Lamia Achour

Mark G H Scott

Hamasseh Shirvani

Alain Thuret

Georges Bismuth

Catherine Labbé-Jullié

Stefano Marullo
email: <marullo@cochin.inserm.fr>

Marullo Stefano

Scott Mark

Shirvani Hamasseh

Thuret Alain

Achour Labb -Julli É É Catherine

Lamia

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression

Keywords: Animals, Antigens, CD4, metabolism, Antigens, Surface, metabolism, CHO Cells, Cell Compartmentation, physiology, Cells, Cultured, Cricetinae, Cricetulus, Endoplasmic Reticulum, metabolism, Hela Cells, Humans, Intracellular Space, metabolism, Protein Binding, Protein Transport, Receptors, CCR5, metabolism

come   L'archive ouverte pluridisciplinaire

Introduction

The CC chemokine receptor 5 (CCR5) is a seven membrane-spanning domain, G protein-coupled receptor (GPCR), which regulates chemotaxis and effector functions of T-lymphocytes, macrophages, and dendritic cells (reviewed in ). Upon activation by its cognate 1 chemokines, CCL5 (former Regulated upon Activation, Normal T cell Expressed and Secreted, RANTES), CCL3 and CCL4 (former macrophage inflammatory proteins 1 and 1 ) , CCR5 leads to cellular signaling through G proteins as well as G protein-independent α β 2 3 pathways . In addition to its physiological functions, CCR5 is also the main coreceptor of the human immunodeficiency virus (HIV), in 4 association with CD4 , a cell-surface glycoprotein that participates in molecular complexes involved in both T cell development and 5

antigen recognition by T cells. CD4 is a single-membrane spanning domain receptor interacting with class II major histocompatibility complex molecules and with its cognate ligand IL-16. So far, no specific physiological function has been attributed to the CCR5-CD4 6 complex.

In the context of immune cell chemotaxis , recent studies have shown that the sustained (hours) lymphocyte motility within in vivo lymph nodes, is driven by the lymphoid chemokine-rich environment and mediated by the activation of chemokine receptors . 7 Paradoxically, studies in reconstituted models have shown that stimulated chemokine receptors, including CCR5, are rapidly desensitized (seconds to minutes) via phosphorylation and -arrestin translocation before being internalized and eventually degraded , . In fact, an β 1 8-10 adaptive mechanism to escape inactivation and maintain prolonged reactivity upon sustained stimulation has been reported for in vivo other GPGRs, based on the retention of important stocks of functional receptors within intracellular compartments (reviewed in ).

11

Initially described for protease-activated receptors (such as the thrombin receptor ), this phenomenon was subsequently reported for 12 dopamine D1 receptors in tubular renal cells , and for -opioid receptors in neurons . The list of GPCRs displaying a predominant 13 δ 14 intracellular localization is rapidly growing . A common feature for most of these prevalently intracellular GPCRs is their capacity of 11 interacting with a vast array of membrane-associated or cytoplasmic proteins that facilitate their translocation to the cell surface . Often, 15

these proteins possess their own functions, in addition to aiding GPCR expression at the cell surface . Among them, type-I 11 single-transmembrane domain proteins with a large N-terminal extracellular domain and a short C-terminus are particularly frequent. This group includes Receptor-activity-modifying-proteins (RAMPs), which facilitate cell surface expression of a Class-B GPCRs ; the 16

Receptor Transporting Proteins (RTPs) and REEP1 (Receptor Expression Enhancing Protein 1), which permit functional cell surface targeting of odorant and taste receptors ; the M10 MHC class-I molecules which are necessary for the correct trafficking of 17 18 pheromone receptors to the plasma membrane ; the MC2R accessory protein (MRAP), which interacts with the melanocortin 2 receptor, 19

regulating its trafficking from the endoplasmic reticulum (ER) to the cell surface . 20

In the current study we investigated the subcellular distribution of endogenous CCR5 receptor in human T lymphocytes and in a monocytic cell line and found that this receptor is predominantly intracellular. We also report that CCR5 and CD4 can associate within intracellular compartments and that this association enhances the export of CCR5 to the cell surface.

Material and Methods

Cell lines and transfections

THP-1 cells were a kind gift from Philip Strange lab (Reading, UK). Cell culture media were from GIBCO and chemicals from SIGMA unless otherwise specified. CHO-K1 cells were cultured in F12 medium, HeLa P4R5 in Dulbecco modified minimal essential medium (DMEM) and THP-1 cells in RPMI 1640, in an atmosphere of 5 CO . Media were supplemented with 10 fetal bovine serum % 2 % (FBS), 100 U/ml Penicillin and 100 g/ml Streptomycin. Transfections of CHO-K1 and HeLa P4R5 cells were performed using μ GeneJuice-Transfection Reagent (Novagen).

Plasmid expression constructs

Constructs for BRET experiments were described previously . Briefly, CCR5 and CXCR4 coding regions were amplified from their 21 respective cDNAs using appropriate sense and antisense primers, containing unique Hind III or Bam H1 sites, respectively. The fragments were then subcloned in frame between the Hind III and Bam H1 sites of a plasmid for YFP (Clontech) or for a humanized form of Renilla Luciferase (Biosignal; PerkinElmer Life Sciences). The plasmid for CCR5 Cter-YFP was obtained by the amplification of a shorter Δ fragment (corresponding to met1-gly301 CCR5) using a more proximal antisense primer. For CD4-Luc and CD4-YFP constructs, the stop codon of the CD4 sequence was suppressed and replaced by a restriction site using site directed mutagenesis (QuickChange site-directed mutagenesis kit, Stratagene). The coding regions of all constructs were entirely sequenced. The GABABR1-YFP and OBR-YFP constructs were kind gifts from Michel Bouvier (Montreal, Canada) and Ralf Jockers (Paris, France), respectively. The constructs for GFP-tagged wild-type VPU and phosphorylation mutant VPU were kind gifts from Florence Margottin (Paris, France). The plasmid for 2/6 22 ER-DsRed was purchased from Clontech BD Biosciences.

Antibodies

Mouse monoclonal antibodies were as follows: 2D7 anti-human CCR5 (555991, BD Pharmingen) directed against the second extracellular loop of CCR5, OKT4 anti-human CD4 (for FACS and immunofluorescence, 140048, eBioscience), 12G5 anti-CXCR4 (ab 21555, Abcam), 1F6 anti-human CD4 (for immunoblot experiments, NCL-L-CD4-1F6, Novocastra), anti-GFP (11-814-460-001, Roche). Rat monoclonal antibodies were as follows: 1/85a Alexa-Fluor 647-conjugated anti-human CCR5 (313712, Biolegend), which recognizes

+

Human T lymphocyte preparation and activation

Human T-cells were isolated from the peripheral blood of healthy volunteers by Ficoll density gradient centrifugation, followed by negative depletion on magnetic beads (Human T lymphocyte Enrichment set-DM, Becton Dickinson). T-cells were activated by incubation for 48 hours with anti-CD3 and anti-CD28 antibody-coated beads (Dynabeads Human CD3/CD28 T Cell Expander Kit, Invitrogen), at a bead-to-cell ratio of 1:3, in RPMI 1640 medium supplemented with pooled human AB serum and antibiotics.

FACS analysis

The proportion of surface CCR5, and CD4 on resting or activated T lymphocytes, THP-1 cells and CHO-K1 cells expressing exogenous receptors was determined by analyzing in parallel, intact and permeabilized cells. For total staining, 1 10 cells were fixed in 1 × 6 paraformaldehyde for 20 min, washed with PBS and permeabilized for 1h at 4 C in PBS, containing 2 FBS and 0,2 Triton-X-100, as % °% % described . Cells were then incubated on ice for 1h in 100 l of PBS, 2 FBS with the 1/85a Alexa Fluor 647-conjugated anti-human 24 μ % CCR5 antibody (1:50) or the OKT4 anti-human CD4 (1:100) and control isotypes at the same dilutions. For CD4, this step was followed by 1h incubation with Cy5-conjugated anti-mouse IgG (1:100). For extracellular staining, cells were incubated on ice for 1h with PBS, 2%

FBS without Triton-X-100 and subsequently incubated with the appropriate antibodies, as above. After washing with PBS, cells were fixed in 2 paraformaldheyde and processed on a Cytomycs FC500 FACS analyzer (Beckman Coulter).

