Coronin-1 expression in T lymphocytes: insights into protein function during T cell development and activation
Béatrice Nal1,2,
Patrick Carroll3,
Elodie Mohr1,
Christophe Verthuy1,
Maria-Isabel Da Silva1,
Odile Gayet3,
Xiao-Jun Guo1,4,
Hai-Tao He1,
Andrés Alcover2 and
Pierre Ferrier1
1 Centre dImmunologie de MarseilleLuminy, INSERMCNRSUniversité de la Méditerranée, Case 906, 13288 Marseille Cedex 9, France 2 Unité de Biologie des Interactions Cellulaires, CNRS URA 2582, Institut Pasteur, Paris 75724, France 3 Institut de Biologie du Développement de Marseille (IBDM), INSERM U382, Marseille 13288, France 4 Institut Méditerranéen de Recherche en Nutrition, UMR-INRA, Marseille 13397, France
Correspondence to: P. Ferrier; E-mail: ferrier{at}ciml.univ-mrs.fr
Transmitting editor: J. Borst
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Abstract
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Coronin has been described as an actin-binding protein of Dictyostelium discoideum, and it has been demonstrated to play a role in cell migration, cytokinesis and phagocytosis. Coronin-related proteins are found in many eukaryotic species, including Coronin-1 in mammals whose expression is enriched in the hematopoietic tissues. Here, we characterize Coronin-1 gene and protein expression in mouse embryonic and adult T lymphocytes. Coronin-1 is expressed throughout T cell ontogeny and in peripheral
ß T cells. Expression varies along thymic cell development, with maximum levels observed in embryonic early thymocytes and, in the adults, the selected TCR
ß+ single-positive thymocytes. Subcellular localization analysis indicates that Coronin-1 is in equilibrium between the cytosol and the cell cortex, where it accumulates in F-actin-rich membrane protrusions induced by polarized activation of TCRCD3-stimulated T cells. These data are consistent with a role of Coronin-1 in T cell differentiation/activation events involving membrane dynamisms and the cortical actin cytoskeleton.
Keywords: actin cytoskeleton, Coronin, T lymphocyte
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Introduction
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The Coronin protein was first identified in the slime mold Dictyostelium discoideum as an actin-binding factor localized to cell surface projections (1). It has been further defined as a WD-repeat protein [WD-repeats are protein motifs thought to be organized into a propeller-like structural domain involved in proteinprotein interactions; (2)], with a role in cell migration, cytokinesis and phagocytosis (35). Coronin-like factors have since been found in many species, from yeast to mammals including the human, bovine and mouse [(6) and references therein; also see (710)]. In mammals, seven paralogues have been described for which the degree of homology ranges beyond 60% amino acid identity.
Coronin-1 [also known as p57, clabp (for coronin-like actin binding protein) or TACO (for tryptophane aspartate-containing coat protein)] was the first mammalian paralogue to be identified and the one that has been studied the most (7,1116). In mouse, the Coronin-1 gene (comprised of 11 exons) maps on chromosome 7 (14) and was described to be preferentially expressed in the hematopoietic tissues (7,11). The corresponding 57-kDa protein contains five consecutive WD-repeat motifs within the N-terminal region, a unique so-called linker region and a leucine zipper domain at the C-terminus (instead of the less-elaborated forms of coiled-coil domains found in other Coronin-like proteins). In neutrophils and macrophages, Coronin-1 interacts with F-actin surrounding the phagocytic vacuoles (12). Within the phagosome coat, Coronin-1/TACO was also proposed to prohibit the fusion of Mycobacterium bovis-containing phagosomes with lysosomes, thus contributing to the long-term survival of the pathogen in host macrophages (7). Phagosomal association of Coronin-1/TACO may depend on cholesterol, a factor that may also be essential for mycobacteria uptake by macrophages (16). However, these data remain controversial as another study led to the conclusion that Coronin-1 is involved in bacterial uptake, but not in phagosome maintenance (17). Clearly, much is still to be learnt on the expression and actual function(s) of Coronin-1 in cells of the various hematopoietic lineages. In this study, we focus on Coronin-1 expression in mouse T lymphocytes. Our results suggest a role for Coronin-1 during T lymphocyte ontogeny, and, specifically, in processes that involve the dynamics of the actin cytoskeleton in response to TCR stimulation and cell activation.
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Methods
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Mice, cell lines, flow cytometric analysis, cell stimulation procedures and immunofluorescence (IF) microscopy
Wild-type BALB/c mice were housed in a specific pathogen-free animal facility in accordance with institutional guidelines. Adult mice were sacrificed for analysis between 3 and 6 weeks of age. Embryos were aged based on a daily vaginal plug assessment, 0.5 day post-coitum (d.p.c.) being the day on which a plug was detected.
