone E7) was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). For anti-actin immunoblotting, anti-actin ascites (mouse monoclonal antibody [mAb] clone C4; Chemicon International, Temecula, 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), respectively, followed by goat-anti-rabbit or goat anti-mouse antisera coupled to horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL) as described previously (Tulp et al., 1994). Blots were exposed to x-ray films (Hyperfilm ECL; GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) after enhanced chemiluminescence reaction (GE Healthcare).
Mammalian Cell Culture and Transfection
The J774A.1 mouse macrophage-like cell line was grown in DMEM (Invitrogen, Paisley, United Kingdom) and 10% fetal calf serum (Invitrogen). Cells (4 x 106) were transfected by electroporation with 40 µg of circular DNA in a volume of 800 µl in 4-mm cuvettes (570 V, 50 µs; Eppendorf multiporator and buffer system; Eppendorf, Hamburg, Germany). After transfection, stable cell lines expressing siRNA constructs were obtained by selection using 1.2 mg/ml G418 (Invitrogen).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of Total Cellular RNA
Total RNA was isolated from 5 x 106 cells of the cell lines J774A.1 and RAW 264.7 by using TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol. Then, 10 µg of total RNA was subjected to a 30-min treatment at 37°C with DNAse I (Ambion, Austin, TX) to remove contaminating genomic DNA. RNA integrity was then checked using the RNA 6000 Nano Assay kit (Agilent Technologies, Palo Alto, CA). RT reactions were set up according to the manufacturer's protocol with 2 µg of DNAse I-treated total RNA, SuperScript III reverse transcriptase (Invitrogen), and random hexanucleotide primers (Promega, Madison, WI). Polymerase chain reactions using coronin-specific primers and cDNA templates from the RT reactions 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 glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following primers were used (Okumura et al., 1998): coronin-1 forward, 5'-AAACCACTTGGGACAGTGGCT-3'; coronin-1 reverse, 5'-CATCCGGGCCCAGCGTCAGCA-3'; coronin-2 forward, 5'-GTGAGCGGTCA GGATGCTAATCCAA-3'; coronin-2 reverse, 5'-TTCTCCCTGCTCCTTGACCAG-3'; coronin-3 forward, 5'-TTTGAGGGGAAGAACGCGGAC-3'; coronin-3 reverse, 5'-AGTGTCTCCCTCTCTGCCCTC-3'; coronin-4 forward, 5'-CGACTAGGGAT TGTCCTCCA-3'; coronin-4 reverse, 5'-GGTCAGGTGAGGTTTCTCCA-3'; coronin-5 forward, 5'-GATCCCCATCACCAAGAATG-3'; coronin-5 reverse, 5'-GGCTGC CGTCTGTATTGAAG-3'; coronin-6 forward, 5'-GTGCTGGACATTGACTGGTG-3'; coronin-6 reverse, 5'-TTGCTTGTGTCCATCTCCTG-3'; coronin-7 forward, 5'-GAGCTGCCAGTGGAGGTACT-3'; and coronin-7 reverse, 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 base pairs; coronin-4, 297 base pairs; coronin-5, 482 base pairs; coronin-6, 551 base pairs; coronin-7, 600 base pairs; and GAPDH, 450 base pairs.
Immunostaining and Imaging
Cells were grown on 10 well Teflon-coated glass slides (Polysciences, Warrington, PA). After fixation (10 min; 3% paraformaldehyde in phosphate-buffered saline [PBS]; 37°C) and permeabilization in 0.1% saponin/2% bovine serum albumin (BSA) in PBS, cells were incubated for 30 min with primary antibodies (anti-coronin 1 antiserum, 1:4000; anti-tubulin, immunoglobulin G [IgG]1 ascites clone E7, 1:5000). After washing (3 x 0.1% saponin/2% BSA in PBS), phalloidin-fluorescein isothiocyanate (FITC) (Invitrogen, Carlsbad, CA) and secondary antibodies (goat-anti-mouse Alexa Fluor 633, goat-anti-rabbit Alexa Fluor 568; Invitrogen) were applied for 30 min at 1:200 dilutions. The slides were washed three times with 0.1% saponin/2% BSA in PBS and three times with PBS and mounted using Fluoroguard antifade mounting medium (Bio-Rad, Hercules, CA) and analyzed using an LSM510 Meta confocal laser-scanning microscope (Carl Zeiss, Jena, Germany) and the corresponding software. Video microscopy was performed as described previously (Gatfield et al., 2000).
