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The Journal of Immunology, 2007, 178, 5769-5778
Copyright © 2007 by The American Association of Immunologists, Inc.
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Coronin Function Is Required for Chemotaxis and Phagocytosis in Human Neutrophils1

Ming Yan2, Caterina Di Ciano-Oliveira, Sergio Grinstein and William S. Trimble3

Programme in Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Coronins are a family of conserved actin-associated proteins that have been implicated in a variety of cellular processes dependent on actin rearrangements. In this study, we show that in primary human neutrophils, coronins-1–4 and -7 are expressed. Coronin-1 accumulates at the leading edge of migrating neutrophils and at the nascent phagosome. Inhibition of coronin function by transduction of a dominant-negative form of the protein leads to inhibition of chemotaxis and a reduction in neutrophil spreading and adhesion. This inhibition appears to correlate with changes in the distribution of F-actin structures within the cell. In addition, phagocytosis is inhibited, but neither secretion nor activation of the NADPH oxidase appears to be affected. Together, these results show that coronins are required for actin-dependent changes in cell morphology that lead to migration and phagocytosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chemotaxis is the directed migration of cells toward a gradient of a chemical attractant. In mammals, chemotaxis plays an important role in a host of biological processes ranging from development to innate immunity. In the case of the immune system, many cells, but most notably neutrophils, efficiently sense chemical gradients to migrate toward the sites of injury or infection. This migratory response requires the development of polarity within the neutrophil and unique actin structures are assembled at the leading edge and rear of the cell to achieve movement. Although many of the regulatory proteins contributing to the polarized accumulation of actin have been described, additional factors are likely to also contribute to this regulation.

One such actin-associated protein that may be important for neutrophil chemotaxis is coronin. Coronin was first described in the slime mold Dictyostelium discoideum as a soluble protein that bound to actin-myosin complexes (1). Loss of the coronin gene product results in cells with impaired chemotaxis and phagocytosis (2). Coronins are conserved from yeast to humans, with at least seven isoforms being expressed in mammals (3), but relatively little is known about the specific roles of the mammalian forms or their functional relationship to the Dictyostelium form.

Recent structural studies of murine coronin-1 have revealed that the protein forms a seven-bladed beta propeller structure similar to the beta subunit of heterotrimeric G proteins (4). beta propellers are comprised of a protein domain called the WD domain, so-named due to the presence of conserved tryptophan and aspartate residues, and the WD domain folds such that it contributes to the formation of two adjacent blades of the propeller (5). The C-terminal extension of coronin-1 includes a coiled coil domain implicated in trimer formation (6). beta-propeller domains often represent interaction surfaces for protein-protein interactions, although it is not currently known what proteins bind to the coronin beta-propeller domains. Coronin-1 has been shown to associate with the Arp2/3 complex (7) and to F-actin (8, 9). In yeast, the Arp2/3 binding was mapped to C-terminal coiled-coil region (10) and electron microscopy indicated that this region bound near the p35 subunit of Arp2/3 to cause a conformational change in the complex (11). The Arp2/3 binding region of mammalian coronin-1 has yet to be determined. The regions of coronin-1 responsible for actin binding have been more controversial. Initial studies suggested that the strongest actin binding site was in the N-terminal 34 aa, while the second and third WD domains also had weak actin-binding capacity (9). More recently, studies have suggested that the C-terminal half of the protein bound and cross-linked actin filaments (12) while deletion of residues 400–416 resulted in a protein that did not bind to the actin cytoskeleton (6).

The precise role of coronin in actin assembly also remains unclear. In yeast, the coronin homolog Crn1p enhances barbed-end assembly, apparently by reducing the lag phase of polymerization (13). In contrast, Dictyostelium coronin associates with the entire length of actin filaments and it has been suggested to speed up depolymerization (14). It has also been suggested that the physical association of coronin with the Arp2/3 complex alters the latter’s activity (10), by causing a conformational change in the complex (11).

Recently, we examined the role of coronin-1 in phagocytosis in the murine macrophage cell line RAW 264.7. Using a previously described dominant-negative fragment of coronin-1 (15) linked to the protein transducing domain of TAT, we were able to acutely inhibit the function of coronin (16). We showed that phagocytosis in this model system depended on functional coronin-1. Moreover, while binding to and signaling from the FcRs appeared normal, and both actin and full-length (FL)4 coronin-1 accumulated at the phagocytic cup, the dominant-negative fragment prevented accumulation of Arp2/3 and completion of phagocytosis.

In this study, we set out to examine the role of coronin in the process of neutrophil chemotaxis. We first show that neutrophils express five of the seven human coronin genes. We then demonstrate that coronin-1 transiently accumulates at the leading edge of migrating neutrophils similar to F-actin. Moreover, by introducing the dominant-negative fragment of coronin-1 into neutrophils, we observed significant changes in their adhesion properties and a nearly complete block in chemotaxis. Phagocytosis was also impaired in these cells, but several other functions such as secretion and NADPH oxidase activation were unaffected. Our results demonstrate that coronin-1 plays a critical role in neutrophil adhesion and cell migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and Abs

All restriction enzymes were from New England Biolabs. DMEM, FBS, PBS, trypsin-EDTA, penicillin/streptomycin, and HEPES-buffered RPMI 1640 were purchased from Wisent. Paraformaldehyde was obtained from Canemco. Human IgG, human plasma fibronectin, and PMA were purchased from Sigma-Aldrich. Human coronin-1 cDNA was a gift from Dr. S. Toyoshima (National Institute of Health Science, Tokyo, Japan). Polyclonal anti-coronin-1 and N-terminal 60 coronin-1 antiserum were provided by Dr. J. Pieters (University of Basel, Basel, Switzerland). The plasmids of pTAT, pTAT-beta-galactosidase, and beta-galactosidase (non-TAT) were provided by Dr. S. F. Dowdy (University of California, San Diego, CA). Cy3-and Cy5-conjugated secondary Abs were obtained from Jackson ImmunoResearch Laboratories. Dihydrorhodamine 1,2,3 (DHR 1, 2, 3), rhodamine-phalloidin, and calcein AM were obtained from Molecular Probes. N-formyl-Met-Leu-Phe (fMLF) was obtained from Fluka Chemie. Collagen-coated Transwell chambers (3.0 µm pore size) were purchased from Corning. Polymorphprep was obtained from Accurate Chemical and Scientific. Polystyrene beads (3 µm diameter) were obtained from Bangs Laboratory. Fluorescent mounting medium was obtained from DakoCytomation.