The mouse cell lines that have been used in this study included the pre-T cell line T3 (18), A20 B lymphoma cells and NIH 3T3 fibroblasts (ATCC TIB-208 and CRL-1658 respectively). The human leukemia T cell line Jurkat, clone J77cl20 (19), was also utilized. Cell culture, staining conditions and flow cytometric analysis were carried out as described previously (20). T cell enrichment from mouse lymph nodes was performed by B cell depletion using CD19 microbeads and CS type columns (Miltenyi Biotec, Paris, France) as recommended by the manufacturer.
For cell activation, lymph node T cells from BALB/c mice were incubated at 37°C for 30 min with MHC class II+ A20 B lymphoma cells that had been pre-pulsed with 5 µg/ml each of Staphylococcus aureus enterotoxin A, B, C3 and E superantigens. Jurkat J77cl20 T cells were activated by incubation with antibody-coated 5-µm microspheres at 4°C for 30 min and then at 37°C for 1 min. When necessary, cells were pre-incubated (37°C for 30 min) with medium containing 10 µM PP1 (Calbiochem, La Jolla, CA) or 2 µM latrunculin A (Sigma-Aldrich, St Quentin Fallavier, France). Following incubation, the cells were set to adhere onto poly-L-lysine-coated coverslips and processed for IF as described by Roumier et al. (21).
Antibodies
Phycoerythrin (PE)-, cytochrome c, allophycocyanin- and PerCP Cyanine 5.5-conjugated mAb against the CD8 (536.7), CD4 (L3T4 RM 4-5), CD44 (Pgp-1), CD25 (PC61), B220 (RA3-6B2), Mac-1 (M1/70) and Gr-1 (RB6-8c5) markers were purchased from PharMingen (San Diego, CA). The
-P400413 Coronin-1 antiserum (see below) was revealed using a FITC-coupled goat anti-rabbit antibody. The anti-human CD3 mAb UCHT1 (IgG1) was used as described previously (21). The mAb against human CD43 (TP1/36/1/1) (22) was a gift from Dr F. Sanchez-Madrid (Hospital Universitario de la Princesa, Madrid, Spain).
RNA preparation, northern blot and RT-PCR analysis, and cDNA cloning procedures
Total RNA extraction from mouse embryos and adult tissues, and northern blot analysis were performed using standard protocols. The Coronin-1 and Hprt probes utilized for northern blot hybridization were prepared by DNA PCR amplification using wild-type mouse thymus cDNA templates. RT-PCR reactions were performed as outlined previously (23) (oligonucleotide primers available upon request). cDNA cloning procedures were carried out using standard protocols of molecular biology.
In situ hybridization assays
In situ hybridization was performed as described by Carrier et al. (24). The RNA probes were prepared by in vitro transcription using the Coronin-1 cDNA in the presence of digoxigenin-coupled UTP (Boehringer Mannheim, Meylan, France), as recommended by the manufacturer. Revelation of the hybridized probes was performed using an alkaline phosphatase-coupled anti-digoxigenin antibody and the NBT/BCIP-specific substrates (Boehringer Mannheim).
Production of the Coronin-1 antiserum and western blot procedures
The
-P400413 antiserum was produced in rabbits (Eurogentec Bel, Seraing, Belgium) by immunization with an ovalbumin-coupled Coronin-1 peptide (amino acid residues 400413). For western blotting, thymocyte extracts in Laemmli lysis buffer were separated by electrophoresis through a 10% polyacrylamide gel and transferred onto a PVDF membrane (Millipore, Bedford, MA). After saturation (in PBS x 1, 0.1% Tween 20, 5% milk), the membrane was incubated with the
-P400413 antiserum, and the reaction was revealed using a horseradish peroxidase-coupled anti-rabbit antiserum and the ECL western blotting revelation reagent (Amersham Pharmacia Biotech, Little Chalfont, UK).
Isolation of detergent insoluble membrane components (DIM)
DIM were isolated as described previously (25). Briefly, thymocytes (2 x 108) were gently sonicated (5-s, 5 W, Vibracell; Bioblock Scientific, Illkirch, France) in 1 ml of ice-cold sonication buffer (25 mM HEPES, 150 mM NaCl, 1 mM EGTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml chymostatin and 5 µg/ml
2-macroglobulin). The post-nuclear supernatant was prepared and subjected to solubilization with 1% Brij 98 at 37°C for 5 min. Samples were diluted with 2 ml of sonication buffer containing 2 M sucrose, chilled on ice and laid at the bottom of a step sucrose gradient (0.9, 0.8, 0.75, 0.7, 0.6, 0.5, 0.4, 0.2 M sucrose; 1 ml each) in sonication buffer. After centrifugation at 38,000 r.p.m., 4°C for 16 h in a SW41 rotor (Beckman Instruments), 1-ml fractions (nos 18) were harvested from the top of the gradient; the last fraction (no. 9) contains 3 ml. DIM are comprised within pooled fractions nos 25; the heavy (H) material (i.e. the bulk of solubilized materials at the bottom of the gradient) corresponds to pooled fractions nos 8 and 9.