%

To study the impact of CD4 co-expression on CCR5 targeting at the cell surface, CHO-K1 cells were co-transfected with plasmids coding for CCR5-Luc (at fixed concentration) and CD4-YFP (at increasing concentrations). 36h after transfection, cells were incubated on ice for 1h with 0.5 g/ml of the 2D7 anti-human CCR5 antibody, followed by incubation with Cy5-conjugated anti-mouse IgG (1:100).

μ

To determine the effect of CD4 co-expression on CXCR4 targeting at the cell surface, CHO-K1 were co-transfected with plasmids coding for CXCR4-Luc (at fixed concentration) and CD4-YFP (at increasing concentrations). FACS experiments were conducted, as described above for CCR5, by incubating cells for 1h with 0.5 g/ml 12G5 anti-human CXCR4 antibody, followed by incubation with μ Cy5-conjugated anti-mouse IgG (1:100).

The level of endogenous CCR5 at the cell surface of THP-1 cells after CD4 silencing was measured with the 1/85a Alexa Fluor 647-conjugated anti-CCR5 antibody as described above. The proportion of surface CCR5 was evaluated by analyzing both intact and permeabilized cells.

For each analysis, 10000 cells were sorted and the data analyzed using the Cytomics RXP software.

Immunofluorescence

CHO-K1 cells were seeded on glass cover slips in six-well plates (2 10 per well), transfected with the indicated plasmids, and

× 5
processed for immunofluorescence, 48h after transfection. Subsequently cells were fixed in 4 paraformaldehyde for 20 min at 4 C or in

% °methanol
for 5 min at 20 C, according to the antibody used. After quenching with 50mM NH Cl in PBS for 15 min, cells were washed

- °4
with PBS and then incubated with PBS-bovine serum albumin (BSA) 1 containing or not 0,1 saponin and labeled with the indicated

% %
antibodies.

The subcellular localization of endogenous CCR5 and CD4 in T lymphocytes and THP-1 cells was analyzed by immunofluorescence in cells maintained in suspension. After fixation in 4 paraformaldehyde for 20 min at room temperature and quenching (as described % above) cells were incubated with PBS-BSA1 or PBS-BSA1 containing 0.05 of Tween 20, for permeabilization . Cells were then % % % 25 incubated for 1h with the 2D7 anti-human CCR5 or the OKT4 anti-human CD4 antibodies. Following incubation with appropriate secondary antibodies, cells were washed with PBS and mounted on slides.

All images were obtained using a confocal Leica DMIRE2 microsccope and analyzed using the Metamorph 7 software.

Immunoprecipitation and immunoblotting

Cells growing in 100mm dishes were lysed in 50mM HEPES, pH 7.4, 250mM NaCl, 2mM EDTA, 0.5 NP40, 10 glycerol, 0.5

% % %
deoxycholic acid (lysis buffer), containing Complete protease inhibitors (Roche), on ice for 1h. After centrifugation at 13000 g for

™ ×
20min, the supernatant was collected and protein concentration determined (BCA protein assay, Pierce). Supernatants were incubated with anti-GFP antibody (2 g) for 3h at 4 C and then incubated with protein G-Sepharose beads for 1h at 4 C. After centrifugation, the μ °°immuno-bead-bound material was washed three times with lysis buffer. The immunoprecipitates were resolved in 10 SDS-PAGE gels.

%

After transfer on nitrocellulose, blots were probed with the appropriate antibodies, all used at 1:1000 dilution. Immunoblots were revealed by luminescence (ECL, Amersham Bioscience).

ERK activation studies

CHO-K1 grown in 6-well plates were transfected with the plasmid encoding CCR5-Luc with or without the plasmid for CD4-YFP.

After overnight starvation, cells were detached with EDTA 10mM in PBS, centrifuged (1400 g for 5 min) and resuspended in Hank

× '
s-balanced salt solution. Aliquots were subjected to luciferase activity measurements to determine average CCR5-Luc concentration. After addition of 5 M coelenterazine h (Interchim), emitted light was measured in a Mithras LB940 reader (Berthold Instruments). Other μ aliquots of cells were stimulated with MIP-1 (Preprotech) for various periods of time, chilled on ice, centrifuged (8000 g for 1min at 4

β × °C)
and analyzed for ERK activation as described . Briefly, after resuspension in ice-cold 50 mM Tris-HCl pH 6.8, containing 2.5 mM 26 orthovanadate, 2.5 mM EDTA and a cocktail of protease inhibitors, cells were lysed in 2X Laemmli sample buffer. Proteins were denatured (5min at 95 C) separated by 10 SDS-PAGE and transferred on nitrocellulose membranes. Membranes were probed with °% Phospho-p44/42 MAP kinase or MAP kinase 1-2 antibodies and autoradiograms quantified using the Image J 1.38X software.

BRET assays

CHO-K1 cells (5 10 per well of a 6-well plates) were transfected with 30ng of plasmid DNA coding for the BRET donor (CD4-Luc) under the same experimental conditions. For better readability, results were expressed in milli-BRET units (mBRET), 1 mBRET corresponding to the BRET ratio multiplied by 1000. BRET ratios were plotted as a function of (YFP-YFP )/YFP / Rluc/Rluc , where

[ 0 0 ] [ 0 ]
YFP is the fluorescence signal at 530nm after excitation at 485 nm, and Rluc the signal at 485 nm after addition of coelenterazine h. YFP 0 and Rluc correspond to the same values in cells expressing the Rluc fusion protein alone. In some experiments, cells were incubated 14h 0 before the BRET assay with Brefeldin A at 2 g/ml.

μ

RNA interference

THP-1 cells were transfected using the AMAXA nucleofector system, in Amaxa solution V (program U010), with 0.5 M of siRNA ™ μ oligonucleotides (S1: 5 -GAACUGACCUGUACAGCUUUU-3 ; S2: 5 -GAGCGGAUGUCUCAGAUCAUU-3 ; S3: 5

′ ′ ′ ′ ′
-UAACUAAAGGUCCAAUCCAAUU-3 ) targeting human CD4 or an unrelated oligonucleotide (5 -UAGCGACUAAACACAUCAA-3 ,

′ ′ ′
all from Dharmacon). For each experiment 2 rounds of transfection were performed: 2 10 cells were transfected a first time, cultured for

× 6
72h, resuspended and transfected again using the same protocol. Assays were conducted 7 days after the first transfection. For RNA interference experiments on human T cells, 4 10 freshly purified T lymphocytes were transfected using the AMAXA nucleofector

× 6 ™
system, in Amaxa solution T (program U14) with 1 M of S1 and S3 oligonucleotides, as indicated above. After nucleofection, cells were μ cultured in RPMI medium supplemented with 10 FBS, antibiotics and Interleukin-7 (5ng/ml) for 72h and then activated for 48h with % anti-CD3 and anti-CD28 antibody-coated beads as described above.

Statistical Analysis

Data are expressed as a mean value SEM. Data were analyzed by using Student s t test and p < 0.05 was considered statistically

± '
significant.

