coronin-1 expression in t lymphocytes: insights into protein function during t cell development and...

doi: 10.1093/intimm/dxh022

Coronin-1 expression in T lymphocytes:insights into protein function during T celldevelopment and activation

BeÂatrice Nal1,2, Patrick Carroll3, Elodie Mohr1, Christophe Verthuy1,Maria-Isabel Da Silva1, Odile Gayet3, Xiao-Jun Guo1,4, Hai-Tao He1,AndreÂs Alcover2 and Pierre Ferrier1

1Centre d`Immunologie de Marseille±Luminy, INSERM±CNRS±UniversiteÂ de la MeÂditerraneÂe, Case 906,13288 Marseille Cedex 9, France2UniteÂ de Biologie des Interactions Cellulaires, CNRS URA 2582, Institut Pasteur, Paris 75724, France3Institut de Biologie du DeÂveloppement de Marseille (IBDM), INSERM U382, Marseille 13288, France4Institut MeÂditerraneÂen de Recherche en Nutrition, UMR-INRA, Marseille 13397, France

Keywords: actin cytoskeleton, Coronin, T lymphocyte

Abstract

Coronin has been described as an actin-binding protein of Dictyostelium discoideum, and it hasbeen demonstrated to play a role in cell migration, cytokinesis and phagocytosis. Coronin-relatedproteins are found in many eukaryotic species, including Coronin-1 in mammals whose expressionis enriched in the hematopoietic tissues. Here, we characterize Coronin-1 gene and proteinexpression in mouse embryonic and adult T lymphocytes. Coronin-1 is expressed throughout Tcell ontogeny and in peripheral ab T cells. Expression varies along thymic cell development, withmaximum levels observed in embryonic early thymocytes and, in the adults, the selected TCRab+

single-positive thymocytes. Subcellular localization analysis indicates that Coronin-1 is inequilibrium between the cytosol and the cell cortex, where it accumulates in F-actin-rich membraneprotrusions induced by polarized activation of TCR±CD3-stimulated T cells. These data areconsistent with a role of Coronin-1 in T cell differentiation/activation events involving membranedynamisms and the cortical actin cytoskeleton.

Introduction

The Coronin protein was ®rst identi®ed in the slime mold

Dictyostelium discoideum as an actin-binding factor localized

to cell surface projections (1). It has been further de®ned as a

WD-repeat protein [WD-repeats are protein motifs thought to

be organized into a `propeller-like' structural domain involved

in protein±protein interactions; (2)], with a role in cell migration,

cytokinesis and phagocytosis (3±5). 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 (7±10)]. 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-con-

taining coat protein)] was the ®rst mammalian paralogue to

be identi®ed and the one that has been studied the most

(7,11±16). 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 ®ve

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 inter-

acts 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 de-

pend 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

Correspondence to: P. Ferrier; E-mail: [email protected]

Transmitting editor: J. Borst Received 24 March 2003, accepted 21 October 2003

International Immunology, Vol. 16, No. 2, pp. 231±240 ã 2004 The Japanese Society for Immunology

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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, speci®cally, in processes that

involve the dynamics of the actin cytoskeleton in response to

TCR stimulation and cell activation.

Methods

Mice, cell lines, ¯ow cytometric analysis, cell stimulationprocedures and immuno¯uorescence (IF) microscopy

Wild-type BALB/c mice were housed in a speci®c pathogen-

free animal facility in accordance with institutional guidelines.

Adult mice were sacri®ced 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 ®broblasts (ATCC TIB-208 and CRL-1658 respect-

ively). The human leukemia T cell line Jurkat, clone J77cl20

(19), was also utilized. Cell culture, staining conditions and

¯ow 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 mg/ml each

of Staphylococcus aureus enterotoxin A, B, C3 and E

superantigens. Jurkat J77cl20 T cells were activated by

incubation with antibody-coated 5-mm microspheres at 4°Cfor 30 min and then at 37°C for 1 min. When necessary, cells

were pre-incubated (37°C for 30 min) with medium contain-

ing 10 mM PP1 (Calbiochem, La Jolla, CA) or 2 mM

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 de-

scribed by Roumier et al. (21).