Isolation of Membrane and Cytosol Fractions
To examine subcellular actin distribution biochemically, cells were sedimented at 300 x g for 5 min and resuspended in 10 volumes of homogenization buffer (10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, and 0.25 M sucrose, pH 7.4). Homogenization of cells was performed by mechanical disruption using a syringe and 22-gauge needle. The postnuclear supernatant was prepared by centrifugation (240 x g; 15 min; 4°C) and subsequently subjected to ultracentrifugation (100,000 x g; 30 min). Membranes (pellet) and cytosol (supernatant) were then analyzed for actin distribution by SDS-polyacrylamide gel electrophoresis (PAGE) and subsequent immunoblotting using anti-actin antibody. Protein equivalents were loaded on SDS-PAGE.
Isolation of a Cytoskeleton-containing Detergent-insoluble Fraction
To isolate a cytoskeleton-containing detergent-insoluble fraction, cells were sedimented at 300 x g for 5 min and resuspended in 10 volumes of ice-cold cytoskeletal isolation buffer (1% Triton X-100 in 80 mM PIPES, pH 6.8, 5 mM EGTA, and 1 mM MgCl2). The samples were then spun in a tabletop centrifuge at 3000 x g for 2 min at 4°C. Pellet and supernatant were analyzed for actin distribution by SDS-PAGE and subsequent immunoblotting using anti-actin antibody. Cell equivalents rather than protein equivalents were loaded on SDS-PAGE.
Analysis of Fluid Phase Uptake and Macropinocytosis by Immunofluorescence
Fluid phase uptake and macropinocytosis were analyzed by seeding macrophages (20,000 cells per well in a 10 well Teflon slide) and allowing these cells to adhere for 4 h in serum-free DMEM at 37°C and 5% CO2 to induce serum starvation. The cells were then stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; for fluid phase uptake) or 100 ng/ml epidermal growth factor (Cell Signaling Technology, Danvers, MA; to analyze macropinocytosis) 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 cells were reincubated at 37°C and 5% CO2 for 20 min to allow uptake of FITC-dextran, shifted to 4°C, fixed with 3% paraformaldehyde, and analyzed using confocal microscopy (LSM510 Meta; Carl Zeiss). The number of FITC-positive vacuoles per cell (n = 20–25) and the percentage of cells having internalized FITC macropinosomes was quantitated (n = 100).
Analysis of Lamellipodia
Macrophages (20,000 cells in 10% fetal bovine serum [FBS] supplemented DMEM) were seeded per well in a 10-well Teflon slide and allowed to adhere 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 µm 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 µl 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 Laboratories/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 times in PBS, and fixed (3% paraformaldehyde in PBS for 10 min at 37°C). To visualize internalized RBCs, slides were stained with rabbit-anti-sheep RBC IgG (diluted 1:2000), phalloidin-FITC, and goat anti-rabbit IgG Alexa Fluor 568 (Invitrogen).
The average number of internalized RBC per J774 macrophage was determined using a fluorescence microscope (LSM510 Meta; Carl Zeiss). Data are means ± SD of three experiments, with 90–130 cells counted in each case.