Results

CCR5 is predominantly retained within intracellular compartments

To determine the cellular distribution of CCR5, both purified T lymphocytes from healthy donors and cells of the human THP-1 monocytic cell line, expressing endogenous CCR5 were analysed by FACS ( , and Sup. Table 1). In freshly prepared Figure 1A unstimulated T lymphocytes, the proportion of surface CCR5 was very low, 95 of total receptors being intracellular, as measured by ≈ % fluorescence analysis of intact or permeabilized cells with a CCR5-specific antibody. As shown in previous studies , surface CCR5 27 increased after stimulation with anti-CD3 and anti-CD28 antibodies (from 5 to 11 ). In THP-1 cells, CCR5 was also principally

% ≈ %
present within intracellular compartments, with about 90 of the receptor being found within internal compartments. These observations % were obtained with 2 different antibodies recognizing distinct epitopes (Sup. Table 1). The same procedure was used to measure in parallel the distribution of the CD4 molecule. As expected, in both T lymphocytes and monocytes, CD4 was predominantly expressed at the cell surface (up to 92 in activated T lymphocytes). To confirm these findings, immunofluorescence confocal microscopy studies were % conducted on the same cells subjected (P) or not (NP) to membrane permeabilization ( ). Whereas the CD4 staining was Figure 1B comparable in permeabilized and non-permeabilized cells, the major proportion of CCR5 appeared to be intracellular in both T lymphocytes and THP-1 cells. The Jurkat T cell line, which does not express CCR5, was used as negative control for staining. Colocalization studies were also conducted and demonstrated that endogenous CCR5 colocalized with both Golgi (Giantin) end ER (Bip) markers in THP-1 cells, although additional subcellular localizations cannot be excluded. CCR5 distribution was finally analyzed in transiently transfected CHO cells, which will be used in this study as a reconstitution model. As found for the endogenous receptor in T lymphocytes and THP-1 cells, in CHO cells exogenous CCR5 was predominantly intracellular under our experimental conditions, whether wild-type receptor, or a form tagged at the C-terminus with a yellow variant of the green fluorescent protein (YFP), were expressed (Sup.

Figure 1 and Sup. Table 1). Confocal microscopy analyses of transfected CHO cells confirmed that both wild-type CCR5 and CCR5-YFP colocalized with a panel of both Golgi and ER markers (Sup ). From the data above it appears that both endogenous and Figures 2 exogenous CCR5 are prone to intracellular retention.

CCR5 and CD4 associate in the ER

As indicated in the introduction, the cell surface expression of many predominantly intracellular GPCRs can be markedly enhanced by the association with other proteins, which interact with the receptors in the ER and/or the Golgi apparatus, and remain associated with them, once the receptors have reached the cell surface , . We thus investigated whether CD4, in addition to its proper immunological 11 15 functions, might also interact with CCR5 in the ER and facilitate its export to the cell surface. For this purpose, we made use of a natural mutant of CCR5, lacking its C-terminal intracellular tail , that is completely retained in the ER , as supported by the complete absence 28 29 of cell-surface detection by antibodies in immunofluorescence and FACS experiments ( ). CHO cells were transiently Figure 2A,B transfected with a construct encoding CD4, in addition to plasmids for either wild type or truncated CCR5, harbouring a N-terminal HA epitope and a C-terminal YFP (HA-CCR5-YFP and HA-CCR5 Cter-YFP constructs, respectively). In these cells, similar amounts of CD4 Δ were co-immunoprecipitated with both the full length and truncated forms of CCR5 (

) and both HA-CCR5 Cter-YFP and Figure 2D Δ HA-CCR5-YFP colocalized with CD4 within intracellular compartments (

). We confirmed that the CD4-CCR5 association in Figure 2C CHO cells, evidenced by co-IP studies, was not due to non-specific hydrophobic interaction, by showing that mixing cell-lysates expressing either molecule did not allow co-immunoprecipitation (Supp. Figure 3). CD4 CCR5 interaction was further examined in live CHO cells using bioluminescence resonance energy transfer (BRET). CD4 fused to luciferase (CD4-Luc) was expressed in the Renilla presence of increasing concentrations of BRET acceptors, consisting of either HA-CCR5-YFP or HA-CCR5 Cter-YFP. In both cases, Δ hyperbolic saturation curves were obtained, indicating specific interactions (

). BRET signals markedly depend on the relative Figure 2E distance of donor and acceptor , explaining why maximal BRET values were different in wild-type CCR5 and the truncated mutant. In 30 contrast, the value of the YFP:Luc ratio, for which half-maximal BRET was obtained, was comparable, indicating that these two forms of CCR5 display the same propensity to associate with CD4. Free YFP and the GABABR1 receptor fused to YFP (GABABR1-YFP) were used as negative controls. Although GABABR1 is constitutively retained in the ER like HA-CCR5 Cter-YFP, only a weak 31 Δ non-saturable interaction with CD4-Luc could be measured. Finally, in the presence of free YFP, only a linear non-specific BRET signal was obtained. Bystander BRET signals may occur upon random collisions in cells expressing non-physiological high levels of BRET pairs . To rule out this possibility, we verified that the levels of BRET donor and acceptor proteins at BRET saturating concentration were 30 comparable or below those of endogenous CD4 and CCR5 in human monocytic THP-1 cells (Sup. Figure 4). To confirm that the measured BRET signals are a result of interactions occurring in the ER, cells were pre-treated with brefeldin A (BFA), a drug, which induces Golgi apparatus disassembly and redistribution of Golgi-derived vesicles to the ER. The Golgi apparatus was indeed dismantled after BFA treatment (

), and FACS analysis could not detect almost any surface receptor in cells expressing HA-CCR5-YFP ( ). Figure 3A Figure 3B Despite these effects, BRET values remained comparable whether or not cells were treated with BFA ( ). Together, these data are Figure 3C consistent with the hypothesis that CCR5 and CD4 associate within intracellular compartments, likely in the ER, before they are exported to the cell surface.

Association with CD4 in the ER enhances CCR5 export to the cell surface

We next examined whether CD4 might enhance the proportion of surface CCR5 in a reconstituted CHO-K1 cell model. A fixed concentration of a plasmid coding for luciferase-tagged CCR5 (CCR5-Luc) was co-transfected with increasing amounts of a CD4-YFP construct. Surface CCR5 values, measured by FACS with an anti-CCR5 antibody in cells co-expressing CD4-YFP, were plotted as a function of the CD4-YFP-associated fluorescence (

). In co-transfected cells, surface CCR5 progressively increased with increasing Figure 4 levels of CD4-YFP to reach a maximum, corresponding to an augmentation of more than 100 , and then returned gradually to basal % values for higher levels of CD4-YFP (

). The descending portion of the curve was not due to a decreased number of CD4 Figure 4B molecules that could reach the cell surface, since in the same cells, surface CD4 increased linearly with increasing amounts of total CD4-YFP (Sup. Figure 4B). Throughout the experiments we verified that the amount of CCR5-Luc remained constant by measuring luciferase-associated activity. In control experiments, with either free YFP or with the single transmembrane domain leptin receptor fused to YFP (OBR-YFP), no enhancement of surface CCR5 was observed (

). As for BRET experiments, the effect of CD4 on Figure 4A andB surface CCR5 expression was achieved at physiological concentrations of the two proteins (Sup. Figure 4A). HIV can use a second chemokine co-receptor, in conjunction with CD4, to infect target cells . This receptor, CXCR4, also displays a predominant intracellular 32 distribution in human T lymphocytes and THP-1 monocytes (reference and data not shown) and can form stable complexes with CD4, 33 which are detectable by co-immunoprecipitation experiments ( and ). However, contrasting with what was observed for Figure 4C 34 CCR5, its surface expression was not positively modulated by CD4 in transfected cells ( ). Figure 4D The functional significance of the enhancement of surface CCR5, due to the presence of CD4, was estimated by measuring ERK1-2 activation. CHO cells expressing identical amounts of CCR5-Luc, determined by comparing luciferase activity, and a concentration of CD4-YFP corresponding to its maximal effect on CCR5 export, were stimulated or not with non-saturating concentrations of MIP-1 for β increasing periods of time up to 10 min (

). The maximal activation of the MAP kinase doubled in cells co-expressing CD4 and, in Figure 5 addition, it was also reached much faster (compare lanes 3 (time 1min) in and corresponding histograms in ).