Antibodies

Phycoerythrin (PE)-, cytochrome c, allophycocyanin- and

PerCP Cyanine 5.5-conjugated mAb against the CD8 (53±

6.7), 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 a-P400±413

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, andcDNA cloning procedures

Total RNA extraction from mouse embryos and adult tissues,and northern blot analysis were performed using standardprotocols. The Coronin-1 and Hprt probes utilized for northernblot hybridization were prepared by DNA PCR ampli®cationusing wild-type mouse thymus cDNA templates. RT-PCRreactions were performed as outlined previously (23) (oligo-nucleotide primers available upon request). cDNA cloningprocedures were carried out using standard protocols ofmolecular biology.

In situ hybridization assays

In situ hybridization was performed as described by Carrieret al. (24). The RNA probes were prepared by in vitrotranscription using the Coronin-1 cDNA in the presence ofdigoxigenin-coupled UTP (Boehringer Mannheim, Meylan,France), as recommended by the manufacturer. Revelationof the hybridized probes was performed using an alkalinephosphatase-coupled anti-digoxigenin antibody and the NBT/BCIP-speci®c substrates (Boehringer Mannheim).

Production of the Coronin-1 antiserum and western blotprocedures

The a-P400±413 antiserum was produced in rabbits(Eurogentec Bel, Seraing, Belgium) by immunization with anovalbumin-coupled Coronin-1 peptide (amino acid residues400±413). For western blotting, thymocyte extracts in Laemmlilysis buffer were separated by electrophoresis through a 10%polyacrylamide gel and transferred onto a PVDF membrane(Millipore, Bedford, MA). After saturation (in PBS 3 1, 0.1%Tween 20, 5% milk), the membrane was incubated with the a-P400±413 antiserum, and the reaction was revealed using ahorseradish peroxidase-coupled anti-rabbit antiserum and theECL western blotting revelation reagent (AmershamPharmacia Biotech, Little Chalfont, UK).

Isolation of `detergent insoluble membrane' components(DIM)

DIM were isolated as described previously (25). Brie¯y,thymocytes (2 3 108) were gently sonicated (5-s, 5 W,Vibracell; Bioblock Scienti®c, Illkirch, France) in 1 ml of ice-cold `sonication' buffer (25 mM HEPES, 150 mM NaCl, 1 mMEGTA, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 2 mg/mlchymostatin and 5 mg/ml a2-macroglobulin). The post-nuclearsupernatant was prepared and subjected to solubilization with1% Brij 98 at 37°C for 5 min. Samples were diluted with 2 ml ofsonication buffer containing 2 M sucrose, chilled on ice andlaid 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 sonicationbuffer. After centrifugation at 38,000 r.p.m., 4°C for 16 h in aSW41 rotor (Beckman Instruments), 1-ml fractions (nos 1±8)were harvested from the top of the gradient; the last fraction(no. 9) contains 3 ml. DIM are comprised within pooledfractions nos 2±5; the heavy (H) material (i.e. the bulk ofsolubilized materials at the bottom of the gradient) corres-ponds to pooled fractions nos 8 and 9.

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Results

Coronin-1 gene expression in adult and embryonic mousetissues

The Coronin-1 gene was reported to be preferentiallyexpressed in hemato/lymphopoietic tissues including thethymus and spleen (7,11,13). To con®rm and extend these®ndings, we ®rst performed northern blot analysis of tissuesfrom adult wild-type mice using a cDNA probe speci®c formouse Coronin-1 (Nal, unpublished data). Indeed, wedetected Coronin-1 RNA predominantly in the thymus, spleenand lymph nodes, whereas no, or much weaker, signal wasfound in other, mostly non-hematopoietic, tissues (includingliver, testis, kidney, brain, muscle, heart and intestine; Fig. 1A).That the thymus signal is contributed to a large extent by Tcells was con®rmed in parallel analysis of isolated thymocytes.In separate RT-PCR experiments, we found very low levels ofCoronin-1 expression in the developing mouse embryo at 7.5d.p.c. (E7.5) and E8.5, and increased signals from E9.5onwards (Fig. 1B)Ðan expression pro®le that parallels thetimings of development and expansion of the hemato/lymphopoietic cell lineages during mouse embryogenesis(26).