Isolation of human neutrophils

Human blood was obtained from healthy volunteers. Donations were obtained following guidelines approved by the Hospital for Sick Children Research Ethics Board. The neutrophils were purified using a Polymorphprep gradient separation procedure according to the manufacturer’s instructions. Isolated neutrophils were resuspended in PBS containing 1 mM calcium chloride, 1 mM magnesium chloride, and 10 mM glucose at a concentration of ~1 x 106 cells/ml and kept at 4°C before use normally within 1–2 h (and never >6 h) of isolation. To ensure that cell viability was not compromised during preparation, the responsiveness to fMLF (chemotaxis and spreading, described below) was tested in all cases and nonresponsive preparations were discarded. Giemsa stain assay was used to evaluate the purity of the neutrophils, according to the manufacturer’s protocol. Briefly, 30 µl of freshly isolated neutrophils were loaded on the glass slide and a thin film was made using an applicator stick. Air-dried films were fixed by dipping the film in a Coplin jar containing absolute methanol for 10 min. Staining with Giemsa solution (1/20 dilution, v/v) was conducted for 20 min. Films were rinsed with distilled water and allowed to air dry. The slide was mounted and the neutrophils and other cell types were examined using a Leica DB LB2 microscope.

DNA constructs, protein purification, and RT-PCR amplification

TAT-WD and TAT protein purification were described previously (16). To generate the construct GST-WT-coronin, the WT-cor-GFP construct was digested with XhoI and AgeI, subcloned into the pTAT vector, and then digested with XhoI and NcoI and resubcloned into the pGEX-KG vector containing the GST tag. GST-coronin was expressed and affinity purified by a standard procedure. Briefly, the pGEX-coronin construct was transformed into DH5{alpha}, and grown in Luria Bertani broth to OD = 0.6 at 37°C and induced with 0.2 mM isopropyl beta-D-thiogalactoside (IPTG) for 4 h. Cell pellets were lysed in a French Press in STE (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 5 µg/ml leupeptin (pH 8.0)), and centrifuged at 12,000 x g for 30 min. The supernatant was applied to a glutathione-Sepharose 4B (Qiagen) column. The GST-coronin fusion protein was eluted with STE containing 20 mM reduced glutathione. Total RNA from white blood cells or neutrophils was extracted using Qiamp RNA blood mini kit (Qiagen) according to the manufacturer’s protocol. RT-PCR was used to amplify partial cDNAs of all seven coronins by using the one-step RT-PCR kit (Qiagen). All RT-PCR products were detected on a 1% agarose gel and confirmed by DNA sequence analysis. PCR primers of all seven human coronin genes were follows as (using the nomenclature in Ref. 5 ; coronin-1 sense 5'-GCTGCACGAGCGGAGGTGTG-3' and antisense 5'-ATCCGAGCTGGGAGTGCCAC-3'; coronin-2 sense 5'-TCCTTCCGCAAAGTGGTCCG-3' and antisense 5'-AGGCTGTCCAGGCGGTACAG-3'; coronin-3 sense 5'-CAGATTTGTTGCCATAATCATAGAG-3' and antisense 5'-TGATAATGGCATTATCACAGCC-3'; coronin-4 sense 5'-ACAAAGGAGTCTGTCATCACAAG-3' and antisense 5'-GCCATGGAATTGAAGATAGG-3'; coronin-5 sense 5'-CCATCACCAAGAATGTGCAC-3' and antisense 5'-GCAGTCAATCATCTTCACCG-3'; coronin-6 sense 5'-ATCCGGCCAGGACGCCGAACC-3' and antisense 5'-ACCAGCTCGCACAGCATGTTC-3'; coronin-7 sense 5'-GCCAGCTGCTCCTATATGAAG-3' and antisense 5'-TAGTC CCACTCGTCCTCGTC-3'.

Measurement of endogenous coronin concentration by immunoblotting

A total of 1.1 x 106 neutrophils was lysed in 1 ml of boiling 1% SDS lysis buffer, and passed five-time through a 27-gauge needle. A total of 2.5 µl of the neutrophil lysate (roughly 3000 cell equivalents) was subjected to SDS-PAGE on a 10% polyacrylamide gel. To generate a standard curve, purified GST-coronin from 0 to 10 ng was electrophoresed the same gel. Western blotting was then conducted to detect endogenous human neutrophil coronin-1 and purified GST-coronin using a polyclonal coronin-1 antiserum (1/10,000). Multiple exposures were generated to ensure that signals were obtained within a linear range. Densitometric scans of bands were performed and the concentration of endogenous coronin was interpolated from within the linear range of recombinant GST-coronin protein.

Transduction with TAT-beta-gal or TAT-WD protein

All TAT-WD and TAT-beta-galactosidase transductions were performed as described previously (16). Briefly, neutrophils and HeLa cells were first washed in PBS, treated with TAT-beta-gal or TAT-WD protein at a final concentration of 200 nM in PBS containing 1 mM calcium chloride, 1 mM magnesium chloride, and 10 mM glucose, then incubated at 37°C for 30 min, without CO2. To monitor beta-galactosidase activity, the cells treated with TAT-beta-gal were fixed for 5 min at 4°C and then incubated for 24 h at 37°C in a histochemical staining solution containing 3 mM ferrocyanide, 3 mM potassium ferricyanide, 10% DMSO, 2 mM MgCl2 and 1 mg/ml 5-bromo-4-chloro-3-indolyl beta-D-galactoside in PBS. Samples were examined using an Axiovert 200 M light microscope (Carl Zeiss) with a Microcolor RGB filter.