=

Figure 5A

Figure 5B We next examined whether the effect of CD4 on CCR5 surface targeting also occurs in cells expressing endogenous receptors. We reasoned that a substantial decrease of CD4 molecules in these cells might affect the density of CCR5 at the cell surface. A panel of 3 interfering RNAs (siRNAs) was used, which targeted the CD4 sequence. THP-1 cells were submitted to 2 consecutive nucleofections and tested after a week to achieve sufficient inhibition of CD4 synthesis and decay of CCR5 molecules already present at the cell surface before the effect of siRNAs. A significant reduction of 70 80 of total CD4 was obtained with 2 specific siRNAs, compared to the control -% ( ). In the cells showing reduced CD4, surface CCR5 was significantly decreased by 25 50 , compared to the control and to Figure 6A,B -% cells expressing the ineffective siRNA (

). Unexpectedly, cells treated with the 2 siRNAs active on CD4 expression showed a Figure 6C,D significant enhancement of 20 60 of total CCR5 (Sup ) suggesting a possible feedback regulation. These results support the -%

hypothesis that CD4 molecules can modulate the proportion of surface CCR5 in cells expressing both molecules. In a physiopathological context, the HIV-1-encoded Vpu protein was reported to induce rapid degradation of CD4 shortly after its synthesis via the 35 ER-associated protein degradation pathway . In line with our findings above, the specific targeting of CD4 molecules to the proteasome 36 by overexpressed Vpu should affect the surface expression of CCR5. A stable Hela cell line, expressing both CCR5 and CD4, was transiently transfected with either wild type GFP-tagged Vpu or a GFP-fused mutant Vpu that has lost its capacity of down-regulating CD4

or free GFP (Sup ). After transfection, surface expression of CCR5 was quantified by FACS in cells expressing comparable 36 Figure 6 levels of green fluorescence. Confirming the hypothesis, expression of Vpu-GFP induced comparable and significant reduction of both total CD4 and surface CCR5, whereas the control mutant Vpu was ineffective.

Finally, the contribution of CD4 to the cell surface expression of CCR5 was explored in primary human cells. T cells were purified from PBMCs, nucleofected with appropriate siRNAs, activated to enhance the cell surface expression of CCR5 and analyzed for both total CD4 content and proportion of cell surface CCR5 ( ). In line with the results obtained with THP-1 cells, for all 3 tested donors, Figure 6E the siRNA-mediated reduction of CD4 was associated with a decreased proportion of CCR5 at the cell surface compared to the control, ranging from 30 to 65 . Primary monocytes purified from one donor were also examined with the same assay and displayed a 75 % % reduction in the proportion of surface CCR5 (not shown).

Discussion

Although previously unreported, the predominant intracellular localization observed here for CCR5 was described for other GPCRs and might constitute a more general feature than previously appreciated. The physiological significance of this phenomenon may vary as a function of the receptor and the tissue where it occurs. For example, thrombin receptors are irreversibly activated by cleavage, internalized and degraded in lysosomes, whereas intracellular receptors are protected from activation by the protease. Replenishment of plasma membrane thrombin receptors is thus correlated with recovery of thrombin responsiveness . In neurons, only a small fraction of -opioid 12

δ
receptors is localized at the neuronal plasma membrane , consistent with their low physiological involvement in acute response . 37 38 Cell surface translocation of -opioid receptors from intracellular compartments might account for the enhanced effect of drugs, δ specifically targeting this receptor, during chronic pain . Similarly, what we describe here for CCR5 may represent a mechanism for 37 maintaining a sustained sensitivity of leukocytes to chemokine guidance within tissues.

From a mechanistic point of view, two important issues must be addressed to fully understand this phenomenon, namely, what are the molecular tethers, which retain receptors within intracellular compartments, and what are the intracellular events that allow their release.

So far, only two proteins directly and specifically involved in the control of GPCR cell surface export via intracellular retention have been identified. The second extracellular loop of the protease-activated PAR2 receptor was shown to interact with the N-terminal domain of the Golgi-resident type I transmembrane protein p24A, and to be retained in the Golgi because of this interaction. Upon activation of cell surface PAR2, the small G protein ARF1 is recruited in its GDP-bound form, to Golgi membranes, where a specific exchange factor activates ARF1. This process results in the dissociation of PAR2 from p24A and receptor sorting to the plasma membrane . In a 39 different context, during development, a GPCR-retaining protein was reported to control the surface receptor availability of Frizzled, a GPCR, which promotes caudalizing signals. This ER-resident protein, Shisa, is specifically expressed in head ectoderm, where it binds to and inhibits cell surface trafficking of Frizzled. Shisa-mediated receptor retention thus constitutes a mechanism to control head-tail polarity .

Other recent examples of regulatory retaining proteins have been reported for growth-factor receptors , the gamma secretase 40 41 complex or the glutamate transporter , suggesting that this field may rapidly evolve. [START_REF] Spasic | Rer1p competes with APH-1 for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway[END_REF] 43

A clear picture of the cellular mechanisms, which allow the release of intracellular GPCRs and their cell surface translocation is still missing. Various cellular signals were reported to elicit acute effects, such as the rise in intracellular calcium by either release from

" "
intracellular stores or direct opening of ion channels, in the case of -opioid receptors . Similarly, in tubular renal cells, dopamine D1 δ 44 receptors can be recruited from cytosolic stores to the plasma membrane upon agonist activation of cell surface receptors or via atrial 13 natriuretic peptide-dependent heterologous activation . Interestingly, the cell surface targeting of dopamine D1 receptors is also 45 regulated via the interaction with DRIP78, a putative two-transmembrane domain protein, resident of the ER, which binds to a FXXXFXXXF motif found in the C-terminus of the receptor . The potential link between signal-mediated export of dopamine D1 46 receptors and interaction with DRIP78 has not been established yet. Nevertheless, DRIP78 is an example of an emerging paradigm in the field, namely that GPCR translocation to the plasma membrane from intracellular compartments often requires their interaction with non-conventional chaperones and escorts proteins. In addition to classical chaperones, such as calnexin, calreticulin and Bip, which regulate folding and export of proteins engaged in the secretory pathway , a vast array of membrane-associated or cytoplasmic proteins 47 has been identified, which interact with GPCRs within intracellular compartments and facilitate their cell surface expression. The list of these proteins, which also include RAMPs, RTPs, REEP, MRAP, and M10 MHC class-I molecules, already mentioned in the introduction, is rapidly growing . Based on the data reported in the present study the CD4 molecule may be a new member of this list. 11 CCR5-CD4 association has been known for a long time, as these molecules combine to form a receptor for HIV at the cell surface of mononuclear blood cells . Although still a matter of debate, studies based on different experimental approaches have suggested the 5 existence of a physical, constitutive, virus-independent, CCR5-CD4 association at the cell surface of both transfected , and primary 34 48 cells , . The association of HIV co-receptors is likely mediated by interactions involving the extracellular globular domain of CD4 and 49 50 the extracellular regions of CCR5 , , , but it has not yet been determined whether these interactions occur once the HIV co-receptors 34 48 51 have reached the plasma membrane or if they are established within intracellular compartments before their export to the cell surface. Our data strongly support the hypothesis that CCR5-CD4 interaction occurs within intracellular compartments, likely in the ER.

Facilitating the export of CCR5 to the cell surface is clearly not the principal biological function of CD4 and it is possible that the surface expression of CCR5 is enhanced by the interaction with other proteins, which eventually interact with CCR5 along the biosynthetic pathway. In addition, it remains to be established whether CCR5 might also be rapidly mobilized by cell signalling events, as for the dopaminergic and opioid receptors mentioned above. From a mechanistic point of view, CD4-CCR5 association in the ER might facilitate further interactions with components of the biosynthetic pathway, likely via the carboxyterminal tail of CCR5. Indeed, although not involved in the interaction with CD4, the CCR5 tail was reported to contain a bipartite motif critical for CCR5 cell surface expression , 52

and we confirmed that the CCR5 Cter mutant was completely retained within intracellular compartments, even in the presence of excess Δ CD4 (not shown). Interestingly, the effect of CD4 on surface CCR5 was concentration-dependent, displaying a bell-shaped pattern comparable to that observed for calnexin and the ER-membrane-associated protein, DRIP78 on the maturation and trafficking of dopaminergic receptors , . The existence of an optimal concentration of chaperones or escort proteins likely reflects the requirement of 46 53 a specific stoichiometry between these proteins, the receptors, and other components of the biosynthetic machinery. In the case of CD4, an alternative possible explanation may be that, at high concentrations, CD4 itself can saturate the export machinery.

For many years, no functional explanation was proposed of how HIV may have selected a composite receptor to infect human immune cells. Recently, a plausible explanation for the formation of the second major HIV receptor, constituted by CD4 and CXCR4 on T lymphocytes, came from the observation that CXCR4 associates with the T cell receptor to signal in T cells . Thus, the stable proximity 54 of CXCR4 and CD4 at the cell surface of lymphocytes likely arises from the bridging of these proteins via a common interactor, the T cell receptor. Interestingly, based on our findings, a different explanation emerges for CCR5: the physiological constitutive association of CCR5 and CD4 in the ER of blood mononuclear cells, and the subsequent translocation of the resulting complexes at the plasma membrane might be promoted by the escort properties of CD4 for CCR5.