To further map Coronin-1 gene expression and the celltypes involved, we carried out in situ hybridizations oftransversal cross-sections of mouse embryos at differentstages of their development using a Coronin-1 RNA±digoxigenin antisense probe (Fig. 1C). At E10.5, Coronin-1expression is primarily detected in the developing ventralspinal cord where the ®rst motor neurons originate, as well asin individual cells scattered throughout the embryo that maycorrespond to isolated macrophages (Fig. 1C, left panel;individual stained cells were generally visible throughout allthe stages and cross-sections that were analyzed). Coronin-1RNA labeling of neuronal structures is also evident onembryonic sections at E12.5, e.g. within the ventral spinalcord motor neurons and cells of the sensory dorsal rootganglia (Fig. 1C, middle panel). At this stage, however,Coronin-1 expression is simultaneously detected in the liver inwhich fetal hematopoiesis essentially takes place (26).Subsequently, at E14.5, Coronin-1 expression was still foundwithin the motor neurons and dorsal root ganglia cells, and, ata higher level, within the hematopoietic tissues with especiallystrong signals now visible in lymphoid cells of the embryonicthymus (clearly distinct from macrophages or epithelial cells;Fig. 1C, right panel). Observation at a higher magni®cation(340) of thymus hybridizations at E14.5 (a stage that closelyfollows initial colonization by embryonic T cell precursors)indicates that Coronin-1 expression is diffuse among theseearly [mostly CD4±CD8± double-negative (DN)] thymocytes,with limited zones of more prominent cell staining (Fig. 1D,arrows). Likewise, a diffuse, heterogeneous cell-stainingpro®le for Coronin-1 RNA was observed on cross-sections ofan adult thymus (Fig. 1E). Thus, foci of more intensivelystained thymocytes were found to mainly distribute (i) withinthe upper cortex and subcapsular zone, two areas where DN Tcells undergo progressive developmental changes and, withinthe ab lineage, the pre-TCR-based selection process (b-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 underwentsuccessful TCR±MHC interaction [i.e. the positively selectedTCRab+, CD4+CD8±/CD4±CD8+ single-positive (SP) thymo-cytes] ®rst appear (28±30). Also noteworthy were the frequentimages of intensively stained thymocytes juxtaposed with anon-labeled cell (e.g. Fig. 1E, 340 magni®cation).

Overall, these experiments demonstrate that the Coronin-1gene is expressed throughout the T cell life: Coronin-1expression increases in parallel with ongoing thymus embry-ogenesis and, in the adult, is abundant within the T cell-richtissues (i.e. thymus, lymph nodes). Furthermore, they stronglysuggest that, over a basal level of Coronin-1 expression,developing thymocytes occasionally display an accumulationof Coronin-1 RNA messages, possibly in the situations ofprivileged cellular interactions (e.g. with specializedcomponents 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 haveproduced a Coronin-1 antiserum by injection of an ovalbumin-coupled peptide in rabbits. The immunizing peptide waschosen within the linker region of the protein, between theN-terminal WD-repeats and C-terminal leucine zipper (aminoacids 400±413). Figure 2(A) shows the utilization of the a-P400±

413 antiserum in western blot analysis of a cell lysate from adultmouse thymus. Incubation of transferred membranes with thea-P400±413 antiserum, but not the pre-immune serum, yields aprominent band at 57 kDa as expected for Coronin-1 (Fig. 2A,lanes 1 and 2). The band is abrogated after the antiserum wasincubated with the immunizing peptide prior to membranelabeling (Fig. 2A, lane 3). The speci®city of the a-P400±413

reagent was further con®rmed in western blotting assays usinga lysate from human HeLa cells (Coronin-1±) that have beentransfected with a mouse Coronin-1 expression construct(data not shown).

We have used the a-P400±413 antiserum for cyto¯uorometricanalysis of intracellular Coronin-1 expression in thymic andperipheral T cell populations from wild-type mice, as de®nedby parallel staining for the CD4 and/or CD8 cell-surfacemarkers. We readily detected intracellular Coronin-1 in thevast majority of DN, DP and SP thymocytes, and of CD4+CD8±

and CD4±CD8+ lymph node cells (Fig. 2B and C; note that nosignal was detected in control staining using the antiserumpre-incubated with the P400±413 peptide), con®rming itsexpression throughout thymocyte development and the per-ipheral abT cells. The proportion of Coronin-1+ cells variedfrom ~75±80% in DN thymocytes and the peripheral T cells upto >99% in the DP and SP thymic cell subsets; with asubpopulation of Coronin-1±/lo cells especially well-individua-lized in the DN subset. Highest and lowest levels of intra-cellular Coronin-1 (as indicated by comparison of mean¯uorescence values) were observed in SP and DP thymocytesrespectively (Fig. 2C, upper histograms). Further analysis ofDN thymocytes using the CD44 and CD25 markers demon-strated >99% Coronin-1+ cells at the CD44+CD25±,CD44+CD25+ or CD44±CD25+ DN cell stages, whereas20±30% Coronin-1±/lo cells readily accumulate among themore developed CD44±CD25± DN cells (i.e. the stage that

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immediately follows b selection; Fig. 2C, middle histograms).