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Results
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Coronin-1 gene expression in adult and embryonic mouse tissues
The Coronin-1 gene was reported to be preferentially expressed in hemato/lymphopoietic tissues including the thymus and spleen (7,11,13). To confirm and extend these findings, we first performed northern blot analysis of tissues from adult wild-type mice using a cDNA probe specific for mouse Coronin-1 (Nal, unpublished data). Indeed, we detected Coronin-1 RNA predominantly in the thymus, spleen and lymph nodes, whereas no, or much weaker, signal was found in other, mostly non-hematopoietic, tissues (including liver, testis, kidney, brain, muscle, heart and intestine; Fig. 1A). That the thymus signal is contributed to a large extent by T cells was confirmed in parallel analysis of isolated thymocytes. In separate RT-PCR experiments, we found very low levels of Coronin-1 expression in the developing mouse embryo at 7.5 d.p.c. (E7.5) and E8.5, and increased signals from E9.5 onwards (Fig. 1B)an expression profile that parallels the timings of development and expansion of the hemato/lymphopoietic cell lineages during mouse embryogenesis (26).

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Fig. 1. Analysis of Coronin-1 gene expression. (A) Total RNA (15 µg) from the indicated adult mouse lymphoid and non-lymphoid tissues was analyzed by northern blot using a 32P-labeled Coronin-1 cDNA probe (Cor.-1, top panel) and, following membrane stripping, for hybridization to an Hprt control probe (bottom panel). Th, Sp, LN, Li, Te, Kd, Br, Mu, He and Int: adult mouse thymus, spleen, lymph nodes, liver, testis, kidney, brain, muscle, heart and intestine respectively; Thc, adult mouse isolated thymocytes. (B) RT-PCR assays using total RNA (2 µg) from whole mouse embryos at the indicated d.p.c. stages (E7.5E14.5) and Coronin-1 or ß-actin specific oligonucleotide primers (top and bottom panels respectively). Assays were performed in the presence (+) and absence () of reverse transcriptase to control for contamination by genomic DNA. (C) In situ hybridization for Coronin-1 expression using transversal cross-sections of mouse embryos at d.p.c. 10.5, 12.5 and 14.5 (E10.5, E12.5 and E14.5), and a Coronin-1 RNAdigoxigenin antisense probe (magnification: x16); DRG, dorsal root ganglia; mn, motor neuron. (D) Image of an antisense probe hybridized to a E14.5 transverse cross-section, focusing on the thymus image (left); hybridization with the control sense probe is shown on the right (magnification: x40). Arrows indicate zones of Coronin-1 prominent cell staining. (E) Antisense hybridization of an adult thymus cross-section focusing on the thymic cortex (C) and medulla (M) areas (left panel: magnification: x16; middle and right panels: magnification: x40). Arrowheads indicate images of close juxtaposition between stained thymocytes and a non-labeled cell.
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To further map Coronin-1 gene expression and the cell types involved, we carried out in situ hybridizations of transversal cross-sections of mouse embryos at different stages of their development using a Coronin-1 RNA digoxigenin antisense probe (Fig. 1C). At E10.5, Coronin-1 expression is primarily detected in the developing ventral spinal cord where the first motor neurons originate, as well as in individual cells scattered throughout the embryo that may correspond to isolated macrophages (Fig. 1C, left panel; individual stained cells were generally visible throughout all the stages and cross-sections that were analyzed). Coronin-1 RNA labeling of neuronal structures is also evident on embryonic sections at E12.5, e.g. within the ventral spinal cord motor neurons and cells of the sensory dorsal root ganglia (Fig. 1C, middle panel). At this stage, however, Coronin-1 expression is simultaneously detected in the liver in which fetal hematopoiesis essentially takes place (26). Subsequently, at E14.5, Coronin-1 expression was still found within the motor neurons and dorsal root ganglia cells, and, at a higher level, within the hematopoietic tissues with especially strong signals now visible in lymphoid cells of the embryonic thymus (clearly distinct from macrophages or epithelial cells; Fig. 1C, right panel). Observation at a higher magnification (x40) of thymus hybridizations at E14.5 (a stage that closely follows initial colonization by embryonic T cell precursors) indicates that Coronin-1 expression is diffuse among these early [mostly CD4CD8 double-negative (DN)] thymocytes, with limited zones of more prominent cell staining (Fig. 1D, arrows). Likewise, a diffuse, heterogeneous cell-staining profile for Coronin-1 RNA was observed on cross-sections of an adult thymus (Fig. 1E). Thus, foci of more intensively stained thymocytes were found to mainly distribute (i) within the upper cortex and subcapsular zone, two areas where DN T cells undergo progressive developmental changes and, within the
ß lineage, the pre-TCR-based selection process (ß-selection) into more mature CD4+CD8+ double-positive (DP) cells [(27) and references therein]; and also (ii) within the thymic medulla, an area in which T cells that underwent successful TCRMHC interaction [i.e. the positively selected TCR
ß+, CD4+CD8/CD4CD8+ single-positive (SP) thymocytes] first appear (2830). Also noteworthy were the frequent images of intensively stained thymocytes juxtaposed with a non-labeled cell (e.g. Fig. 1E, x40 magnification).