R

  the N-terminal extremity of CCR5, Alexa-Fluor 647-conjugated rat IgG2a, k isotype control (400526, Biolegend), 3F10 23 R anti-hemagglutinin (anti-HA) (1-867-423, Roche). Rabbit polyclonal antibodies were as follows: anti-giantin (ab24586, Abcam) anti-TGN46 (ab16052, Abcam), anti-GPR78/BiP (ab21685, Abcam), anti-Phospho-p44/42 (Thr202/Tyr204) MAP Kinase (9101, Cell Signalling), anti ERK 1-2 (06-182, Cell Signalling), Cy3-conjugated anti-goat immunoglobulin (305-167-003, Jackson Immunoresearch), peroxidase-conjugated anti-mouse IgG (315-035-003, Jackson Immunoresearch), peroxidase-conjugated anti-goat IgG (28169, Anaspec). Goat polyclonal antibodies were as follows: anti-calnexin (C-20) (sc-6465, Santa Cruz Biotechnology), anti-actin (I-19) (sc-1616, Santa Cruz Biotechnology), peroxidase-conjugated anti-rabbit IgG (111-035-003 Jackson Immunoresearch), Cy5-conjugated anti-mouse IgG (610-110-121, Rockland). Donkey polyclonal antibodies were as follow: Cy3-conjugated anti-rabbit IgG (711-167-003 Jackson Immunoresearch), Cy3-conjugated anti-rat IgG (712-166-153 Jackson Immunoresearch), Alexa-Fluor 488-conjugated anti-mouse IgG (H R L) (A-21202, Molecular Probes, Invitrogen).

× 5 and

 5 increasing amounts of BRET acceptor plasmids (CCR5-YFP, CCR5 Cter-YFP, GABABR1-YFP; 10 1000ng per well) or YFP (0.5 Δ --100ng per well). 24h after transfection, cells were washed in PBS, detached using PBS-EDTA 10mM, centrifuged (1400 g for 5min), × resuspended in Hank s-balanced salt solution and distributed in 96-well plates (Perkin-Elmer plates; 1 10 cells per well). After addition of ' × 5 the luciferase substrate, coelenterazine h (5 M final concentration), luminescence and fluorescence were measured simultaneously (at 485 μ and 530nm, respectively) in a Mithras LB940 plate reader. The BRET ratio was calculated as: (emission at 530nm/emission at 485 nm) [ -(background at 530nm/background at 485nm) , where background corresponds to signals in cells expressing the Rluc fusion protein alone ]

Figure 1 Figure 2

 12 Figure 1 Subcellular distribution of the human CCR5 receptor (A) FACS analyses showing human resting T lymphocytes from healthy donor or THP-1 monocytes were stained for surface (left panels) and total (after cell permeabilization, right panels) CCR5 (upper panels) or CD4 (lower panels), using 1/85a anti-human CCR5 antibody or OKT4 anti-human CD4, respectively, and analysed by flow cytometry. Green and red histograms correspond to specific labelling for CCR5 and CD4, respectively. Gray histograms are the isotypic control for each antibody. (B) Human resting T lymphocytes, THP-1 and Jurkat cells (negative control) were stained for surface (non-permeabilized, NP) and total (permeabilized P) CCR5 or CD4, using 2D7 anti-human CCR5 or OKT4 anti-human CD4 antibodies. Images were obtained by confocal microscopy. Scale bar: 10 M. (C) THP-1 cells, expressing μ endogenous receptor, were stained with the 2D7 anti-human CCR5 antibody and antibodies directed against Bip, an ER marker, or Giantin, a Golgi marker (see methods), and analysed by confocal microscopy. Colocalization of CCR5 (green) with Bip or Giantin (both in red) is shown in orange (Merge). Scale bar: 10 M.μ

  Figure3A).

Figure 3 Figure 4

 34 Figure 3 CCR5-CD4 association is not affected by Brefeldin A treatment CHO-K1 cells were transfected with the plasmids coding for CD4-Luc and HA-CCR5-YFP or HA-CCR5 Cter-YFP at concentrations Δ yielding sub-maximal BRET (indicated by an asterisk in). Fourteen hours before BRET analysis cells were subjected or not to a figure2EBrefeldin A (BFA) treatment (see methods). (A) BFA-treated and untreated cells were stained for the Golgi marker Giantin; DAPI was added to label nuclei. Scale bar: 10 M. (B) FACS analysis of CCR5 cell surface expression in cells treated or not with BFA. The grey filled μ histogram is the isotypic control, the dotted open histogram corresponding to the specific signal (C) BRET analysis of CD4-CCR5 (wild-type and truncated) interaction in CHO-K1 cells after BFA treatment. Procedure and BRET controls were as in . Figure2E

Figure 5 8 -

 58 Figure 5 CD4 expression enhances CCR5-dependent ERK activation (A) CHO-K1 cells were transfected with the plasmid for CCR5-Luc with or without the plasmid for CD4-YFP at concentrations, which correspond to the asterisk in . Cells were serum-starved overnight and then stimulated with MIP1 (5 10 M) for the indicated Figure 3B β × 8 durations of time. The luciferase signal was measured to monitor the total amount of CCR5. Immunoblotting was performed with the indicated antibodies. Equal amounts of lysates were loaded in each lane and a representative immunoblot of 3 independent experiments is shown. Total ERK represents the loading control. (B) Quantification of phosphorylated ERK 1-2 (normalized with total ERK) from 3 independents experiments. indicate significant difference from untreated cells; indicate significant difference between cells expressing or * ‡ not CD4; and : p<0.05, : p<0.01, and : p<0.001.

Figure 6

 6 Figure 6CD4 silencing negatively modulates CCR5 surface expression in THP-1 and primary T cells (A) Western blot analysis of CD4 from THP-1 cells nucleofected with siRNAs directed against CD4 (S1, S2 and S3) or scrambled control. CD4 was detected using the 1F6 anti-human CD4 antibody. (B) Quantification of CD4 after treatment with indicated siRNAs (n 3, in = triplicates). Asterisks indicate statistical significance (p<0.001) with cells treated with control siRNA. (C) FACS analysis of cell surface CCR5 using the 1/85a Alexa-fluor 647-conjugated anti-human CCR5. Grey open histograms of S1, S2 and S3-treated cells are compared to the control (scramble siRNA) histogram in black. Grey filled histogram corresponds to isotypic control. (D) The effect of CD4 inhibition on surface CCR5 was measured as follows: the proportion of surface CCR5 was calculated by FACS (by comparing the signal in unpermeabilzed and permeabilized cells) in 3 independent experiments, in triplicates ( p<0.01; p<0.001); the values were compared to the proportion of ** *** surface CCR5 in THP-1 nucleofected with control scramble siRNA. (E) Left panel: Representative western blot analysis of CD4 from donor-purified, activated (see methods) primary T cells, nucleofected with 2 siRNAs targeting CD4 sequences (S1 and S3); note that, as for THP-1 cells, the S3 siRNA did not reduce CD4 concentration in T cells. Right panel: effect of CD4 inhibition on surface CCR5 in T cells from 3 donors. The proportion of surface CCR5 was calculated by FACS, by comparing the signal in unpermeabilzed and permeabilized cells, and values obtained in cells nuleofected with the S1 siRNA were compared to the proportion of surface CCR5 in cells nucleofected with the ineffective S3 siRNA.