Of note, CD4±CD8± lymph node cells also contain >90%

Coronin-1+ cells, likely re¯ecting expression in other hemato/

lymphopoietic cell lineages including macrophages and,

Fig. 1. Analysis of Coronin-1 gene expression. (A) Total RNA (15 mg) from the indicated adult mouse lymphoid and non-lymphoid tissues wasanalyzed by northern blot using a 32P-labeled Coronin-1 cDNA probe (Cor.-1, top panel) and, following membrane stripping, for hybridizationto 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 mg) fromwhole mouse embryos at the indicated d.p.c. stages (E7.5±E14.5) and Coronin-1 or b-actin speci®c oligonucleotide primers (top and bottompanels respectively). Assays were performed in the presence (+) and absence (±) of reverse transcriptase to control for contamination bygenomic DNA. (C) In situ hybridization for Coronin-1 expression using transversal cross-sections of mouse embryos at d.p.c. 10.5, 12.5 and14.5 (E10.5, E12.5 and E14.5), and a Coronin-1 RNA±digoxigenin antisense probe (magni®cation: 316); DRG, dorsal root ganglia; mn, motorneuron. (D) Image of an antisense probe hybridized to a E14.5 transverse cross-section, focusing on the thymus image (left); hybridizationwith the control sense probe is shown on the right (magni®cation: 340). 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: magni®cation:316; middle and right panels: magni®cation: 340). Arrowheads indicate images of close juxtaposition between stained thymocytes and anon-labeled cell.




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possibly, B, gd T and NK lymphocytes as well (Nal and Verthuy,

unpublished data).Parallel cyto¯uorometric analyses of thymic cell subsets

from wild-type and from MHC class I- or MHC class II-de®cient

mice [in which positive selection of respectively CD8+ and

CD4+ abT cells is impaired (29)] have con®rmed these

pro®les, and, speci®cally, 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

CD44±CD25± DN compartment, we found that this subpopula-

tion is mostly comprised of TCRb±, TCRgd± T cells [as judged

from low levels of intracellular staining for TCRb (<10 versus

>85% in their Coronin-1+ counterparts) and TCRgd (<1.5%),

Fig. 2. Analysis of Coronin-1 protein expression in T lymphoid cells. (A) The anti-Coronin-1 a-P400±413 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 incubationof the a-P400±413 antiserum with the P400±413 immunizing peptide is also shown (lane 3). Antiserum labeling was revealed using goat anti-rabbitsecondary antibodies coupled to horseradish peroxidase. (B and C) Flow cytometric analysis of intracellular Coronin-1 expression. Totalthymocytes or lymph node cells from BALB/c mice were ®rst stained using cytochrome c- and PE-conjugated anti-CD8 and anti-CD4 mAb, orallophycocyanin- 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 thea-P400±413 antiserum (revealed with a FITC-coupled, goat anti-rabbit secondary antibody). Flow cytometric analyses are shown for CD4/CD8surface 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 indicatepercentages (P) and mean ¯uorescence values (M) of Coronin+ cells. Controls for intracellular Coronin-1 speci®city used the a-P400±413

antiserum pre-incubated with the P400±413 immunizing peptide (C, ®lled curves).




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and from concurrent intracellular staining for Thy 1 (>90%) andCD3e (>79%)], whereas staining for markers speci®c of Blymphocytes, macrophages or dendritic cells was marginal(data not shown).

Subcortical localization of Coronin-1 in T cells andinteraction with the actin cytoskeleton

IF microscopy analyses using the a-P400±413 antiserum con-®rmed Coronin-1 expression in mouse primary thymocytes

and peripheral T lymphocytes (and, to a lesser level, in primarymotor neurons as well), and in several mouse cell lines ofhematopoietic origin (including the cell thymoma EL-4, thepre-T and pre-B cells T3 and 18.81, the B cell hybridoma