Complement Receptor-mediated Phagocytosis of RBCs
Three hundred microliters of a 10% suspension of sheep red blood cells (Cappel Laboratories/ICN) were washed twice with PBS and incubated with 1:200 diluted rabbit anti-sheep RBC immunoglobulin M (IgM) (CEDARLANE Laboratories, Burlington, ON, Canada) in 1 ml of PBS at room temperature. RBCs were washed twice in PBS and resuspended in 50 µl of PBS. Fifty microliters of C5-deficient human serum (Sigma Chemie, Deisenhofen, Germany) were added, and the RBC suspension was incubated for 20 min at 37°C. RBCs were washed again three times in PBS, and then they were resuspended in DMEM. J774 macrophages grown on 10-well Teflon-coated glass slides (Polysciences) were serum starved for 2 h in DMEM at 37°C and 5% CO2, and they were activated with 100 nM PMA in DMEM for 20 min. Subsequently, 800,000 RBCs were added per 10,000 J774 macrophages, and phagocytosis was allowed to occur for 1 h at 37°C and 5% CO2. Slides were exposed to ice-cold distilled water for 15 s to lyse RBCs that were not internalized, they were washed three times in PBS, and then they were fixed (3% paraformaldehyde in PBS for 10 min at 37°C). To visualize internalized RBCs, slides were stained as described above.
The average number of internalized RBC per J774 macrophage was determined using a fluorescence microscope (LSM510 Meta; Carl Zeiss). Data are means ± SD of three experiments, with 30–50 cells counted in each case.
Determination of Superoxide Production
J774 macrophages (1 x 106) resuspended in 1 ml of 10% DMEM were either 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 µM concentration for 15 min. The cells were 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 488 nm and emission measured at FL-1.
Cell Migration Assay
Macrophages were harvested, washed twice with DMEM/10 mM HEPES, pH 7.3, and seeded at a density of 4 x 105 cells/well into the upper part of the Transwell chamber (8-µm pore size, 6.5 mm in diameter; Corning Life Sciences, Acton, MA) containing 0.6 ml of DMEM/10 mM HEPES, pH 7.3, with or without chemoattractant (either 200 ng/ml macrophage chemoattractant protein 1 or 10% zymosan-activated human serum) in the lower well. As a control, the cells were treated with 4 µM latrunculin B. After 4 h of incubation at 37°C, the Transwell filters were fixed in 3% paraformaldehyde and wiped on the top with a cotton swab to remove nonmigrated cells. Filter membranes were stained with propidium iodide solution (2 µg/ml PBS), excised, and mounted onto glass slides using Fluoroguard antifade mounting medium (Bio-Rad). Migrated cells were counted in five to eight optical fields per filter by using a confocal microscope (LSM510 Meta; Carl Zeiss) at 63x magnification.
Mycobacterial Survival
Mycobacterial survival was essentially performed as described previously (Walburger et al., 2004). In brief, 5 x 104 J774 cells in 200 µl of DMEM supplemented with 10% FBS was seeded per well in a 96-well plate (Corning Life Sciences), and they were allowed to adhere for 2 h at 37°C and 5% CO2. Then, 100 µl of Mycobacterium bovis bacillus Calmette-Guérin (BCG) washed three times in fresh DMEM resuspended to a final OD of 0.02 was replaced gently over the adhered cells, and it was allowed to infect the macrophages for 1 h at 37°C and 5% CO2. Free bacteria were removed by three washes and treatment with 200 µg/ml amikacin for 1 h. To initiate the chase, 200 µl of fresh medium was added per well and chased for the times indicated. At the end of chase, the medium was removed, and the macrophages were lysed by addition of 100 µl of incorporation media (7H9 medium with 10% DS supplement, 0.15% saponin, and 10 µCi/ml tritiated uracil) to release the intracellular mycobacteria, and then the macrophages were further incubated for 24 h at 37°C and 5% CO2. Mycobacteria were lysed by addition of 20 µl of 1 N NaOH and incubation at 50°C for 30 min. Proteins from the lysate were precipitated with 80 µl of 50% trichloroacetic acid, and the supernatant was harvested using a FilterMate harvester (PerkinElmer Life and Analytical Sciences) with Unifilter-96, GF/C filter. The incorporated counts were measured using a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences) according to the manufacturer's protocol.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Maniak et al., 1995). To analyze the localization of coronin 1 in J774 macrophages, cells were analyzed by immunofluorescence microscopy. Coronin 1 strongly colocalized with F-actin in pseudopods of resting J774 macrophages (Figure 1D), lamellipodia/leading edges (Figure 1H), and membrane ruffles (Figure 1L) that extend from the upper part of PMA-activated cells. Thus, coronin 1 is enriched in structures where the F-actin cytoskeleton is being remodeled similarly to the localization of coronin in Dictyostelium.