Cell spreading

Neutrophils, either treated with TAT-beta-gal, TAT-WD, or untreated, were plated on 20 µg/ml fibronectin-coated coverslips with or without 10–7 M fMLF for 3 min at room temperature (RT) to allow cell spreading. The cells were then fixed in 4% paraformaldehyde for 1 h and washed three times with PBS. The area of each cell was outlined and measured using Image J software. Data of the two-dimensional surface areas per cell are means ± SE of three independent experiments with at least 30 cells counted in each case. The t tests were used for pairwise comparisons and p < 0.05 was considered to be statistically significant.

Adhesion

Adhesiveness of neutrophils was measured by allowing ~10,000 neutrophils to briefly adhere at 37°C/well of a fibronectin (20 µg/ml) coated 96-well U-shaped plate. Purified human neutrophils were labeled with calcein AM for 10 min following three washes with PBS. Neutrophils were then pretreated for 30 min with TAT-WD or TAT-beta-gal as control, and allowed to adhere in PBS containing 1 mM calcium chloride, 1 mM magnesium chloride, and 10 mM glucose for 10 min at 37°C before removing nonadhesion cells by spinning plate upside down at 100 x g for 1 min. The bound cells were quantified in a fluorescence plate reader (Spectra MSX Gemini EM) at Ex 497/Em 517, then measured again after removing the nonadherent cells by centrifugation. The adhesive index was calculated as a ratio of the second measurement to the first. Data are means ± SE of three independent experiments. Asterisk (*) indicates p < 0.05 with at least 6 wells counted in each.

Immunofluorescence and phalloidin staining

To detect endogenous coronin-1 protein distribution, neutrophils treated with 10–7 M fMLF were allowed to spread on 25-mm fibronectin-coated coverslips at 37°C. The cells were fixed after 0 and 1 min in 4% paraformaldehyde for 1 h, and permeabilized for 15 min using 0.1% Triton X-100 containing 100 mM glycine. The neutrophils were immunologically stained with rabbit anti-coronin-1 (1/5000) for 1 h, washed, then incubated with Cy3 donkey anti-rabbit secondary Ab (1/2000) for another 1 h. To determine cell volumes, a z-series of images were captured using a Quorum spinning disk confocal microscope (Quorum Technologies) and Volocity software (Improvision) was used to determine total and lamellipodium volume and sum pixel intensity for each cell. In all cases, subthreshold intensities were used to ensure that the signals were not saturated.

To label F-actin, rhodamine-phalloidin was diluted to 0.4 U/ml in PBS and incubated with cells for 30 min. Coverslips were washed in PBS and mounted using DakoCytomation fluorescent mounting medium. Cells were imaged using a Zeiss LSM510 laser scanning confocal microscope.

Phagocytosis

Suspended neutrophils were left untreated (control) or either treated with TAT-beta-gal or TAT-WD protein for 30 min before exposure to polystyrene beads opsonized with human IgG and prestained with Cy2-conjugated anti-human Ab. Cells were then allowed to adhere for 5 min in ice-cold HEPES-buffered RPMI to coverslips coated with fibronectin. Unbound beads were washed away with ice-cold PBS. The binding index was calculated, using a phase contrast microscopy, as the average number of remaining beads associated with each cell. Alternatively, neutrophils were warmed to 37°C and allowed to undergo phagocytosis for 5 min. After phagocytosis, neutrophils were fixed in 4% paraformaldehyde at 4°C for 1 h without permeabilization and labeled with Cy3 Ab. The phagocytic index was calculated as the number of internalized beads, which are Cy2 positive and Cy3 negative. The index data are means ± SE of three independent experiments, with at least 30 cells counted in each case.

Neutrophil migration

The calcein AM-labeled neutrophils were treated with either TAT-beta-gal, TAT-WD protein for 20 min with rotation at room temperature. 50 µl of suspended cells (~10,000 cells) in RPMI 1640 without serum were loaded on a Transwell membrane (3-µm pore size and 6-mm diameter) in a 12-well plate. To keep a stable chemotaxis gradient, 10 µl of low melting agarose gel containing 10–6 M fMLF was coated on the bottom wells of chemotaxis chamber. Neutrophils were allowed to migrate across the Transwell membrane into the lower well along a gradient of chemoattractant in RPMI 1640 without serum at 37°C for 30 min intervals. The chamber was then moved to another well for the next time period and the number of calcein-AM positive cells that migrated into the lower well was summed. The data represent the mean value (SEM) from at least three independent experiments. To determine the concentration dependence of this effect, varying concentrations of fMLF ranging from 2 x 10–5 to 2 x 10–8 M were coated on the bottom wells of the chamber and neutrophils were allowed to migrate at 37°C for 2 h.

Oxidase analysis

Neutrophils were treated either with TAT-beta-gal, TAT-WD protein, or left untreated (control) for 30 min with rotation at room temperature. To measure NADPH oxidase activity, DHR 1,2,3 (up to 2 µM) was added for another 20 min while rotating at room temperature. PMA (2 µM) or nothing (control) was added and cells were incubated for 10 min at 37°C. Neutrophils were then fixed in 4% paraformaldehyde for 1 h and washed three times with PBS. Oxidase activity was measured as the average fluorescence of 10,000 cells (at least 104 gated events were recorded in each experiment) by using flow cytometric analysis. The index data are means ± SE of three independent experiments.

Alternatively, superoxide (the initial product formed by the NADPH oxidase) was measured over time by determining superoxide dismutase-inhibitable cytochrome c reduction (17). Briefly, a total of 125 µl of neutrophils in each assay well were pretreated either with TAT-beta-gal, or TAT-WD protein (~10,000 cells), then resuspended in PBS supplemented with divalent cations and glucose (0.9 mM CaCl2, 0.5 mM MgCl2, and 7.5 mM glucose) (PBSG). 75 µM of cytochrome C with or without superoxide dismutase (60 µg/ml) was then added for 10 min at 37°C. The neutrophils were then incubated with or without PMA (2 µM) before placing the 96-well microplate into the reading chamber with agitation at 37°C. The absorbance at 550 nm was quantified in a microplate reader (Versa Max) in kinetic mode for 20 min, acquiring readings at 10-s intervals.