Fig. 3. Analysis of Coronin-1 localization and association with `raft'microdomains in mouse T lymphocytes. (A) IF microscopy analysisof Coronin-1 localization using the a-P400±413 antiserum (revealed asin Fig. 2B) in mouse T3 pre-T cells in the absence (left panel) or inthe presence of the immunizing peptide (central panel), and in NIH3T3 ®broblasts (right panel). (B) IF confocal analysis of the relativelocations of Coronin-1 and F-actin in enriched T cells from mouselymph nodes. Cells were adhered to ®bronectin-coated coverslipsand were labeled with the a-P400±413 antiserum followed by TexasRed-coupled goat anti-rabbit secondary antibody and Alexa-488-coupled phalloidin. Arrows indicate areas of cell membraneprotrusions to which Coronin-1 or F-actin preferentially accumulates.(C) Western blot analysis of low-density DIM and the heavy densitycellular fractions prepared from mouse primary thymocytes. Proteinsin DIM and a quarter of the heavy fractions were concentrated, andresolved on SDS±PAGE, followed by western blotting. Proteins wererevealed using the a-P400±413 antiserum or, as positive and negativecontrols for raft isolation, antibodies against the p56lck tyrosinekinase (p56lck) or the Rab5 small GTPase (Rab5) respectively. (Dand E) Mouse primary lymph node T cells were incubated for 10 minat 37°C with B lymphoma cells A20 that had been pre-pulsed withenterotoxin superantigen (D) or, as a negative control, with mediumalone (E). Coronin-1 staining was carried out as in (B), left panel. Adifferential interference contrast image was taken for each cellconjugate (right panels; bar = 5 mm).

Fig. 4. IF confocal analysis for the relative locations of F-actin andCoronin-1 in human Jurkat T cells. Cells were labeled with Alexa-488-coupled phalloidin and the a-P400±413 antiserum followed byrhodamine-coupled goat anti-rabbit secondary antibodies (A±E).Arrows indicate areas of cell membrane protrusions to which F-actinor Coronin-1 preferentially accumulates; arrowheads point to themerging of the two images. (A) Example of a cell displayinglamellipodium-like membrane protrusion after adhesion to poly-L-lysine coverslips. (B±E) cells were activated for 1 min with anti-CD3(UCHT1)-coated polystyrene beads in the absence (B) or in thepresence of the src tyrosine kinase inhibitor PP1 (D); as a control foranti-CD3 speci®city, Jurkat cells were also incubated with anti-CD43-coated beads (C). In (E), the cells were treated with the actinpolymerization inhibitor latrunculin A (Lat.) before activation. DIC:differential interference contrast images. (F) Cell±bead conjugatesdisplaying polarized accumulation of Coronin-1 and F-actin werequanti®ed by counting under a ¯uorescence microscope.




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1H11, and the macrophage line J774), but not in NIH 3T3®broblasts (Fig. 3A and B, left panel and data not shown). In allpositively stained cells, Coronin-1 was found to be cytosolic,and enriched in subcortical areas and/or membrane protru-sions. Pre-incubation of the antiserum with the P400±413

peptide consistently resulted in the lack of cell staining (e.g.Fig. 3A, middle panel).