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

[START_REF] Cahill | Morinville A Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization[END_REF]

Ackowledgements:

The authors thank Jean Liotard for his skilled technical assistance and Pierre Bourdoncle for his valuable assistance in cell-imaging experiments. This work was supported by grants from the Institut National de la Sant et de la Recherche M dicale (INSERM), the Centre

performed research; GB contributed new reagents and analyzed data; CLJ designed the research, performed research and analyzed data; SM designed the research


J Weimmerskirch-Aubatin

M Stoffel

X Devaux

A Bouche

G Beainy

E Talbot

P Pareige

Y Fagot-Re ´vurat

M Vergnat

H Rinnert

Observation of a nanoscale phase separation in blue-emitting Ce-doped SiO 1.5 thin films †

Both the optical and structural properties of Ce-doped SiO 1.5 thin films were investigated. The Ce-related blue luminescence, which can be seen even at room temperature for as-grown films, exhibits a rather complex evolution with the annealing temperature. In particular, a strong decrease is observed when the films are annealed at 900 1C. Structural characterizations combining scanning transmission electron microscopy and atom probe tomography reveal the formation of Si-and Ce-rich clusters at this temperature, thus demonstrating that the decreasing Ce-related luminescence is due to concentration induced quenching. For annealing temperatures higher than 900 1C, the Ce-related luminescence increases. The different structural characterizations provide clear experimental evidence of a phase separation occurring at the nanoscale between pure Si nanocrystals and Ce-rich clusters having a stoichiometry close to the cerium silicates Ce 2 Si 2 O 7 or Ce 4.667 (SiO 4 ) 3 O. The latter compounds are optically active thereby explaining the increased Ce-related luminescence observed at the highest annealing temperature.

Introduction

Since bulk Si is an indirect bandgap semiconductor, getting light out of Si-based materials is a challenging issue, not only in the infrared range for telecommunication applications but also in the visible range for displays and white light electroluminescent diodes. At the nanometer scale, the charge carriers are confined in three dimensions, and pure Si nanocrystals embedded in either a SiO 2 1 or a Si 3 N 4 2 matrix are shown to emit light in the visible-near infrared range even at room temperature. Moreover, it was shown that a good control of the Si nanocrystal size can be achieved in SiO/SiO 2 multilayers thus allowing us to tune their emission wavelength in the red-near infrared range. 3,4 The optical properties of Si-based materials containing Si nanocrystals can be further adjusted by doping the SiO x (0 o x o 2) matrix with optically active lanthanide ions. The first studies have concerned the Er 3+ doping of SiO x thin films, which is of potential interest for telecommunications. 5 More recently, other authors have also studied the doping of SiO x thin films with Nd 3+ ions for photovoltaic applications. 6,7 In both cases, an efficient energy transfer was found to occur between Si nanocrystals and Er 3+ or Nd 3+ ions, thereby enhancing the rare earth based luminescence. If many results were reported for light emission in the red and infrared range, few results were published for a blue emission in Si-based materials. Among all rare earth elements, Ce is of particular interest since it is characterized by an allowed electric dipolar 5d-4f transition giving rise to emission in the violet-blue range. This property may be of interest for the development of blue light emitting diodes fully compatible with the Si technology. Morshed et al. obtained violet-blue luminescence in CeO 2 films epitaxially grown on Si(111) substrates by pulsed laser deposition and subsequently annealed at 1000 1C in Ar ambient. The luminescence was tentatively attributed to the formation of a Ce 6 O 11 compound. Later, Choi et al. 9 reported violet/blue luminescence in cerium oxide films grown by radio-frequency sputtering directly on Si(001) substrates. The luminescence was attributed to the formation of cerium silicates. Similar observations were made by Kepinski et al. 10 in CeO 2 -SiO 2 films prepared by the sol-gel method and by Li et al. 11 in Ce-doped silicon oxide thin films grown by electron cyclotron resonance plasma enhanced chemical vapor deposition. By investigating Ce-doped SiO 1.5 films prepared by evaporation, we found that Ce 3+ ions are mainly directly excited with only a negligible influence of Si nanocrystals. 12 Finally, strong blue luminescence has also been reported recently in Ce-doped SiO thin films prepared by evaporation. 13 While extensive work has already been performed particularly regarding the optical properties of rare earth doped SiO x thin films, several critical issues still remain unsolved. In particular, the spatial localization of the rare earth ions with respect to the Si nanocrystals has not yet been addressed. For example, it is not yet clear whether the rare earth ions are located in or at the surface/periphery of the nanocrystals. Moreover, the influence of This journal is © The Royal Society of Chemistry 2015 the rare earth ions on Si nanocrystal formation has not yet been studied.

In this study, we investigated the optical and structural properties of Ce-doped SiO 1.5 thin films. For all the Ce contents investigated, the Ce-related luminescence follows a similar evolution as a function of the annealing temperature. At 900 1C, a strong decrease of the PL intensity is observed. The structural characterization tools show that both Ce and Si form clusters located at the same position, thus explaining the decreasing luminescence by concentration induced quenching. For annealing temperatures beyond 980 1C, we observe at some locations the beginning of a separation between Ce-rich clusters and Si nanocrystals. At 1100 1C, we report the first observation of a phase separation occurring at the nanoscale between pure Si nanocrystals and Ce-rich clusters having a stoichiometry close to the cerium silicates Ce 2 Si 2 O 7 or Ce 4.667 (SiO 4 ) 3 O. These compounds are known to be luminescent thereby explaining the reappearance of the Ce-related luminescence at this temperature.

Experimental

The SiO 1.5 films were obtained by co-evaporation on Si(001) or fused silica substrates held at room temperature of SiO powder from a thermal cell and SiO 2 from an e-beam gun in an ultrahigh vacuum chamber. The Ce doping was performed by using a Knudsen cell heated at B1400 1C. The Ce flux was monitored by a quartz microbalance and the Ce content defined by [Ce]/ ([Si] + [O] + [Ce]) was varied between 0.3% and 4%. The film thickness was 20 nm for the samples investigated by transmission electron microscopy and 200 nm for all other samples considered in this study. The samples were post-grown annealed in either a tubular oven at temperatures between 500 1C and 900 1C or in a rapid thermal annealing furnace in a N 2 atmosphere at 1100-1200 1C. In the case of a tubular oven, the temperature was ramped up with a rate of 10 1C min À1 up to the desired temperature prior to cooling down, while for the rapid thermal annealing furnace all samples were kept at 1100-1200 1C for 5 min. The steady state photoluminescence (PL) was excited with a 325 nm He-Cd laser and subsequently analyzed by a monochromator equipped with a 600 grooves per mm grating and measured by a photomultiplier tube operating in the UV-visible range. A Xe lamp (maximum power 300 W) was used as an excitation source for the photoluminescence excitation (PLE) measurements. The response of the detection system was precisely calibrated by a tungsten wire calibration source. The Raman spectra were collected in backscattering geometry using a 532 nm laser diode as an excitation source. The scattered light was dispersed by a grating with 1800 grooves per mm and detected by a multichannel charge coupled device (CCD) camera cooled at 200 K. All Raman measurements were performed on samples deposited on fused silica substrates in order to get rid of the contribution of the transverse optical phonon line of the Si substrate. The X-ray photoelectron spectra were measured using the non-monochromatized AlK a radiation at 1486.6 eV. Prior to the measurements, the samples were subjected to a 45 min Ar + ion bombardment in order to remove potential impurities/ contaminants from the surface. The photoelectrons were collected by a Scienta SES 200 hemispherical analyzer. The overall resolution of the setup was about 0.8 eV. The structural properties of the samples were studied by high-resolution transmission electron microscopy (HRTEM) and scanning transmission electron microscopy (STEM) with a JEOL ARM200F. STEM images were simultaneously recorded using high angle annular dark field (HAADF) and bright field (BF) detectors and completed with local energy-dispersive X-ray spectroscopy (EDS). To avoid any phase precipitation due to ion or electron beam damages, the samples were mechanically thinned up to the electron transparency by the tripod method. Both TEM and STEM investigations were performed at 80 kV. The 3D mapping of the distribution of atoms in the sample was obtained by laserassisted atom probe tomography (APT) analyses. The tips required for the APT analysis were prepared according to a process used on similar materials as reported elsewhere. 14,15 The analyses were carried out at 80 K using a CAMECA LAWATAP instrument with an amplified Ytterbium doped laser emitting at a wavelength of 343 nm providing a 350 fs pulse duration and a 100 kHz repetition rate with an energy per pulse of around 50 nJ. The infrared absorption spectra are obtained using a Fourier transform infrared absorption spectrometer (FTIR). The films are analyzed at normal incidence by direct transmission. The acquisition of the spectra is performed in absorbance mode. The spectra of the thin films are recorded after subtraction of the reference spectrum measured on a Si substrate. In order to compare the obtained results, a baseline is subtracted from each spectrum to eliminate the interference fringes caused by multiple reflections in the substrate.