Secretion assay

Neutrophils were treated either with TAT-beta-gal, TAT-WD protein, or left untreated (control) for 30 min with rotation at room temperature. To measure CD63 externalization as a measure of secretion, the cells were treated with fMLF (10–7 M), Ionomycin (5 µM), cytochalasin D (1 µM) and fMLF (10–7 M) in combination, or control in PBS containing 1 mM CaCl, 1 mM MgCl, and 10 mM glucose for a further 10-min incubation at 37°C. Neutrophils were then fixed in 4% paraformaldehyde for 1 h and washed three times with PBS. The neutrophils were incubated with mouse anti-CD63 (1/100) for 1 h, washed, then incubated with goat anti-mouse Alexa 488 secondary Ab (1/1000) for another 1 h. CD63 secretion was measured by using flow cytometric analysis as above.

Purification of GST-FL and WD coronin

Bl21(DE3) LysS cells transformed with either GST, GST-FL-coronin, or GST-WD-coronin were induced with 0.5 mM IPTG for 4 h at 30°C. Pelleted cells were frozen at –80°C and then lysed in STE buffer (10 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA) with the following additions: 1% Triton X-100, 1% L-sarcosine, 5 mM DTT, 200 µg/ml lysozyme, 5 mM benzamidine, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. The lysate was passed through a French Press (1000 {psi} x 2) and subsequently centrifuged at 12,000 rpm for 10 min. The supernatant was then passed through a glutathione-Sepharose 4B column (Amersham) and GST fusion proteins were eluted with 10 mM reduced glutathione in 50 mM Tris-Cl (pH 8.0) buffer (elution buffer).

Pyrene actin assay

Purified Arp2/3 complex, a GST-fusion of the verprolin-cofilin-acidic (VCA) domain of N-WASp, and rabbit skeletal-muscle G-actin labeled with pyrene with 20% efficiency were obtained from Cytoskeleton Inc. Actin polymerization was analyzed by the method of Uruno et al. (18). Briefly, G-actin was diluted to 0.4 mg/ml (10 µM) in G-buffer (5 mM Tris-Cl (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, and 0.5 mM DTT) and nucleation centers were removed by centrifugation at 200,000 x g for 2 h. Polymerization was initiated by adding 100 µl of pyrene G-actin to 200 µl of 1.5x polymerization buffer (7.5 mM Tris-Cl (pH 7.5), 1.5 mM EGTA, 0.15 mM CaCl2, 4.5 mM NaN3, 75 mM KCl, 3 mM MgCl2, 0.75 mM DTT, and 0.3 mM ATP). The final actin concentration was ~3.3 µM. Polymerization was then monitored by measuring the increase in fluorescence using a SpectraMAX Gemini EM fluorometer (Molecular Devices) with 365 ± 9 nm excitation and 407 ± 9 nm emission filters. Where indicated, elution buffer or purified proteins (Arp2/3, 30 nM; GST-VCA, 100 nM; GST-FL coronin, 300 nM; GST-WD coronin, 300 nM; and GST, 300 nM) were added to 1.5x polymerization buffer before the addition of pyrene G-actin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Several isoforms of coronin are expressed in neutrophils

Coronin is an actin-binding protein that has been implicated in chemotaxis in Dictyostelium. However, its role in this process in other organisms has not been defined. We were particularly interested in investigating the role of coronin proteins in the chemotaxis of mammalian cells, and primary human neutrophils provide an excellent model of chemotactic migration in vitro. To understand coronin function in neutrophils, we first set out to characterize the expression patterns of seven coronin genes in the human neutrophil. To do so, we created pairs of primers specific for each human isoform and performed RT-PCR on mRNA derived from human neutrophils or from pooled white cells from blood as a control. As can be seen in Fig. 1a, PCR products can be seen for all isoforms of coronin in a mixture of human white blood cells. The identity of all of the products was subsequently confirmed by DNA sequencing. In contrast to the white blood cell mixture, neutrophils purified to >97% based on Giemsa staining (data not shown) expressed transcripts for five of the isoforms, while coronins-5 and -6 were not detected (Fig. 1b). Unfortunately, due to the lack of specific Abs, we were unable to determine whether the protein is expressed for each coronin isoform. However, because coronin-1 has been the best characterized to date, and because reagents exist for its analysis, we subsequently focused on this isoform.


Figure 1
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  		FIGURE 1. Comparison of RNA expression of all white blood cells and neutrophils in seven coronin genes. Total RNA from white blood cells (a) or neutrophils (b) was extracted using the QiAmp RNA blood mini kit (Qiagen). RT-PCR were used to amplify partial cDNAs of all seven coronins in human neutrophils by using the one step RT-PCR kit (Qiagen). RT-PCR products of neutrophils and whole white blood cells were fractionated verified on a 1% agarose gel and verified by DNA sequencing. Products were electrophoresed on the same gels, but separated in the figure for presentation in order. Molecular mass markers are indicated at the right. Coronin numbering follows that of Rybakin and Clemen (5 ).

 
Coronin-1 accumulates at the leading edge of migrating human neutrophils

If coronin-1 is involved in chemotaxis in neutrophils, we would expect that it would undergo changes in distribution during cell migration. We therefore set out to define the distribution of coronin-1 in resting human neutrophils and in neutrophils that have been exposed to the chemoattractant peptide fMLF. As shown in Fig. 2a, in resting neutrophils that have recently adhered to glass coverslips, coronin-1 appears evenly distributed around the cell with higher concentrations near the plasma membrane. Following exposure to fMLF, the neutrophils become highly polarized and coronin-1 concentrates in the lamellipodium at the leading edge of the cell (Fig. 2b) where actin accumulation is also found (not shown).