Coronin-like proteins have been inferred to display aprimordial actin-binding function that has been adapted formany uses in the course of evolution (6). Moreover, inmacrophages, Coronin-1 has been reported to associatewith the phagosome membrane in a cholesterol-dependentmanner (16). These ®ndings, coupled to our data of T cellexpression and subcortical localization (see above),prompted us to investigate whether, in mouse T cells,Coronin-1 associates with plasma membrane structuresknown to play a role in T cell selection/activation processes,including (i) the cholesterol- and sphingolipid-rich membranemicrodomains (lipid `rafts') (25,31,32), and/or (ii) F-actin withinthe T cell cytoskeleton (33,34). To test the ®rst possibility, weused the a-P400±413 antiserum in western blot analysis of thelow-density DIM versus heavy (H) density fractions preparedfrom mouse primary thymocytes [these two fractions containlipid rafts and the bulk of solubilized cellular materialsrespectively (25)]. We found a very limited amount ofCoronin-1 in DIM (Fig. 3C; compare with DIM-associatedp56lck tyrosine kinase and DIM-excluded Rab5 small GTPase-positive and -negative controls). Also, thymocyte stimulationupon treatment with anti-CD3e antibodies did not result in asigni®cant increase of Coronin-1 into the DIM fraction (data notshown). We conclude that Coronin-1 is not a predominantconstituent of the thymic `raft' microdomains, although a weakand/or transient interaction with these membrane structurescannot be formally excluded. Next, we probed the associationbetween Coronin-1 and F-actin in mouse primary T lympho-cytes by two-color IF staining and confocal microscopy, usingthe a-P400±413 antiserum and the F-actin-speci®c markerphalloidin respectively. Lymph node-enriched T cells from aBALB/c mouse were plated on ®bronectin-coated coverslips inorder to facilitate cell adhesion and the formation of F-actin-rich membrane protrusions. Thus, Coronin-1/F-actin co-local-ization was readily detected in several areas within the T cellsubcortical zone and in membrane protrusions (Fig. 3B). Ofnote, in most cells, co-localization was not uniform as Coronin-1 generally was concentrated in some, but not all, F-actin-richareas (Fig. 3B and data not shown). To further investigate therelationship between Coronin-1 and F-actin dynamics, werepeated the latter experiments using human Jurkat T cells.Jurkat T cells are larger in size than mouse primary Tlymphocytes or thymocytes and undergo readily visiblemorphological changes upon adhesion or activation (21,35).When adhered on a poly-L-lysine-coated surface, these cellsproduce lamellipodia-like membrane protrusions typical ofmigrating cells (e.g. Fig. 4A). Again, Coronin-1 and F-actinwere found to accumulate together within these structures,although co-localization was not complete (see the `merge'panel). Compared to F-actin's homogeneous distribution,Coronin-1 was generally more concentrated within longitudinalzones across the lamellipodia. Altogether, these observationsare consistent with a recruitment of Coronin-1 in the T cell

subcortical areas that are actively engaged in local actinpolymerization.

Coronin-1 subcortical re-localization in response toTCR±CD3 stimulation

To more precisely investigate the involvement of Coronin-1 inthe membrane dynamics of activated T cells, we analyzedCoronin-1 subcortical distribution in response to TCR±CD3stimulation. We ®rst used human Jurkat T cells because, in ourhands, these cells appear as a suitable experimental systemfor a detailed microscopic analysis on Coronin-1 re-localiza-tion (also see below). Speci®cally, we asked whether Coronin-1 could polarize in a F-actin-dependent fashion in response toTCR±CD3 stimulation, as demonstrated for several otherproteins 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 confocalmicroscopy as described above. Figure 4(B and F) shows thatCoronin-1 accumulates in membrane protrusions produced atthe contact site with the stimulatory anti-CD3-coated beadand, signi®cantly, overlaps with F-actin. Importantly, neitherCoronin-1 nor F-actin re-localization was observed when usingbeads coated with mAb directed to other cell-surface mol-ecules (anti-CD43, -ICAM-2 or -ICAM-3 coated-beads havebeen tested; Fig. 4C and data not shown), implying that theobserved Coronin-1/F-actin re-localization processes arespeci®c of CD3 stimulation. Furthermore, we found thatthese processes are strongly inhibited when the experimentsare carried out in the presence of PP1 (Fig. 4D), an inhibitor ofsrc family protein tyrosine kinases that blocks TCR±CD3-mediated signaling (21). Finally, prior treatment of Jurkat Tcells with latrunculin A [a compound that disrupts actinmicro®laments (37)] also resulted in the abrogation ofCoronin-1 polarization (Fig. 4E). These results strongly arguethat, in this experimental model, Coronin-1 recruitmenttowards the subcortical T cell areas depends on F-actinpolymerization induced by TCR signaling.

The cytoskeleton polarization processes described aboveare reminiscent of those occurring in T cells at their sites ofcontact with activatory antigen-presenting cells (APC) (38). Toascertain that Coronin-1 can re-localize towards the APCcontact zone within primary T lymphocytes, we analyzedmouse lymph node T cells that were mixed with B lymphomacells after the latter have been pre-pulsed with a mix ofenterotoxin superantigens or, as a negative control, withmedium alone. Strikingly, in the presence of superantigen-pre-pulsed APC, a signi®cant fraction of the T cells emittedmembrane protrusions towards the APC contact zone andthese were markedly enriched in Coronin-1 (Fig. 3D). Theseimages were not observed for the T cell±APC conjugates thatwere formed following lymphoma cell incubation in theabsence of superantigens (Fig. 3E). Consistent results wereobtained in similar experiments that used human peripheralblood T cell blasts or Jurkat T cells and superantigen-pulsedAPC of human origin [data not shown and (39)]. Indeed, underour experimental conditions, Coronin-1 re-localization and cellshape changes were more readily observed in human com-pared to mouse T lymphocytes (Nal and Alcover, unpublishedobservations).