Overall, these experiments demonstrate that the Coronin-1 gene is expressed throughout the T cell life: Coronin-1 expression increases in parallel with ongoing thymus embryogenesis and, in the adult, is abundant within the T cell-rich tissues (i.e. thymus, lymph nodes). Furthermore, they strongly suggest that, over a basal level of Coronin-1 expression, developing thymocytes occasionally display an accumulation of Coronin-1 RNA messages, possibly in the situations of privileged cellular interactions (e.g. with specialized components of the thymic stroma during cell-differentiation/selection processes).
Coronin-1 protein expression in T lymphocytes
To extend our expression studies to the protein level, we have produced a Coronin-1 antiserum by injection of an ovalbumin-coupled peptide in rabbits. The immunizing peptide was chosen within the linker region of the protein, between the N-terminal WD-repeats and C-terminal leucine zipper (amino acids 400413). Figure 2(A) shows the utilization of the
-P400413 antiserum in western blot analysis of a cell lysate from adult mouse thymus. Incubation of transferred membranes with the
-P400413 antiserum, but not the pre-immune serum, yields a prominent band at 57 kDa as expected for Coronin-1 (Fig. 2A, lanes 1 and 2). The band is abrogated after the antiserum was incubated with the immunizing peptide prior to membrane labeling (Fig. 2A, lane 3). The specificity of the
-P400413 reagent was further confirmed in western blotting assays using a lysate from human HeLa cells (Coronin-1) that have been transfected with a mouse Coronin-1 expression construct (data not shown).

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Fig. 2. Analysis of Coronin-1 protein expression in T lymphoid cells. (A) The anti-Coronin-1 -P400413 antiserum (lane 1), or control pre-immune serum (lane 2), was used in western blot analysis of a cell lysate from adult mouse thymus; a control experiment after prior incubation of the -P400413 antiserum with the P400413 immunizing peptide is also shown (lane 3). Antiserum labeling was revealed using goat anti-rabbit secondary antibodies coupled to horseradish peroxidase. (B and C) Flow cytometric analysis of intracellular Coronin-1 expression. Total thymocytes or lymph node cells from BALB/c mice were first stained using cytochrome c- and PE-conjugated anti-CD8 and anti-CD4 mAb, or allophycocyanin- and PerCP Cyanine 5.5-conjugated anti-CD44 and anti-CD25 mAb and, in addition, PE-conjugated anti-CD8, -CD4, -CD3, -B220, -Mac-1 and -Gr1 mAb; then, following membrane permeabilization by treatment with saponin 0.3%, cells were stained using the -P400413 antiserum (revealed with a FITC-coupled, goat anti-rabbit secondary antibody). Flow cytometric analyses are shown for CD4/CD8 surface staining of total thymocytes and lymph node cells (B, left and right histograms), for CD44/CD25 surface staining of DN thymocytes (B, middle histogram) and for intracellular Coronin-1 staining of cells in the indicated subsets (C, gating on the PE window). Numbers indicate percentages (P) and mean fluorescence values (M) of Coronin+ cells. Controls for intracellular Coronin-1 specificity used the -P400413 antiserum pre-incubated with the P400413 immunizing peptide (C, filled curves).