Results and discussion

Fig. 1(a) displays a typical room temperature (RT) steady state PL spectrum of an as-deposited SiO 1.5 thin film doped with 3% Ce (red solid line). We observe strong blue luminescence, which can be seen even with the naked eye (see the inset of Fig. 1(a)).

The PL band is rather broad and extends from 330 nm to 530 nm with a maximum centered at B380 nm. The luminescence may be due to either the defects of the SiO 1.5 matrix or the allowed electric dipolar 5d-4f transition of isolated Ce 3+ ions. The former hypothesis can, however, be ruled out since an as-grown and undoped SiO 1.5 film does not show any luminescence near 400 nm. Moreover, the PL intensity increases with the Ce content in the films (see also Fig. 1(b)). We can therefore unambiguously attribute the observed luminescence to the allowed electric dipolar 5d-4f transition of Ce 3+ ions. In order to determine the excitation mechanism of Ce 3+ ions, we further perform PLE measurements. In this case, the detection wavelength is fixed at 450 nm (this wavelength has been chosen since detection is possible for both as-grown and annealed samples for which the PL signal shifts to higher wavelengths; see also the ESI †) and the excitation wavelength is varied between 250 nm and 370 nm. The PLE spectrum, which is shown in Fig. 1(a) as a black solid line, is characterized by a single peak centered at B300 nm. This peak is typical for the excitation of the 5d levels of Ce 3+ ions starting from the fundamental 2 F 5/2 level, thus showing that the Ce 3+ ions are mainly directly excited. In the following, we will concentrate our attention on the Ce-related luminescence and its evolution with the annealing temperature. Fig. 1(b) displays the evolution of the integrated Ce-related luminescence as a function of the annealing temperature. It is noteworthy that the evolution follows the same trend independently of the Ce concentration in the films. One can easily distinguish three different regimes. Firstly, from RT up to 500 1C, the integrated luminescence increases. Secondly, from 500 1C up to 900 1C, the integrated luminescence decreases and eventually reaches a minimum at 900 1C. Thirdly, at higher annealing temperatures up to 1200 1C, the integrated Ce-related luminescence increases. It is important to note that the reappearance of the PL and the existence of a minimum of PL intensity are not due to the annealing treatment method. Indeed, we carefully checked that a similar qualitative evolution is obtained when the samples are annealed using only the RTA furnace with a minimum of PL intensity occurring at a higher annealing temperature, i.e. 1100 1C. A similar qualitative evolution has already been reported by Li et al. 11 in Ce-doped silicon oxide films but the evolution has not been investigated in detail. Interestingly, as the annealing temperature increases, the Ce-related luminescence band shifts to higher wavelength (see ESI †). Let us consider the first regime, i.e. from RT up to 500 1C. It is well known that annealing heals out point defects thus leading to a decreased density of non-radiative recombination centers. Consequently, the luminescence increases as observed in Fig. 1(b). We will now consider the second regime corresponding to annealing temperatures between 500 1C and 900 1C. Li et al. 11 have interpreted the observed decrease by the precipitation of Ce 3+ ions but the existence of Ce rich clusters is unclear and the chemical composition of the clusters remains unknown. Different hypotheses can be put forward to explain the decreased PL intensity. The first one is a modification of the valence of Ce ions evolving from Ce 3+ ions to Ce 4+ ions. The latter are known to be not optically active which could possibly result in a quenching of the luminescence. The second one is that Ce ions diffuse and form clusters leading to a concentration induced quenching of the luminescence. In order to test the possibility of valence changes, X-ray photoelectron spectroscopy (XPS) measurements of the Ce 3d core level were carried out. The different valence states are characterized by well-defined peaks appearing at different binding energies, thus allowing discrimination between Ce 3+ and Ce 4+ . 16,17 Fig. 2 shows the Ce 3d core levels measured on a 3% Ce-doped SiO 1.5 thin film annealed at 500 1C (Fig. 2(a)) and at 900 1C (Fig. 2(b)). The obtained spectra look qualitatively similar to those previously measured on thin CeO 2 films deposited on either Si(111) substrates 17 or thin Pt foils. 16 After subtraction of a Shirley background, 18,19 both spectra were fitted with Gaussian-Lorentzian functions to de-convolve multiple peaks that are closely spaced in binding energy. The spin-orbit splitting between the Ce 3d 3/2 and the Ce 3d 5/2 peaks was fixed at 18.3 eV as in previous studies. 16,17 The fitting was performed by using the chemical shifts from the literature 16,17 and by varying only the height and the full width at half maximum (FWHM) of the peaks. The ratio between the Ce 3+ and Ce 4+ related peak intensities was then used to determine the amount of Ce ions in the (III)/(IV) oxidation state. In both cases, the contribution of Ce 3+ ions is dominant and only a moderate increase of the Ce 4+ content is observed when the annealing temperature increased from 500 1C to 900 1C. We can thus rule out a valence state change as the main origin of the decreased PL intensity observed at 900 1C. To test whether Ce clusters may form, STEM measurements were performed.

Fig. 3 (a) shows a HAADF image of a 3% Ce-doped SiO 1.5 thin film annealed at 900 1C and the corresponding bright field image is shown Fig. 3(b). One can clearly identify a high density of clusters which exhibit a high contrast (bright on HAADF and dark on BF). This observation suggests that they are composed of a heavy element which in our case is Ce. Spatially resolved EDS investigations (Fig. 3(c) and(d)) show that the clusters contain Ce while the surrounding matrix is Ce-free. Moreover, the analysis of the HRSTEM images shows that these clusters are amorphous. The demonstration of the formation of Ce-rich clusters allows us to conclude that the decreased PL intensity observed at 900 1C is the result of concentration induced quenching. If we assume that all Ce ions are randomly distributed in the as-grown films, the observation of Ce clusters in the 900 1C annealed sample indicates that a significant Ce diffusion must have taken place during annealing. We cannot exclude the possibility that other atomic species of lower mass like Si may also diffuse during high temperature annealing. In order to test the possibility of Si diffusion, we have performed atom probe tomography on the 3% Ce-doped SiO 1.5 thin film annealed at 900 1C.

Fig. 4(a) shows a typical slice view (48.8 Â 48.8 Â 5 nm 3 ) taken from the 3D reconstructed volume representing Si atoms for the sample annealed at 900 1C. Oxygen atoms are not represented for clarity reasons. The cartography clearly reveals an inhomogeneous Si distribution with the presence of Si-rich clusters. In contrast, the as-grown sample exhibits a rather uniform Si distribution (not shown here). The presence of Si-rich clusters in the sample annealed at 900 1C thus indicates that the demixtion of the SiO 1.5 film has already started. The excess Si atoms -by comparison with SiO 2 -will diffuse and eventually agglomerate to form the observed clusters as known for undoped SiO x films. 4,20 Fig. 4(b) shows the Ce atoms in the same slice view (48.8 Â 48.8 Â 5 nm 3 ). The cartography gives evidence of the presence of Ce-rich clusters, thus confirming the previous STEM observations. Interestingly, showing both Si-and Ce-related maps (Fig. 4(c)), we found that Ce-rich and Si-rich clusters are located on the same position. Thus, it seems that Ce atoms do not prevent the demixtion of the SiO 1.5 film but instead leads to the formation of Si-clusters containing Ce. Although we have not investigated in detail the diffusion mechanism of both Si and Ce atoms, we can nevertheless suggest that both atomic species will probably migrate to the pores/vacancies contained in the amorphous SiO 1.5 thin films. At the present stage of our investigation, it is not yet clear whether the Si-rich clusters are crystallized or not. To answer this question, Raman measurements have been carried out.