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5% of total cells). In J774 cells that had been transfected with siRNA constructs specific for human coronin 1 (pEGFP-C1::siRNAhCORO1nt594-612), no coronin 1-depleted cells were observed (data not shown). Down-regulation of coronin 1 affected neither the overall cellular morphology nor the tubulin cytoskeleton (Figure 2A). To analyze whether depletion of coronin 1 affects cytoskeletal structures such as the cortical actin cytoskeleton at the single cell level, J774 macrophages transiently transfected with pSUPER::siRNAmCORO1nt198-216 were fixed, permeabilized, and stained for F-actin. Neither the overall morphology of the cells, nor the F-actin cortical structures showed an altered morphology upon coronin 1 depletion (Figure 2B). Additionally, J774 macrophages that were depleted for coronin 1 (stable clones 3–10 and 10–11; see below) were equally capable to spread on glass slides and form pseudopods compared with nondepleted cells (Figure 2C). Together, these data indicate that coronin 1 is not required for cortical F-actin colocalization or pseudopod formation upon cell spreading of J774 macrophages.
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Fluid Phase Uptake, Macropinocytosis, and Lamellipodia Formation in the Absence of Coronin 1
Fluid phase uptake including macropinocytosis ensures the internalization of nonparticulate material in an actin-dependent process (Maniak et al., 1995; Steinman and Swanson, 1995; Araki et al., 1996; Amyere et al., 2000; Johannes and Lamaze, 2002). To analyze a role for coronin 1 in fluid phase uptake and macropinocytosis, coronin 1-depleted or control J774 macrophages that were transiently transfected with pSUPER::siRNAmCORO1nt198-216 and pSUPER::siRNAhCORO1nt594-612, respectively, were incubated with the fluid phase marker FITC-dextran for 20 min after activation with PMA, and the number of FITC-dextran–positive vesicles per cell and the percentage of cells with macropinosomes were analyzed. As seen in Figure 4A, no differences were observed in fluid phase uptake between coronin 1-negative and -positive cells. To analyze macropinocytosis in the presence and absence of coronin 1, coronin 1-expressing or -depleted cells were stimulated with the epidermal growth factor (EGF) before incubation with FITC dextran. As shown in Figure 4, C and D, and Table 1, no qualitative and quantitative differences were observed in the appearance and number of macropinosomes in either coronin 1-expressing and coronin 1-depleted macrophages.
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). 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 coronin 1-positive and -negative macrophages (Figure 4, E and F; see also Supplemental Movies 1–4), suggesting that coronin 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.
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The initial event in the phagocytosis of particles is recognition 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 cell was determined by immunofluorescence microscopy. Although IgG-opsonized RBCs served as cargo for Fc receptor-mediated phagocytosis, treatment of RBCs with rabbit anti-sheep RBC IgM and C5-deficient human serum allowed us to study complement receptor (CR)-mediated uptake. Neither Fc receptor (Figure 5B) nor CR-mediated uptake (Figure 5D) was found to be affected by RNAi-mediated depletion of coronin 1. Therefore, we conclude that coronin 1 is not required for receptor-mediated uptake of sheep RBCs.