Figure 2
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  		FIGURE 2. Distribution and concentration of coronin-1 in human neutrophils. Neutrophils treated with 10–7 M fMLF were loaded on 25-mm fibronectin-coated coverslips. The cells were fixed at 0 (a) and 1 min (b) following adhesion with 4% paraformaldehyde and permeabilization and immunological staining with rabbit anti-coronin-1. A projection of 35 0.15-µm Z-stack images collected using a Quorum-spinning disk confocal microscope is shown, with a single middle slice shown in the insets (a1, b1). Volocity software was used to measure total or lamellipodial volume of each cell and the sum pixel intensity. Scale bar in a, 5 µm. c, The endogenous neutrophil coronin-1 (indicated by open arrow) was quantified by immunoblotting lysate from ~3000 cells (first lane, c) on blots that included defined amounts of rGST-coronin-1 (closed arrow) and probed with Ab specific to coronin-1. A representative of three independent experiments is shown.

 
To determine the degree of recruitment of coronin-1 to the leading edge of the cell, we first estimated the concentration of coronin-1 in neutrophils. A protein lysate from 3000 neutrophils was electrophoresed and blotted together with defined amounts of recombinant coronin-1 and probed with anti-coronin-1 antiserum (Fig. 2c). Densitometric scans of the recombinant proteins were plotted, and the amount of coronin-1 protein in the lysate was interpolated from the linear portion of curves derived from several repeats of this experiment. Based on this approach we were able to estimate that each neutrophil contains ~0.83 pg of coronin-1 protein (Table I). We then estimated cell volumes using three-dimensional reconstruction of confocal images and determined that the average volume of a resting neutrophil was ~360 µm3. Taken together with the protein estimates, this indicates that the concentration of coronin-1 in neutrophils is ~42 µM.


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  		Table I. Determination of coronin-1 concentration in neutrophilsa

 
To determine the concentration of coronin-1 within leading edge lamellipodia we determined the volume of this portion of the cell, again by three-dimensional reconstruction of confocal slices. We then estimated the proportion of the coronin-1 that was localized to the lamellipodium. Cells that had been immunostained with anti-coronin-1 Ab were analyzed and the sum pixel intensities of those cells in which the signal was not saturating were measured. The sum pixel intensity of the entire cell was then compared with that of the lamellipodium to determine the fraction of coronin-1 that was recruited to this structure. We approximate that 31% of the coronin-1 is localized within the lamellipodia, and these structures average a volume of ~35 µm3, resulting in a local coronin-1 concentration of 146 µM (Table I).

Coronin-1 is also transiently recruited to the nascent phagosome

Coronin-1 has been previously shown to be transiently recruited to phagosomes in Dictyostelium (2), in macrophages ingesting opsonized RBC (16), and in both the neutrophil model system HL-60 cells (19) and in primary neutrophils (20) to opsonized zymosan. However, it has also been noted that immunoreactivity to coronin-1 (also called TACO) can be retained at the phagosome following internalization of Mycobacterium species and it has been suggested that this retention may contribute to the inhibition of phagosome maturation (21). We therefore set out to determine whether coronin-1 accumulates at the forming phagosome of adherent neutrophils exposed to opsonized latex beads, and how long it is retained. As can be seen in Fig. 3(top panels) both F-actin and coronin-1 appear at the phagosomal cup within 1 min of interaction with opsonized particles, even before it has sealed (arrows). Within 3 min, many of the particles have been fully engulfed and by this time some particles have begun to shed the coronin-1 from the inner surface of the phagosome (open arrowheads). By 5 min, many of the particles are no longer decorated with coronin-1 (open arrows). In all cases the time course of coronin-1 accumulation and release mirrored that of F-actin.


Figure 3
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  		FIGURE 3. Accumulation of coronin and actin at the phagocytic cup. Suspended neutrophils were mixed with human IgG-opsonized beads and allowed to adhere to 20 µg/ml fibronectin-coated coverslips for 5 min in ice-cold RPMI 1640. The neutrophils were warmed to 37°C to allow phagocytosis to proceed for 1 min (a–c), 3 min (d–f), or 5 min (g–i), then were fixed. After fixation, neutrophils were stained with rhodamine-phalloidin to detect F-actin (a, b, and g) and antiserum for coronin-1 (b, e, and h). c, f, and i, Differential interference contrast images of the cells. Filled arrowheads indicate the beads at unsealed phagocytic cups, open triangles indicate recently sealed phagosomes, and open arrowheads indicate phagosomes that no longer stain for coronin-1. Scale bar, 5 µm.

 
TAT transduction of dominant-negative coronins inhibits chemotaxis

The distribution of coronin-1 at the leading edge of migrating cells and at the phagocytic cup suggests that it may participate in regulating actin turnover in these dynamic processes. However, neutrophils are refractory to transfection, limiting the number of approaches that can be used to gain insights into protein function. We have previously described a dominant-negative form of coronin-1 that inhibited the function of this protein during phagocytosis in the murine macrophage RAW 264.7 cell line. This involved the transduction of a domain of coronin-1 containing the WD repeat sequences into cells acutely through the addition of a membrane permeable TAT transduction domain (TAT-WD) (16). To first confirm that neutrophils would be efficiently transduced, we incubated neutrophils or HeLa cells in the presence recombinant forms of either beta-galactosidase or beta-galactosidase fused to the TAT transduction peptide (TAT-beta-gal) and found that both cell types were readily transduced by the TAT-beta-gal but not by beta-galactosidase alone (data not shown).

We then assayed the ability of dominant-negative coronin-1 to inhibit cell migration. Neutrophil migration was measured across a permeable Transwell membrane. Transwell chambers were placed in culture dishes containing fMLF and the number of neutrophils able to migrate across the Transwell membrane into the dish was counted. Transwell chambers were moved to fresh dishes after each 30-min interval to ensure a gradient was maintained, and the cumulative number of cells that migrated was determined. As shown in Fig. 4a, treatment of neutrophils with TAT-WD significantly inhibited their ability to migrate across the membrane, compared with control cells that had been treated with TAT-beta-gal. Because we coated the base of the dishes with fMLF embedded in soft agar to maintain a standing gradient, we could not be sure of the precise concentration of fMLF that the cells received, and because sensitivity to fMLF follows a bell-shaped curve, it remained possible that TAT-WD did not inhibit chemotaxis, but simply altered the sensitivity to fMLF. We therefore repeated the experiment using different starting concentrations of fMLF and found that while the control cells were sensitive to fMLF over a defined concentration range, the TAT-WD-treated neutrophils were unable to chemotax at any of the fMLF concentrations tested (Fig. 4b).