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We have used the
-P400413 antiserum for cytofluorometric analysis of intracellular Coronin-1 expression in thymic and peripheral T cell populations from wild-type mice, as defined by parallel staining for the CD4 and/or CD8 cell-surface markers. We readily detected intracellular Coronin-1 in the vast majority of DN, DP and SP thymocytes, and of CD4+CD8 and CD4CD8+ lymph node cells (Fig. 2B and C; note that no signal was detected in control staining using the antiserum pre-incubated with the P400413 peptide), confirming its expression throughout thymocyte development and the peripheral
ßT cells. The proportion of Coronin-1+ cells varied from
7580% in DN thymocytes and the peripheral T cells up to >99% in the DP and SP thymic cell subsets; with a subpopulation of Coronin-1/lo cells especially well-individualized in the DN subset. Highest and lowest levels of intracellular Coronin-1 (as indicated by comparison of mean fluorescence values) were observed in SP and DP thymocytes respectively (Fig. 2C, upper histograms). Further analysis of DN thymocytes using the CD44 and CD25 markers demonstrated >99% Coronin-1+ cells at the CD44+CD25, CD44+CD25+ or CD44CD25+ DN cell stages, whereas 2030% Coronin-1/lo cells readily accumulate among the more developed CD44CD25 DN cells (i.e. the stage that immediately follows ß selection; Fig. 2C, middle histograms). Of note, CD4CD8 lymph node cells also contain >90% Coronin-1+ cells, likely reflecting expression in other hemato/lymphopoietic cell lineages including macrophages and, possibly, B, 
T and NK lymphocytes as well (Nal and Verthuy, unpublished data).
Parallel cytofluorometric analyses of thymic cell subsets from wild-type and from MHC class I- or MHC class II-deficient mice [in which positive selection of respectively CD8+ and CD4+
ßT cells is impaired (29)] have confirmed these profiles, and, specifically, the increasing levels of intracellular Coronin-1 as thymocytes develop from DP cells to CD4+CD8lo or CD8+CD4lo cells and, ultimately, to CD4+CD8 or CD8+CD4 SP cells. Finally, focusing on the Coronin-1/lo cells within the CD44CD25 DN compartment, we found that this subpopulation is mostly comprised of TCRß, TCR
T cells [as judged from low levels of intracellular staining for TCRß (<10 versus >85% in their Coronin-1+ counterparts) and TCR
(<1.5%), and from concurrent intracellular staining for Thy 1 (>90%) and CD3
(>79%)], whereas staining for markers specific of B lymphocytes, macrophages or dendritic cells was marginal (data not shown).
Subcortical localization of Coronin-1 in T cells and interaction with the actin cytoskeleton
IF microscopy analyses using the
-P400413 antiserum confirmed Coronin-1 expression in mouse primary thymocytes and peripheral T lymphocytes (and, to a lesser level, in primary motor neurons as well), and in several mouse cell lines of hematopoietic origin (including the cell thymoma EL-4, the pre-T and pre-B cells T3 and 18.81, the B cell hybridoma 1H11, and the macrophage line J774), but not in NIH 3T3 fibroblasts (Fig. 3A and B, left panel and data not shown). In all positively stained cells, Coronin-1 was found to be cytosolic, and enriched in subcortical areas and/or membrane protrusions. Pre-incubation of the antiserum with the P400413 peptide consistently resulted in the lack of cell staining (e.g. Fig. 3A, middle panel).
Coronin-like proteins have been inferred to display a primordial actin-binding function that has been adapted for many uses in the course of evolution (6). Moreover, in macrophages, Coronin-1 has been reported to associate with the phagosome membrane in a cholesterol-dependent manner (16). These findings, coupled to our data of T cell expression and subcortical localization (see above), prompted us to investigate whether, in mouse T cells, Coronin-1 associates with plasma membrane structures known to play a role in T cell selection/activation processes, including (i) the cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) (25,31,32), and/or (ii) F-actin within the T cell cytoskeleton (33,34). To test the first possibility, we used the
-P400413 antiserum in western blot analysis of the low-density DIM versus heavy (H) density fractions prepared from mouse primary thymocytes [these two fractions contain lipid rafts and the bulk of solubilized cellular materials respectively (25)]. We found a very limited amount of Coronin-1 in DIM (Fig. 3C; compare with DIM-associated p56lck tyrosine kinase and DIM-excluded Rab5 small GTPase-positive and -negative controls). Also, thymocyte stimulation upon treatment with anti-CD3
antibodies did not result in a significant increase of Coronin-1 into the DIM fraction (data not shown). We conclude that Coronin-1 is not a predominant constituent of the thymic raft microdomains, although a weak and/or transient interaction with these membrane structures cannot be formally excluded. Next, we probed the association between Coronin-1 and F-actin in mouse primary T lymphocytes by two-color IF staining and confocal microscopy, using the
-P400413 antiserum and the F-actin-specific marker phalloidin respectively. Lymph node-enriched T cells from a BALB/c mouse were plated on fibronectin-coated coverslips in order to facilitate cell adhesion and the formation of F-actin-rich membrane protrusions. Thus, Coronin-1/F-actin co-localization was readily detected in several areas within the T cell subcortical zone and in membrane protrusions (Fig. 3B). Of note, in most cells, co-localization was not uniform as Coronin-1 generally was concentrated in some, but not all, F-actin-rich areas (Fig. 3B and data not shown). To further investigate the relationship between Coronin-1 and F-actin dynamics, we repeated the latter experiments using human Jurkat T cells. Jurkat T cells are larger in size than mouse primary T lymphocytes or thymocytes and undergo readily visible morphological changes upon adhesion or activation (21,35). When adhered on a poly-L-lysine-coated surface, these cells produce lamellipodia-like membrane protrusions typical of migrating cells (e.g. Fig. 4A). Again, Coronin-1 and F-actin were found to accumulate together within these structures, although co-localization was not complete (see the merge panel). Compared to F-actins homogeneous distribution, Coronin-1 was generally more concentrated within longitudinal zones across the lamellipodia. Altogether, these observations are consistent with a recruitment of Coronin-1 in the T cell subcortical areas that are actively engaged in local actin polymerization.