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). To analyze a role for coronin 1 in chemotaxis in J774 macrophages, coronin 1-deficient J774 macrophages were placed in the upper reservoir of a Transwell chamber, and they were subjected to a chemotactic gradient from the lower chamber containing either 10% zymosan-activated human serum or 200 ng/ml monocyte chemoattractant protein-1 (MCP-1) in cell culture medium. Alternatively, the lower chamber contained medium alone (no gradient) or cells treated with actin-depolymerizing agent latrunculin B serving as internal controls. Chemotaxis was assessed by analyzing the number of cells that had traversed the filter by staining with propidium solution either after 4 h (Figure 6A) or by analysis after 1, 2, and 4 h (Figure 6, B and C). As shown in Figure 6, A–C, although the siRNA expression slightly reduced the chemotactic activity of macrophages independently of coronin 1, coronin 1-depleted and the control siRNA macrophages (clone 21) displayed similar chemotactic activity, suggesting that coronin 1-deficient J774 macrophages were not adversely affected in cell locomotion as induced by chemotactic stimuli.
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). To analyze the capacity of coronin 1 to regulate NADPH oxidase activity, coronin 1-deficient and control macrophage cell lines were assayed for constitutive and stimulated superoxide production by incubation with the chromogenic substrate 2,7-dichlorodihydrofluorescein diacetate in the presence or absence of PMA. Superoxide production in resting and PMA-stimulated cells was not influenced by the presence or absence of coronin 1, excluding a role for coronin 1 in the regulation of NADPH oxidase activity (Table 3).
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). To analyze whether mycobacterial trafficking is affected in J774 cells lacking coronin 1 expression, cells were infected with M. bovis BCG for 1 h, followed by a 3-h chase. As shown in Figure 7, A and B, although in J774 cells expressing the control siRNA construct the mycobacteria remained outside lysosomes, in coronin 1-negative macrophages mycobacteria were largely transported to lysosomes, to a similar degree as M. bovis BCG
pkng, a mycobacterial mutant that lacks the capacity to withstand lysosomal delivery (Walburger et al., 2004; Nguyen et al., 2005; Scherr et al., 2007). As expected, on the basis ofthe intracellular localization of mycobacteria in the coronin 1-negative cell lines, the proliferation of mycobacteria was severely affected. Whereas in control J774 cells, mycobacteria readily survived and proliferated, in the absence of coronin 1 expression mycobacteria failed to proliferate, similar to mycobacteria lacking the essential intracellular survival factor PknG (Figure 7D). Together, these data indicate that coronin 1 specifically modulates the intracellular transport of pathogenic mycobacteria.
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) was assessed by RT-PCR. As shown in Figure 8, coronins 1, 2, 3, 6, and 7 were found to be expressed in the macrophage cell lines J774 and RAW264.7, whereas coronins 4 and 5 were not expressed in these cell types. Consistent with these results, no coronin 4 or 5 was detected by immunoblotting by using the lysates of coronin 1-expressing or -depleted cells. In addition, we were unable to detect a specific signal using antisera raised against coronin 6 (data not shown). However, as shown in Figure 8C, the amounts of coronins 2, 3, and 7 were unaltered upon coronin 1 depletion, suggestion that coronin 1 depletion did not result in the modulation of the expression of these other coronin isoforms.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data presented here are in sharp contrast to recent work claiming a role for coronin 1 in phagocytosis on the basis of TAT-mediated transduction of the coronin 1 WD domain (Yan et al., 2005). However, in our hands, expression of exclusively the coronin 1 WD domain, either alone or as a fusion protein, resulted in aggregation of the resulting product due to misfolding in the absence of the C-terminal region (Gatfield et al., 2005; data not shown). Therefore, the observed reduction by Yan et al. (2005) might result from expressing or transducing misfolded proteins inside J774 macrophages, which may compromise cellular functions such as spreading, membrane ruffling, and phagocytosis.
Notably, the results presented here, as far as we know, provide the first description of macrophage cell lines derived using RNAi technology in which a complete gene knockdown has been achieved. In addition, the phenotype of the RNAi-mediated coronin 1-depleted macrophage cell lines are fully consistent with the phenotype of macrophages derived from coronin 1-deficient mice (Jayachandran et al., 2007), excluding any artifacts introduced into the experimental system either through RNAi-mediated down-regulation or gene-targeting technology.