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In summary, our protein studies have con®rmed thatCoronin-1 is expressed throughout T cell development andin mature T cells with, in the former situation, quantitativechanges in given thymocyte subsets that can be related tothose observed for Coronin-1 gene expression by in situhybridization of thymus cross-sections (see discussion).Furthermore, results from IF analysis strongly argue thatCoronin-1, possibly through the dynamic recruitment fromcytosolic stocks, participates in cellular processes involvingmembrane/cytoskeleton reorganization events in T lympho-cytes, notably those induced by the stimulation of the TCR±CD3 complex.

Discussion

T cell ontogeny requires continuing cross-talk between thedeveloping lymphocytes and cells of the thymic stroma (40).Likewise, to effect their immune functions, T lymphocytes mustdevelop 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) andadhesion molecules (e.g. integrins) with their ligands, trigger-ing cell activation for further differentiation, and/or to set off animmune response. Recently, it has become evident that T cellactivation relies on regulated changes at the plasma mem-brane and underlying cytoskeleton, sustaining highly dynamicreceptor re-distribution, cell polarization and mobility, andincreased cell±cell adhesion (33,34). Numerous factors havebeen reported to be involved in these processes, although thelist may be far from complete. Our data establishing thatCoronin-1, in addition of being expressed throughout the T celllife, shows quantitative variations in de®ned thymocytesubpopulations, and focalized recruitment to the actincytoskeleton upon membrane mobilization and/or TCR±CD3stimulation, argue for a functional role along these regulatorynetworks.

To our knowledge, this study represents the ®rst detailedanalysis of Coronin-1 expression in T cells. However, we stressthat this factor most certainly shows a broader spectrum ofexpression within various lympho/hematopoietic cell lineages,including neutrophils and macrophages [as described else-where (7)], and B lymphocytes (Nal, unpublished data).Furthermore, Coronin-1 expression appears not to be re-stricted to the hematopoietic tissues, as shown here mostnotably in developing motor neurons (e.g. Fig. 1C). Whether,depending on the cell lineage, Coronin-1 exerts a similar ordistinct function remains an open question.

Although Coronin-1 was detected throughout T cell onto-geny/development and in peripheral ab T lymphocytes, levelsof gene/protein expression appear to vary depending on thesubpopulation and stage of cell differentiation. For example,higher Coronin-1 gene expression occurs in embryoniccompared to adult thymocytes (Fig. 1 and Da Silva et al.,unpublished results) and, among the latter, highest intra-cellular Coronin-1 protein levels are observed in SP thymo-cytes, while intermediate and lowest levels are found in the DNand DP subsets respectively (Fig. 2C). Given the images of insitu hybridization of embryonic and adult thymus cross-sections (Fig. 1D and E), these differences could re¯ect (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) ®ne-tuned controls at the RNA level(expression and/or stability; although changes in proteinstability due to a post-translational modi®cation(s) are notexcluded). In this respect, using DNA array hybridizations tosearch for genes that are expressed during thymus ontogenyin mouse embryos (42), we found that Coronin-1 expressionculminates at E16.5 and E17.5 (with lower levels at E18.5)Ðadeveloping period that marks synchronous DN±DP cell tran-sition (and b-selection) within the original wave of embryonic Tcell precursors (43,44). Accordingly, Coronin-1 expressionand/or RNA processing could be the target(s) of a regulatorypathway that would be stimulated in response to T cellactivation.

In mammalian cells, including the immune system, alterna-tive splicing mechanisms contribute in generating additionalcomplexity to the basal transcription unit (45). Indeed, follow-ing mouse thymocyte library screening, cloning and sequen-cing, we have found evidence for the production of distinctRNA isoforms of mouse Coronin-1 that differ by the differentialusage of 5¢ untranslated regions owing to alternative splicing(Nal et al., unpublished data). Notably, besides the RNAisoform of mouse Coronin-1 previously reported by Kung andThomas (14), we have identi®ed a novel isoform that carries adistinct untranslated ®rst exon [we propose to refer the twoalternate sequences, located 1488 and 795 bp upstream theATG start, as exons 1a (E1a) and 1b (E1b) respectively; thecorresponding genomic sequence has been deposited underGenBank accession no. AF427040]. Signi®cantly, using a RT-PCR strategy, we found a stronger signal for E1a usagecompared to that of E1b in all the tissues tested (includingthymus, lymph nodes, spleen, brain and kidney), indicatingthat Coronin-1 expression in mouse T cells mostly involves theE1a-containing RNA isoform. One intriguing possibility wouldbe that expression of Coronin-1 alternative isoforms may servea regulatory function, as has been suggested for many genesin mammals (45).