Figure 4
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  		FIGURE 4. Effects of TAT-WD on migration of neutrophils through Transwell filters. a, The calcein AM (1:1000) labeled neutrophils were treated with either TAT-beta-gal or TAT-WD protein for 20 min with rotation at room temperature. A total of 50 µl of suspended cells in RPMI 1640 were loaded in each Transwell suspended in a well of a 12-well plate. Cells were allowed to migrate across the Transwell membrane into a lower well chamber along a gradient of fMLF in RPMI 1640 at 37°C for four 30-min intervals. The number of calcein-positive cells that had migrated into the lower wells was counted using a x40 objective on an inverted fluorescent microscope after moving the Transwell into the next well. The data represent the mean value (SEM) from three independent experiments. b, Similar experiments were performed using different concentrations of fMLF. In this case the cells were collected at a single time point (2 h). Data are from three independent experiments. In the presence of TAT-WD, no significant migration was measured at any concentration of fMLF used.

 
TAT-WD inhibits adhesion and spreading of neutrophils

To gain insights into the mechanisms behind the inhibition of chemotaxis, we determined whether the neutrophils were altered in their ability to adhere to the substratum. We had previously shown that inhibition of coronin-1 function in RAW 264.7 cells resulted in their rounding and detachment from the substratum. To measure adherence, we allowed neutrophils to bind to fibronectin-coated multiwell plates for 30 min, then dislodged weakly adherent cells with a light centrifugal force away from the substratum. As shown in Fig. 5, TAT-WD-treated neutrophils were much less able to adhere tightly to the substratum.


Figure 5
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  		FIGURE 5. Effects of TAT-WD on adhesion of neutrophils. Calcein AM-labeled neutrophils were treated with TAT-WD or TAT-beta-gal for 30 min at 37°C, and were loaded on fibronectin (20 µg/ml) coated 96-well U-shape wells for 10 min at 37°C to allow cell spreading. The number of bound cells was first measured by putting the plate into a fluorescence plate reader (Spectra MSX Gemini EM) to measure the basal fluorescence signal of each well at Ex 497/Em 517. Adhesion was then measured by measuring the fluorescent signal under the same conditions after removing nonadherent cells by spinning the plate upside down at 100 x g for 1 min. The adhesive index was calculated at a ratio of second to first measurement. Data are means ± SE of three independent experiments. *, p < 0.05 with at least six wells counted in each.

 
Because the TAT-WD-treated cells were less adherent, we asked whether they were less able to spread on the substrate. Neutrophils treated with either TAT-beta-gal, TAT-WD or left untreated, were allowed to spread on fibronectin-coated coverslips in the absence or presence of fMLF for 3 min, then photographed (Fig. 6, a–f). The surface areas of the cells were then measured and, as shown in Fig. 6g, there was little difference in area of cells that had not been exposed to fMLF, but spreading in the presence of fMLF was significantly reduced in TAT-WD-treated cells.


Figure 6
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  		FIGURE 6. Effects of TAT-WD on neutrophil spreading. Neutrophils, untreated control (a and d), or treated with either TAT-beta-gal (b and e) or TAT-WD (c and f), were plated on 20 µg/ml fibronectin-coated coverslips with 10–7 M fMLF (a–c) or without fMLF (d–f) for 3 min at RT to allow cell spreading. Scale bar, 20 µm. To determine the extent of spreading, the area of each cell was outlined by using Image J software and tabulated (g). These data are means ± SE of three independent experiments with at least 30 cells counted in each case. *, p < 0.05.

 
To understand the mechanism responsible for the defect in spreading and adhesion, we examined the actin distribution in TAT-transduced neutrophils that had been incubated with or without fMLF for 3 min. In cells transduced with the control TAT-beta-gal protein, the majority of F-actin was detected at the bottom of the cell in punctate structures (Fig. 7a), and in the presence of fMLF F-actin was also recruited to ruffles at the periphery of the cell membrane (Fig. 7b). In contrast, in TAT-WD-treated cells, the F-actin was found around the entire cell membrane, often in blobs or associated with membrane blebs (Fig. 7, c and d) and very little remained associated with the bottom of the cell (Fig. 7, c1 and d1). It therefore appears that TAT-WD altered actin assembly, preventing the formation of adhesive structures needed for binding, spreading, and chemotaxis.


Figure 7
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  		FIGURE 7. Effect of TAT-WD on actin distribution of neutrophils. Neutrophils were treated with either TAT-beta-gal (a and b) or TAT-WD (c and d) protein for 30 min with rotation at room temperature, and allowed to adhere to 20 µg/ml fibronectin-coated coverslips with (a and c) or without (b and d) 10–7 M fMLF for 3 min in RPMI 1640. The cells were fixed, permeabilized, and stained with rhodamine-phalloidin to detect F-actin. TAT-beta-gal-treated cells appeared normal with most actin associated with the basal surface of the cell (a and b). The TAT-WD-transduced cell had dramatically altered actin morphologies in both fMLF and control conditions. TAT-WD-treated cells were rounder and actin was associated with the membrane at all planes of the cell (a section through the middle of the cell is shown in c and d). Inset images taken at the basal surface of the cell (c1, d1) revealed few adhesion structures. Scale bar, 5 µm.