In J774 macrophages, several other coronins are expressed, such as the ubiquitously expressed coronin 2 and 3 molecules; therefore, the function of coronin 1 for the above-mentioned actin-mediated activities might be redundant with these other coronin family members. Interestingly, the yeast Saccharomyces cerevisiae expresses only a single coronin gene (crn1), and deletion of the coronin gene does not have an obvious phenotype (Heil-Chapdelaine et al., 1998; Goode et al., 1999). The yeast coronin protein interacts with the actin-related protein 2/3 (Arp2/3) complex; therefore, it may be involved in the temporal and spatial organization of actin filamentous networks (Humphries et al., 2002). Whether coronin molecules in leukocytes regulate actin filamentous network under specific physiological conditions remains to be established.
What may be the role of coronin 1 in macrophages? One possibility is that coronin 1 functions in the temporal and spatial regulation of the actin cytoskeleton, as was suggested from analysis of T cells from coronin 1-deficient mice (Foger et al., 2006). Given the lack of any phenotype with respect to the actin-related processes investigated in this study, the involvement of coronin 1 may be restricted to specific conditions (Gatfield and Pieters, 2000). Such conditions may include the internalization and intracellular processing of certain pathogens or the internalization of leukocyte specific cell surface receptors (Nal et al., 2004). For example, when pathogenic microbes such as Mycobacterium spp. and Helicobacter pylori enter macrophages, they recruit and retain coronin 1 around the nascent phagosome, thereby preventing fusion of phagosomes with lysosomes (Ferrari et al., 1999; Zheng and Jones, 2003). Given a possible role of actin in regulating membrane transport processes (Guerin and de Chastellier, 2000; Vieira et al., 2002), coronin 1 actively recruited by these pathogens may be involved in the modulation of the actin cytoskeleton thereby influencing intracellular trafficking and survival.
In conclusion, the data presented here show that coronin 1 depletion in J774 macrophages does not influence actin-dependent processes unlike coronin from Dictyostelium, such as phagocytosis, cell motility, or regulation of NADPH oxidase activity. Instead, coronin 1 was specifically required to allow mycobacteria to escape lysosomal delivery and to survive intracellularly. However, the results presented here do not provide a clue as to the normal role for coronin 1 in the absence of a mycobacterial infection. Coronin 1 may be involved in as yet unknown leukocyte-specific processes, and the availability of coronin 1-negative cell lines described in this study may be instrumental in addressing a function for coronin 1 in these important leukocytes.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work. 
Address correspondence to: Jean Pieters (jean.pieters{at}unibas.ch)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Allen, L. A., DeLeo, F. R., Gallois, A., Toyoshima, S., Suzuki, K., and Nauseef, W. M. (1999). Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease. Blood 93, 3521–3530.
Amyere, M., Payrastre, B., Krause, U., Van Der Smissen, P., Veithen, A., and Courtoy, P. J. (2000). Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C. Mol. Biol. Cell 11, 3453–3467.
Araki, N., Johnson, M. T., and Swanson, J. A. (1996). A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol 135, 1249–1260.
Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553.
David, V., Gouin, E., Troys, M. V., Grogan, A., Segal, A. W., Ampe, C., and Cossart, P. (1998). Identification of cofilin, coronin, Rac and capZ in actin tails using a Listeria affinity approach. J. Cell Sci 111, 2877–2884.[Abstract]
de Hostos, E. L. (1999). The coronin family of actin-associated proteins. Trends Cell Biol 9, 345–350.[CrossRef][Medline]
de Hostos, E. L., Bradtke, B., Lottspeich, F., Guggenheim, R., and Gerisch, G. (1991). Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits. EMBO J 10, 4097–4104.[Medline]
de Hostos, E. L., Rehfuess, C., Bradtke, B., Waddell, D. R., Albrecht, R., Murphy, J., and Gerisch, G. (1993). Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility. J. Cell Biol 120, 163–173.