The ®nding of a population of Coronin-1±/lo T cells thataccumulate speci®cally within the CD44±CD25± DN thymiccompartment (20±30% of cells in this subset) is intriguing. Asnoted, the CD44±CD25± DN compartment contains develop-ing, ab-committed pre-T cells that have passed b selection,and, therefore, express a productively (`in-frame') rearrangedTCRb locus and pre-TCR complex, a signi®cant percentage ofthem (~25%) being proliferating cells with >2N DNA content(46). It is unlikely that the Coronin-1±/lo CD44±CD25± DNthymocytes correspond to the latter dividing cells, however, asintracellular TCRb staining is extremely low in this populationcompared to their Coronin-1+ counterpart. Likewise, lack ofanti-TCR gd staining also suggests that they are not gd T cellseither. Recently, in CD44±CD25± DN thymocytes of wild-typemice, a population of TUNEL+ cells has been described(~25% of total CD44±CD25± DN cells) that expresses no TCRgdand no (or insuf®cient) amount of TCRb proteinsÐmost likelyimmature thymocytes unsuccessful in ab or in gd lineagedevelopment that die by apoptotic cell death (47). Thepossibility that the Coronin-1±/lo cells are undergoing apopto-sis is further supported by the observation that their percent-age increases in CD44±CD25± DN thymocytes from




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pre-TCR-de®cient (e.g. Rag±/±) mice (Verthuy and Ferrier,unpublished data), a characteristic also reported for theTUNEL+ cells mentioned above (47). Additional experiments,including the determination of cell cycle/survival criteria of theCoronin-1±/lo CD44±CD25± DN thymocytes, as well as theirpro®les of gene rearrangement/expression at the various TCRloci, should help to de®ne the origin of this population.

Through their interaction with actin, Coronin orthologues areassumed to play a role during dynamic processes related tothe formation and treatment of the phagosomal vesicles inlower eukaryotes (5) and the mammalian macrophages (7).We observed partial co-localization of Coronin-1 and F-actin inmembrane 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 TCR±CD3 signaling and the integrity of the actin cytoskeleton, sinceit is blocked by both the src kinase inhibitor PP1 and the actin-polymerization inhibitor latrunculin A (Fig. 4D and E). Similar tomost WD-repeat-containing proteins, Coronins have no enzy-matic activity (6). Therefore, our ®ndings would be consistentwith a role for Coronin-1, via its conserved WD-repeat-containing domain, of a scaffold protein that recruits speci®csignaling components downstream of the TCR±CD3 complex.Essential scaffolding functions to assemble higher ordermolecular machines are suspected for several WD-repeat-containing proteins in various systems, including receptorsignaling [e.g. (48,49)]. Interestingly, in the situations of T cellactivation analyzed here, Coronin-1 co-localization with F-actin seemed incomplete (Figs 3B and 4B). However, undersimilar conditions, another actin-binding protein, Ezrin, stilloverlapped with F-actin (21). This suggests that, althoughfunctionally linked, Coronin-1 may be less tightly coupled to F-actin than other actin-binding proteins such as Ezrin. Futurestudies, based on the de®nition of Coronin-1-interactingmolecules and the availability of Coronin-1-de®cient mice,will provide further insights into the interacting partner(s) andfunction(s) of this factor in T lymphocytes.

Supplementary data

Supplementary data is available at International Immunology online.

Acknowledgements

We thank Drs S. Allonso (IBDM) and P. Naquet (CIML) for criticalreading of this manuscript. This work was supported by institutionalgrants from INSERM and the CNRS, and by speci®c grants from theAssociation pour la Recherche sur le Cancer (ARC), the Commissionof the European Communities, the Fondation Princesse Grace deMonaco (to P. F.) and the ACI/MinisteÁre de la Recherche (to P. F. andA. A.). B. N. and E. M. were fellows of the Ligue Nationale Contre leCancer 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 fromthe MinisteÁre de la Recherche.

Abbreviations

APC antigen-presenting cellsDIM detergent insoluble membraneDN double negative

DP double positiveIF immuno¯uorescencePE phycoerythrinSP single positive

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