 
TAT-WD also inhibits phagocytosis, but not secretion or oxidase activation

We then examined the effect of TAT-WD on other cellular processes in which actin polymerization may play a role. To examine the role of coronin-1 at the phagocytic cup, neutrophils treated with either TAT-beta-gal or TAT-WD were incubated in the presence of opsonized beads on ice to allow binding to the neutrophils, then warmed to 37°C for 5 min to allow phagocytosis to proceed. The efficiency of phagocytosis was determined by staining the external particles of nonpermeabilized neutrophils with anti-human IgG Ab. As can be seen in Fig. 8, a–d, there were no external beads associated with neutrophils incubated with TAT-beta-gal whereas those cells treated with TAT-WD had ingested significantly fewer particles. These data are presented quantitatively in Fig. 8e. The number of beads associated with each cell was measured as the binding index, and as shown in Fig. 8e, this was not affected by TAT-WD treatment.


Figure 8
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  		FIGURE 8. Phagocytosis was inhibited by TAT-WD in neutrophils. Neutrophils treated with either TAT-beta-gal (a and c) or TAT-WD (b and d) protein were mixed with human IgG-opsonized beads, which was prelabeled with Cy2 Ab, and allowed to adhere to 20 µg/ml fibronectin-coated coverslips for 5 min in ice-cold HEPES-buffered RPMI 1640. Unbound beads were washed away with ice-cold PBS. The binding index was calculated using a phase contrast microscope to count the average number of remaining beads bound per cell (e). Alternatively, neutrophils were warmed to 37°C and allowed to undergo phagocytosis for 5 min. After phagocytosis, neutrophils were fixed in 4% paraformaldehyde at RT for 1 h without permeabilization and labeled with Cy3 Ab. a and b, External and internal beads (Cy2 positive and Cy3 negative), while c and d show external beads (Cy2 positive and Cy3 positive). The phagocytic index was calculated by counting the number of Cy2-positive and Cy3-negative beads associated with cells (e). The index data are means ± SE of three independent experiments, with at least 30 cells counted in each case. *, p < 0.05. Scale bar, 5 µm.

 
In contrast to phagocytosis and chemotaxis, other properties of the neutrophils were unaffected by TAT-WD treatment. For example, CD63 externalization as a marker of secretion was measured by flow cytometry and we found that control cells, TAT-beta-gal and TAT-WD-treated cells had similar surface levels of CD63 following treatment with fMLF alone, with cytochalasin D and fMLF, or following ionomycin treatment (data not shown).

The NADPH oxidase was previously shown to associate in a complex with coronin-1 during phagocytosis (20, 22), raising the possibility that coronin-1 participates in NADPH oxidase assembly and recruitment to the phagosome. We therefore measured oxidase activity by labeling cells with the nonfluorescent precursor dihydrorhodamine 1,2,3, which is converted to a fluorescent product, rhodamine 1,2,3, by reactive oxygen species. Formation of rhodamine 1,2,3 was quantified by flow cytometry. No difference in oxidase activity was observed following activation of the neutrophils with phorbol esters in the presence or absence of TAT-WD (Fig. 9a). In addition, we used a colorimetric assay to measure the kinetics of superoxide production, based on its ability to reduce cytochrome c. As shown in Fig. 9b, no significant difference was noted in the production of superoxide following phorbol ester activation in the presence of TAT-WD or the control TAT-beta-gal. Together, these data indicate that the function of coronin is required for some but not all of the dynamic changes associated with neutrophil activation.


Figure 9
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  		FIGURE 9. Oxidase activity is not affected by TAT-WD. a, Neutrophils were treated with either TAT-betaGal or TAT-WD protein for 30 min before incubation with dihydrorhodamine 1,2,3 (2 µM). The cells were then activated PMA (2 µM) or vehicle for a further 10 min at 37°C, fixed in 4% paraformaldehyde, and measured for oxidase activity by flow cytometric analysis (at least 104 gated events were recorded in each experiment). The index data are means ± SE of three independent experiments. *, p < 0.05. b, The superoxide dismutase-sensitive component of the reduction of cytochrome c was monitored spectroscopically as an increase in absorbance at 550 nm, and presented as nanomoles of superoxide using Beer’s law, as described previously (17 ). Values plotted are the means ± SE of three independent experiments.

 
FL coronin-1, but not TAT-WD, inhibits Arp2/3 function

In vitro studies had previously shown that a small portion of the C terminus of yeast coronin inhibits the activity of Arp2/3 (9). We had previously shown that Arp2/3 accumulation at the nascent phagosome in RAW 264.7 macrophage cells was inhibited by TAT-WD treatment and this correlated with a reduced accumulation of actin (15). To determine whether the WD domain of coronin-1 inhibited Arp2/3, we tested its effect on a pyrene actin polymerization assay. Rather than using the TAT-tagged constructs, we used GST-fusions of the WD domain and FL coronin-1, because the FL protein was extremely insoluble and the GST domain contributed to its solubility. As shown in Fig. 10a, FL coronin-1 is poorly expressed in bacteria, compared with the WD domain alone.


Figure 10
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  		FIGURE 10. Effect of GST-coronin-1 proteins on Arp2/3-dependent actin polymerization. a, Coomassie staining of GST-FL coronin-1 (cor) and GST-WD cor purified from bacterial lysates. b, The polymerization of pyrene-labeled G-actin (final concentration, 3.3 µM) was assessed by measuring the increase in fluorescence intensity over time. Where indicated, Arp2/3 (30 nM) and GST-VCA (100 nM) were added alone ({square}) or together with either buffer control (vehicle, {diamond}, GST (300 nM, {circ}), GST-FL cor (300 nM, •) or GST-WD cor (300 nM, {blacktriangleup}).