Ferrari, G., Knight, A. M., Watts, C., and Pieters, J. (1997). Distinct intracellular compartments involved in invariant chain degradation and antigenic peptide loading of major histocompatibility complex (MHC) class II molecules. J. Cell Biol 139, 1433–1446.
Ferrari, G., Langen, H., Naito, M., and Pieters, J. (1999). A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell 97, 435–447.[CrossRef][Medline]
Foger, N., Rangell, L., Danilenko, D. M., and Chan, A. C. (2006). Requirement for coronin 1 in T lymphocyte trafficking and cellular homeostasis. Science 313, 839–842.
Gatfield, J., Albrecht, I., Zanolari, B., Steinmetz, M. O., and Pieters, J. (2005). Association of the leukocyte plasma membrane with the actin cytoskeleton through coiled coil-mediated trimeric coronin 1 molecules. Mol. Biol. Cell 16, 2786–2798.
Gatfield, J., and Pieters, J. (2000). Essential role for cholesterol in entry of mycobacteria into macrophages. Science 288, 1647–1650.
Gerisch, G., Albrecht, R., De Hostos, E., Wallraff, E., Heizer, C., Kreitmeier, M., and Muller Taubenberger, A. (1993). Actin-associated proteins in motility and chemotaxis of Dictyostelium cells. Symp. Soc. Exp. Biol 47, 297–315.[Medline]
Goode, B. L., Wong, J. J., Butty, A. C., Peter, M., McCormack, A. L., Yates, J. R., Drubin, D. G., and Barnes, G. (1999). Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast. J. Cell Biol 144, 83–98.
Grogan, A., Reeves, E., Keep, N., Wientjes, F., Totty, N. F., Burlingame, A. L., Hsuan, J. J., and Segal, A. W. (1997). Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils. J. Cell Sci 110, 3071–3081.[Abstract]
Guerin, I., and de Chastellier, C. (2000). Pathogenic mycobacteria disrupt the macrophage actin filament network. Infect. Immun 68, 2655–2662.
Heil-Chapdelaine, R. A., Tran, N. K., and Cooper, J. A. (1998). The role of Saccharomyces cerevisiae coronin in the actin and microtubule cytoskeletons. Curr. Biol 8, 1281–1284.[CrossRef][Medline]
Humphries, C. L., Balcer, H. I., D'Agostino, J. L., Winsor, B., Drubin, D. G., Barnes, G., Andrews, B. J., and Goode, B. L. (2002). Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin. J. Cell Biol 159, 993–1004.
Itoh, S., Suzuki, K., Nishihata, J., Iwasa, M., Oku, T., Nakajin, S., Nauseef, W. M., and Toyoshima, S. (2002). The role of protein kinase C in the transient association of p57, a coronin family actin-binding protein, with phagosomes. Biol. Pharm. Bull 25, 837–844.[CrossRef][Medline]
Jayachandran, R., Sundaramurthy, V., Combaluzier, B., Mueller, P., Korf, H., Huygen, K., Miyazaki, T., Albrecht, I., Massner, J., and Pieters, J. (2007). Survival of mycobacteria in macrophages is mediated by coronin 1-dependent activation of calcineurin. Cell 130, 37–50.[CrossRef][Medline]
Johannes, L., and Lamaze, C. (2002). Clathrin-dependent or not: is it still the question? Traffic 3, 443–451.[CrossRef][Medline]
Maniak, M., Rauchenberger, R., Albrecht, R., Murphy, J., and Gerisch, G. (1995). Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag. Cell 83, 915–924.[CrossRef][Medline]
Nal, B., Carroll, P., Mohr, E., Verthuy, C., Da Silva, M. I., Gayet, O., Guo, X. J., He, H. T., Alcover, A., and Ferrier, P. (2004). Coronin-1 expression in T lymphocytes: insights into protein function during T cell development and activation. Int. Immunol 16, 231–240.