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Fig. 4. IF confocal analysis for the relative locations of F-actin and Coronin-1 in human Jurkat T cells. Cells were labeled with Alexa-488-coupled phalloidin and the -P400413 antiserum followed by rhodamine-coupled goat anti-rabbit secondary antibodies (AE). Arrows indicate areas of cell membrane protrusions to which F-actin or Coronin-1 preferentially accumulates; arrowheads point to the merging of the two images. (A) Example of a cell displaying lamellipodium-like membrane protrusion after adhesion to poly-L-lysine coverslips. (BE) cells were activated for 1 min with anti-CD3 (UCHT1)-coated polystyrene beads in the absence (B) or in the presence of the src tyrosine kinase inhibitor PP1 (D); as a control for anti-CD3 specificity, Jurkat cells were also incubated with anti-CD43-coated beads (C). In (E), the cells were treated with the actin polymerization inhibitor latrunculin A (Lat.) before activation. DIC: differential interference contrast images. (F) Cellbead conjugates displaying polarized accumulation of Coronin-1 and F-actin were quantified by counting under a fluorescence microscope.
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Coronin-1 subcortical re-localization in response to TCRCD3 stimulation
To more precisely investigate the involvement of Coronin-1 in the membrane dynamics of activated T cells, we analyzed Coronin-1 subcortical distribution in response to TCRCD3 stimulation. We first used human Jurkat T cells because, in our hands, these cells appear as a suitable experimental system for a detailed microscopic analysis on Coronin-1 re-localization (also see below). Specifically, we asked whether Coronin-1 could polarize in a F-actin-dependent fashion in response to TCRCD3 stimulation, as demonstrated for several other proteins that link TCR signaling to the actin cytoskeleton (21,35,36). To this end, Jurkat T cells were activated with anti-CD3-coated microspheres, and analyzed by IF and confocal microscopy as described above. Figure 4(B and F) shows that Coronin-1 accumulates in membrane protrusions produced at the contact site with the stimulatory anti-CD3-coated bead and, significantly, overlaps with F-actin. Importantly, neither Coronin-1 nor F-actin re-localization was observed when using beads coated with mAb directed to other cell-surface molecules (anti-CD43, -ICAM-2 or -ICAM-3 coated-beads have been tested; Fig. 4C and data not shown), implying that the observed Coronin-1/F-actin re-localization processes are specific of CD3 stimulation. Furthermore, we found that these processes are strongly inhibited when the experiments are carried out in the presence of PP1 (Fig. 4D), an inhibitor of src family protein tyrosine kinases that blocks TCRCD3-mediated signaling (21). Finally, prior treatment of Jurkat T cells with latrunculin A [a compound that disrupts actin microfilaments (37)] also resulted in the abrogation of Coronin-1 polarization (Fig. 4E). These results strongly argue that, in this experimental model, Coronin-1 recruitment towards the subcortical T cell areas depends on F-actin polymerization induced by TCR signaling.
The cytoskeleton polarization processes described above are reminiscent of those occurring in T cells at their sites of contact with activatory antigen-presenting cells (APC) (38). To ascertain that Coronin-1 can re-localize towards the APC contact zone within primary T lymphocytes, we analyzed mouse lymph node T cells that were mixed with B lymphoma cells after the latter have been pre-pulsed with a mix of enterotoxin superantigens or, as a negative control, with medium alone. Strikingly, in the presence of superantigen-pre-pulsed APC, a significant fraction of the T cells emitted membrane protrusions towards the APC contact zone and these were markedly enriched in Coronin-1 (Fig. 3D). These images were not observed for the T cellAPC conjugates that were formed following lymphoma cell incubation in the absence of superantigens (Fig. 3E). Consistent results were obtained in similar experiments that used human peripheral blood T cell blasts or Jurkat T cells and superantigen-pulsed APC of human origin [data not shown and (39)]. Indeed, under our experimental conditions, Coronin-1 re-localization and cell shape changes were more readily observed in human compared to mouse T lymphocytes (Nal and Alcover, unpublished observations).