 
As has been previously seen, actin alone can polymerize but does so with a long lag phase due to its weak nucleating capacity (Fig. 10b). When incubated in the presence of Arp2/3 and its activator, the VCA domain of WASp, nucleation is accelerated, leading to a leftward shift of the curve. As expected, based on the yeast studies, FL GST-coronin1 significantly inhibited Arp2/3-dependent nucleation, although not to the extent seen for the yeast protein. The inability of this protein to completely inhibit actin nucleation is likely due to the insolubility of the mammalian protein and the difficulty expressing it at high concentrations. In contrast, the GST-WD domain had no effect on the ability of Arp2/3 to promote nucleation, despite its much greater solubility. Thus, consistent with the yeast coronin protein, the domains of coronin that inhibit Arp2/3 function lie outside the WD repeats.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we have shown that human neutrophils transcribe five of the seven coronin genes in contrast to whole white blood cell fractions in which transcripts for all seven genes can be detected. This was somewhat surprising because coronins-1 and -7 were thought to be primarily expressed in hemopoietic tissue, coronins-5 and -6 were thought to be mainly in the brain and coronins-2–4 were thought to be broadly expressed (5). The neutrophils transcribe the hemopoietic and generally expressed forms, but fail to transcribe the two forms abundantly expressed in the brain. It would be of interest to know which cells in the white blood cell fraction transcribe coronins-5 and -6. Our PCR analysis was not quantitative, so there could be relatively little mRNAs of some isoforms, but clearly multiple coronins mRNAs are present. If each of these transcripts corresponds to a functional protein product, then it is possible that coronin function may be redundant in neutrophils, although the sequence similarity between the different isoforms is relatively low. Although they may have overlapping functions, at least some coronins are thought to have distinct distributions. For example, mammalian coronin-7 was found to be localized to the Golgi and its localization did not depend on actin polymerization (23).

In the case of coronin-1, we have shown that it undergoes reorganization during the polarization of the neutrophil, accumulating at the leading edge of the migrating cells. We noted that the concentration increased more than three fold at the leading edge of the cell, potentially concentrating coronin-associated molecules that could participate in actin remodeling. This is a region known to be undergoing dynamic changes in actin structure and a great deal of actin nucleation and branching occurs through the action of the Arp2/3 complex. Coronin-1 has previously been shown to associate with Arp2/3 and in yeast the complex is thought to be regulated by its association with crn1p (10). Indeed, we show here that FL recombinant coronin-1 also inhibits Arp2/3-stimulated actin nucleation, although not as efficiently as was observed for the yeast ortholog. This inefficiency is likely to be due to with the limited solubility of the recombinant mammalian coronin-1 protein, rather than to distinct properties of the protein. However, the WD domains had no effect on this activity, suggesting that their role may be limited to recruitment of the complex. Hence, the recruitment of coronin to the leading edge of the cell could be important for the actin nucleation and branching activities conducted by Arp2/3.

Coronin-1 has also been suggested to serve as a bridge between the actin cytoskeleton and the plasma membrane due to its membrane association (6). It should be noted that the surface area of the leading edge of neutrophils is also vastly increased to extensive ruffling and invaginations of this area (24, 25). We can therefore not distinguish whether the concentration reflects an enrichment of the protein within the small cytoplasmic volume of the lamellipod or if its concentration reflects the enrichment of membrane domains to which it may be associated. In either case, the polarized accumulation of coronin-1 would be important in ensuring the actin polymerization also occurs in a polarized manner, facilitating cell migration. When cells were pretreated with coronin their attachment to, and spreading on, the substratum was also impaired because these processes would require dynamic remodeling of actin structures.

We had previously used the dominant-negative TAT-WD coronin-1 fragment to inhibit coronin function in macrophage cells and found that this protein inhibited the phagocytosis at a stage consistent with a role in actin polymerization (16). We show here that phagocytosis in neutrophils is also arrested by treatment of cells with this protein. Hence, coronins appear to have a conserved role in phagocytosis by a variety of cell types.

A failure to observe any effect of the TAT-WD on oxidase activation was somewhat surprising given that coronin-1 was previously shown to associate directly with the p40phox subunit in a complex with p47phox and p67phox (22). In addition, it has been shown that upon treatment of neutrophils with PMA, both p40phox and coronin showed redistribution to perinuclear regions and this was not observed in patients of chronic granulomatosis disease lacking p47phox or p67phox (22). This led the authors to speculate that the phox proteins may contribute to the regulation of the actin cytoskeleton through their interaction with coronin. Allen et al. (20) demonstrated that in patients lacking p47phox, coronin-1 was still recruited to the phagosome in the absence of p67phox and in time course studies showed that in neutrophils from chronic granulomatous disease patients, there was accumulation of p47phox and p67phox in the periphagosomal area, but this association was only transient and was lost upon dissociation of the actin and coronin-1. This raised the possibility that coronin-1 could participate in the stabilization of the NADPH oxidase complex in conjunction with p91phox and p22phox. Although the present study does not directly address this possibility, it shows that the activation of the NADPH oxidase does not depend on the ability of coronin to mediate actin remodeling. However, it remains possible that the association of endogenous coronin-1 to p40phox is not altered in the presence of TAT-WD.

Although we cannot be certain at this time that all of the coronin isoforms are expressed as proteins in neutrophils, their potential plurality raises the possibility that the dominant-negative form of the protein is not only inhibiting coronin-1, but could be generally blocking common functions of all coronins. In the case of macrophages, we were able to use small interfering RNA techniques that allowed us to conclude that coronin-1 function alone was required for phagocytosis (16). Unfortunately, because neutrophils are short-lived and refractory to transfection, we are unable to use similar approaches here. Long-term studies of the functions of coronin isoforms in neutrophils will require either the use of specific dominant-negative forms for each isoform or the generation of tissue-specific isoform knockouts to address their individual roles.


   Acknowledgments
 
We thank Dr. Satoshi Toyoshima for the coronin-1 cDNA, Dr. Jean Pieters for polyclonal anti-coronin-1 Abs, and Dr. Steve Dowdy for pTAT, pTAT-beta-galactosidase, and beta-galactosidase (non-TAT) constructs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Canadian Institutes of Health Research. S.G. is the current holder of the Pitblado Chair in Cell Biology. W.S.T. is a recipient of a Canada Research Chair in Molecular Cell Biology. Back

2 Current address: Division of Nephrology, Duke University Medical Center, Durham, NC 27708. Back

3 Address correspondence and reprint requests to Dr. William S. Trimble, Programme in Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. E-mail address: wtrimble{at}sickkids.on.ca Back

4 Abbreviations used in this paper: FL, full length; fMLF, N-formyl-Met-Leu-Phe; RT, room temperature. Back

Received for publication May 5, 2006. Accepted for publication February 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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