Nguyen, L., Walburger, A., Houben, E., Koul, A., Muller, S., Morbitzer, M., Klebl, B., Ferrari, G., and Pieters, J. (2005). Role of protein kinase G in growth and glutamine metabolism of Mycobacterium bovis BCG. J. Bacteriol 187, 5852–5856.
Okumura, M., Kung, C., Wong, S., Rodgers, M., and Thomas, M. L. (1998). Definition of family of coronin-related proteins conserved between humans and mice: close genetic linkage between coronin-2 and CD45-associated protein. DNA Cell Biol 17, 779–787.[Medline]
Pantaloni, D., Le Clainche, C., and Carlier, M. F. (2001). Mechanism of actin-based motility. Science 292, 1502–1506.
Parente, J. A., Jr, Chen, X., Zhou, C., Petropoulos, A. C., and Chew, C. S. (1999). Isolation, cloning, and characterization of a new mammalian coronin family member, coroninse, which is regulated within the protein kinase C signaling pathway. J. Biol. Chem 274, 3017–3025.
Pieters, J. (2001). Evasion of host cell defense mechanisms by pathogenic bacteria. Curr. Opin. Immunol 13, 37–44.[CrossRef][Medline]
Rybakin, V., and Clemen, C. S. (2005). Coronin proteins as multifunctional regulators of the cytoskeleton and membrane trafficking. Bioessays 27, 625–632.[CrossRef][Medline]
Scherr, N., Honnappa, S., Kunz, G., Mueller, P., Jayachandran, R., Winkler, F., Pieters, J., and Steinmetz, M. O. (2007). From the cover: structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 104, 12151–12156.
Schuller, S., Neefjes, J., Ottenhoff, T., Thole, J., and Young, D. (2001). Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance. Cell Microbiol 3, 785–793.[CrossRef][Medline]
Smith, T. F., Gaitatzes, C., Saxena, K., and Neer, E. J. (1999). The WD repeat: a common architecture for diverse functions. Trends Biochem. Sci 24, 181–185.[CrossRef][Medline]
Steinman, R. M., and Swanson, J. (1995). The endocytic activity of dendritic cells. J. Exp. Med 182, 283–288.
Suzuki, K., Nishihata, J., Arai, Y., Honma, N., Yamamoto, K., Irimura, T., and Toyoshima, S. (1995). Molecular cloning of a novel actin-binding protein, p57, with a WD repeat and a leucine zipper motif. FEBS Lett 364, 283–288.[CrossRef][Medline]
Tailleux, L., Neyrolles, O., Honore-Bouakline, S., Perret, E., Sanchez, F., Abastado, J. P., Lagrange, P. H., Gluckman, J. C., Rosenzwajg, M., and Herrmann, J. L. (2003). Constrained intracellular survival of Mycobacterium tuberculosis in human dendritic cells. J. Immunol 170, 1939–1948.
Tulp, A., Verwoerd, D., Dobberstein, B., Ploegh, H. L., and Pieters, J. (1994). Isolation and characterization of the intracellular MHC class II compartment. Nature 369, 120–126.[CrossRef][Medline]
Vieira, O. V., Botelho, R. J., and Grinstein, S. (2002). Phagosome maturation: aging gracefully. Biochem. J 366, 689–704.[CrossRef][Medline]
Walburger, A., Koul, A., Ferrari, G., Nguyen, L., Prescianotto-Baschong, C., Huygen, K., Klebl, B., Thompson, C., Bacher, G., and Pieters, J. (2004). Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 304, 1800–1804.
Yan, M., Collins, R. F., Grinstein, S., and Trimble, W. S. (2005). Coronin-1 function is required for phagosome formation. Mol. Biol. Cell 16, 3077–3087.
Zheng, P. Y., and Jones, N. L. (2003). Helicobacter pylori strains expressing the vacuolating cytotoxin interrupt phagosome maturation in macrophages by recruiting and retaining TACO (coronin 1) protein. Cell Microbiol 5, 25–40.[CrossRef][Medline]
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