In summary, our protein studies have confirmed that Coronin-1 is expressed throughout T cell development and in mature T cells with, in the former situation, quantitative changes in given thymocyte subsets that can be related to those observed for Coronin-1 gene expression by in situ hybridization of thymus cross-sections (see discussion). Furthermore, results from IF analysis strongly argue that Coronin-1, possibly through the dynamic recruitment from cytosolic stocks, participates in cellular processes involving membrane/cytoskeleton reorganization events in T lymphocytes, notably those induced by the stimulation of the TCRCD3 complex.
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Discussion
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T cell ontogeny requires continuing cross-talk between the developing lymphocytes and cells of the thymic stroma (40). Likewise, to effect their immune functions, T lymphocytes must develop close contacts with APC or tumor cell targets (41). These physical contacts notably involve the interaction of cell-surface receptors (e.g. TCR and/or cytokine receptors) and adhesion molecules (e.g. integrins) with their ligands, triggering cell activation for further differentiation, and/or to set off an immune response. Recently, it has become evident that T cell activation relies on regulated changes at the plasma membrane and underlying cytoskeleton, sustaining highly dynamic receptor re-distribution, cell polarization and mobility, and increased cellcell adhesion (33,34). Numerous factors have been reported to be involved in these processes, although the list may be far from complete. Our data establishing that Coronin-1, in addition of being expressed throughout the T cell life, shows quantitative variations in defined thymocyte subpopulations, and focalized recruitment to the actin cytoskeleton upon membrane mobilization and/or TCRCD3 stimulation, argue for a functional role along these regulatory networks.
To our knowledge, this study represents the first detailed analysis of Coronin-1 expression in T cells. However, we stress that this factor most certainly shows a broader spectrum of expression within various lympho/hematopoietic cell lineages, including neutrophils and macrophages [as described elsewhere (7)], and B lymphocytes (Nal, unpublished data). Furthermore, Coronin-1 expression appears not to be restricted to the hematopoietic tissues, as shown here most notably in developing motor neurons (e.g. Fig. 1C). Whether, depending on the cell lineage, Coronin-1 exerts a similar or distinct function remains an open question.
Although Coronin-1 was detected throughout T cell ontogeny/development and in peripheral
ß T lymphocytes, levels of gene/protein expression appear to vary depending on the subpopulation and stage of cell differentiation. For example, higher Coronin-1 gene expression occurs in embryonic compared to adult thymocytes (Fig. 1 and Da Silva et al., unpublished results) and, among the latter, highest intracellular Coronin-1 protein levels are observed in SP thymocytes, while intermediate and lowest levels are found in the DN and DP subsets respectively (Fig. 2C). Given the images of in situ hybridization of embryonic and adult thymus cross-sections (Fig. 1D and E), these differences could reflect (i) variations in the ratio between high versus moderate/low expressors in the SP/DN versus DP subsets and, as inferred, in the extent of T cell activation in response to selection/stimulation events; and (ii) fine-tuned controls at the RNA level (expression and/or stability; although changes in protein stability due to a post-translational modification(s) are not excluded). In this respect, using DNA array hybridizations to search for genes that are expressed during thymus ontogeny in mouse embryos (42), we found that Coronin-1 expression culminates at E16.5 and E17.5 (with lower levels at E18.5)a developing period that marks synchronous DNDP cell transition (and ß-selection) within the original wave of embryonic T cell precursors (43,44). Accordingly, Coronin-1 expression and/or RNA processing could be the target(s) of a regulatory pathway that would be stimulated in response to T cell activation.
In mammalian cells, including the immune system, alternative splicing mechanisms contribute in generating additional complexity to the basal transcription unit (45). Indeed, following mouse thymocyte library screening, cloning and sequencing, we have found evidence for the production of distinct RNA isoforms of mouse Coronin-1 that differ by the differential usage of 5' untranslated regions owing to alternative splicing (Nal et al., unpublished data). Notably, besides the RNA isoform of mouse Coronin-1 previously reported by Kung and Thomas (14), we have identified a novel isoform that carries a distinct untranslated first exon [we propose to refer the two alternate sequences, located 1488 and 795 bp upstream the ATG start, as exons 1a (E1a) and 1b (E1b) respectively; the corresponding genomic sequence has been deposited under GenBank accession no. AF427040]. Significantly, using a RT-PCR strategy, we found a stronger signal for E1a usage compared to that of E1b in all the tissues tested (including thymus, lymph nodes, spleen, brain and kidney), indicating that Coronin-1 expression in mouse T cells mostly involves the E1a-containing RNA isoform. One intriguing possibility would be that expression of Coronin-1 alternative isoforms may serve a regulatory function, as has been suggested for many genes in mammals (45).
The finding of a population of Coronin-1/lo T cells that accumulate specifically within the CD44CD25 DN thymic compartment (2030% of cells in this subset) is intriguing. As noted, the CD44CD25 DN compartment contains developing,
ß-committed pre-T cells that have passed ß selection, and, therefore, express a productively (in-frame) rearranged TCRß locus and pre-TCR complex, a significant percentage of them (
25%) being proliferating cells with >2N DNA content (46). It is unlikely that the Coronin-1/lo CD44CD25 DN thymocytes correspond to the latter dividing cells, however, as intracellular TCRß staining is extremely low in this population compared to their Coronin-1+ counterpart. Likewise, lack of anti-TCR 
staining also suggests that they are not 
T cells either. Recently, in CD44CD25 DN thymocytes of wild-type mice, a population of TUNEL+ cells has been described (
25% of total CD44CD25 DN cells) that expresses no TCR
and no (or insufficient) amount of TCRß proteinsmost likely immature thymocytes unsuccessful in
ß or in 
lineage development that die by apoptotic cell death (47). The possibility that the Coronin-1/lo cells are undergoing apoptosis is further supported by the observation that their percentage increases in CD44CD25 DN thymocytes from pre-TCR-deficient (e.g. Rag/) mice (Verthuy and Ferrier, unpublished data), a characteristic also reported for the TUNEL+ cells mentioned above (47). Additional experiments, including the determination of cell cycle/survival criteria of the Coronin-1/lo CD44CD25 DN thymocytes, as well as their profiles of gene rearrangement/expression at the various TCR loci, should help to define the origin of this population.
Through their interaction with actin, Coronin orthologues are assumed to play a role during dynamic processes related to the formation and treatment of the phagosomal vesicles in lower eukaryotes (5) and the mammalian macrophages (7). We observed partial co-localization of Coronin-1 and F-actin in membrane protrusions of T cells (Figs 3A and B, and 4A). Moreover, we observed that Coronin-1 redistributes in F-actin-rich areas upon T cell activation by superantigen-pulsed APC (Fig. 3D) or by anti-CD3-coated microspheres (Fig. 4B). Coronin-1 re-localization appears to be dependent on TCRCD3 signaling and the integrity of the actin cytoskeleton, since it is blocked by both the src kinase inhibitor PP1 and the actin-polymerization inhibitor latrunculin A (Fig. 4D and E). Similar to most WD-repeat-containing proteins, Coronins have no enzymatic activity (6). Therefore, our findings would be consistent with a role for Coronin-1, via its conserved WD-repeat-containing domain, of a scaffold protein that recruits specific signaling components downstream of the TCRCD3 complex. Essential scaffolding functions to assemble higher order molecular machines are suspected for several WD-repeat-containing proteins in various systems, including receptor signaling [e.g. (48,49)]. Interestingly, in the situations of T cell activation analyzed here, Coronin-1 co-localization with F-actin seemed incomplete (Figs 3B and 4B). However, under similar conditions, another actin-binding protein, Ezrin, still overlapped with F-actin (21). This suggests that, although functionally linked, Coronin-1 may be less tightly coupled to F-actin than other actin-binding proteins such as Ezrin. Future studies, based on the definition of Coronin-1-interacting molecules and the availability of Coronin-1-deficient mice, will provide further insights into the interacting partner(s) and function(s) of this factor in T lymphocytes.
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Supplementary data
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Supplementary data is available at International Immunology online.
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Acknowledgements
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We thank Drs S. Allonso (IBDM) and P. Naquet (CIML) for critical reading of this manuscript. This work was supported by institutional grants from INSERM and the CNRS, and by specific grants from the Association pour la Recherche sur le Cancer (ARC), the Commission of the European Communities, the Fondation Princesse Grace de Monaco (to P. F.) and the ACI/Ministère de la Recherche (to P. F. and A. A.). B. N. and E. M. were fellows of the Ligue Nationale Contre le Cancer and ARC respectively; and B. N. is now a fellow of ARC. M. I. D. S. was the recipient of a Foreign Associate Scientist position from the Ministère de la Recherche.
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Abbreviations
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APCantigen-presenting cells
DIMdetergent insoluble membrane
DNdouble negative
DPdouble positive
IFimmunofluorescence
PEphycoerythrin
SPsingle positive
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