Fig. 5 displays the Raman spectra of a 3% Ce-doped SiO 1.5 film recorded at different annealing temperatures ranging from 900 1C to 1150 1C. When the film is annealed at 900 1C, the Raman spectrum looks very similar to that of a fused silica substrate indicating that the crystallization of the Si atoms in excess has not yet started. Indeed, the Raman response of amorphous silicon is characterized by a broad and weak peak located at around 480 cm À1 . The absence of the Raman signature of the film is interpreted by the presence of silicon in its amorphous state. In contrast, as the film is annealed at 1025 1C, a small peak emerges at B520 cm À1 , i.e. very close to the position of the transverse optical phonon in bulk crystalline Si (520.5 cm À1 ). We thus conclude that pure Si nanocrystals start to form at temperatures higher than 980 1C. A similar evolution is observed for a 0.7% Ce-doped SiO 1.5 film (not shown here). Moreover, the intensity of the peak observed at 520 cm À1 increases as the annealing temperature increases (see inset of Fig. 5), thus suggesting the formation of a larger number of pure Si nanocrystals. This result is quite surprising since we have shown in Fig. 4(c) that Ce ions have the same locations as Si atoms. One could thus expect the formation of a Ce-Si-O compound rather than the formation of pure Si nanocrystals. To understand further the evolution of the system, in particular the distribution of the Ce ions in the sample annealed at 980 1C, atom probe tomography experiments were further performed on the 3% Ce-doped SiO 1.5 thin film annealed at 980 1C.

Fig. 6(a) shows a typical slice view (33 Â 33 Â 5 nm 3 ) taken from the reconstructed volume representing Si atoms. Similar to the sample annealed at 900 1C, the distribution of Si atoms is still inhomogeneous. The same is valid for Ce ions (Fig. 6(b)) as observed previously (Fig. 4(b)). If one shows both Si-and Ce-related atomic maps (Fig. 6(c)), we can recognize that, at some locations, the Ce-rich regions start to be separated from the Si nanocrystals as shown by the dashed circles in Fig. 6(c). It would now be interesting to understand how the system evolves at even higher annealing temperatures, for instance at 1100 1C where the Ce-related luminescence is found to increase (Fig. 1(b)). We thus perform a similar analysis on the 3% Ce-doped SiO 1.5 thin film annealed at 1100 1C.

Fig. 7(a) shows a typical slice view (33 Â 33 Â 5 nm 3 ) taken from the reconstructed volume and representing both Si atoms (red dots) and Ce atoms (blue dots). While at 980 1C we observe the beginning of a separation between pure Si nanocrystals and Ce-rich clusters, at 1100 1C, the phase separation now seems to be almost complete. Indeed, the center of mass of the Si nanocrystals does no longer coincide with the center of mass of the Ce-rich clusters. This is the first experimental evidence of a phase separation occurring at a length scale below 10 nm in rare earth doped silicon oxide thin films. We then further used atom probe tomography to obtain a better insight into the composition of the Ce-rich clusters. The compositional analysis This journal is © The Royal Society of Chemistry 2015 reveals that these clusters are indeed composed of Ce but also of Si and O with relative amounts 16.3%, 18.2% and 65.5%, respectively. The composition of these clusters appears to be compatible with the formation of cerium silicates of stoichiometry Ce 2 Si 2 O 7 and Ce 4.667 (SiO 4 ) 3 O. The latter compounds are known to be luminescent in the violet/blue range. 9,11 This eventually explains the reappearance of the Ce-related luminescence for samples annealed at 1100 1C (third regime in Fig. 1(b)). Finally, in order to further strengthen our observations, we investigate the structural properties of the 3% Ce-doped SiO 1.5 film annealed at 1100 1C by transmission electron microscopy (TEM). Fig. 7(b) shows a high resolution TEM image providing evidence of a phase separation between a pure Si nanocrystal (circled in red) and a Ce-rich cluster located in its vicinity (circled in blue). The latter is not sufficiently well-oriented to clearly evidence crystal planes on the micrograph. Nevertheless both STEM HAADF and STEM BF have shown that the Ce-rich clusters are crystalline. However, the main grains appear twinned or polycrystalline; this makes a structural determination difficult starting from HRSTEM micrographs. For the few monocrystalline grains that were observed, the coexistence of both crystalline Ce 4.667 (SiO 4 ) 3 O and Ce 2 Si 2 O 7 compounds cannot be excluded in agreement with the Ce-Si-O phase diagram. 21 To further confirm the formation of cerium silicate, we investigate the Ce-doped SiO 1.5 film annealed at 1200 1C by FTIR spectroscopy. Fig. 8 displays the FTIR spectra of Ce-doped SiO 1.5 films annealed at 1200 1C for different Ce concentrations ranging from 0.3% to 4%. All spectra are dominated by welldefined lines at 800 cm À1 corresponding to the symmetric stretching mode of the Si-O-Si bond and at 1080 cm À1 corresponding to the asymmetric stretching mode of the Si-O-Si bond in SiO 2 . 22,23 Apart from these lines, we can further identify a rather broad band between 900 cm À1 and 975 cm À1 (see also the inset of Fig. 8). The intensity of this band increases as the Ce content in the films increases, thus suggesting that this feature is related to the presence of Ce. In the literature, 24,25 several closely spaced peaks have been identified in the range 826-975 cm À1 , which may be due to the formation of the Ce 2 Si 2 O 7 or Ce 4.667 (SiO 4 ) 3 O compounds. Our FTIR investigations thus confirm the formation of a cerium silicate for high annealing temperatures.

Finally, we will discuss the general evolution observed during annealing of Ce-doped SiO 1.5 thin films. The atom probe tomography measurements performed on the as-grown films show that Ce atoms are almost randomly distributed (not shown here). When the samples are annealed at 900 1C, amorphous Ce-rich clusters have been identified by both STEM (Fig. 3) and APT measurements (Fig. 4). This indicates that Ce diffusion is thermally activated in our films. Moreover, as the annealing temperature increases up to 900 1C, a demixtion occurs in the SiO 1.5 films leading to Si atoms in excess in a matrix with a composition reaching SiO 2 . Both Ce and Si atoms will then diffuse and eventually accumulate at the pores/vacancies of the films, thus leading to Ce-and Si-rich clusters as shown in Fig. 4(c). At least up to the annealing temperature of 900 1C, the evolution appears to be essentially governed by kinetics, i.e. by diffusion and clustering of both Si and Ce. When the films are further annealed at higher temperatures, thermodynamics takes over and the Si-O-Ce system moves towards stable compounds.

The formation of cerium silicates is, however, precluded at 900 1C since the thermal budget is still too low. 21 According to the Ce-Si phase diagram, 26 the formation of cerium silicides also appears to be unlikely for the Ce contents and annealing temperatures considered in our study. Moreover, we can also rule out the formation of CeO 2 since we do not observe the wellknown Raman peak at 462 cm À1 in Fig. 5. This conclusion is further supported by the X-ray photoelectron spectroscopy data which do not show a significant increase of the Ce 4+ related peak intensities as the annealing temperature increases. For annealing temperatures above 980 1C, the formation of cerium silicates such as Ce 2 Si 2 O 7 or Ce 4.667 (SiO 4 ) 3 O starts. At this temperature, the compositional analysis based on APT measurements has shown that the Si content in the clusters is about 24%, which is much larger than the amount of Si required to form the cerium silicates (i.e. 18.2% for Ce 2 Si 2 O 7 and 14.5% for Ce 4.667 (SiO 4 ) 3 O). Consequently, the Si atoms in excess will be ejected and will agglomerate to form pure Si nanocrystals in the immediate vicinity of the cerium silicate clusters. We thus expect that thermodynamics is the main driving force leading to the formation of stable phases which in our case are Si, SiO

Conclusions

To summarize, we have investigated both the structural and optical properties of Ce-doped SiO 1.5 thin films. The complex evolution of the Ce-related luminescence with the annealing temperature is well explained thanks to a complementarity of different nanoscale structural characterizations and vibrational spectroscopies. In particular, the strong PL decrease at 900 1C is related to the presence of Ce-rich clusters. At higher annealing temperatures, we were able to identify and characterize a phase separation occurring at a length scale below 10 nm between pure Si nanocrystals and Ce-rich clusters having a stoichiometry close to Ce 2 Si 2 O 7 or Ce 4.667 (SiO 4 ) 3 O. This is the first direct observation of a nanoscale phase separation in a solid state matrix. The cerium silicates are known to be luminescent, thereby explaining the increased Ce-related luminescence at 1100 1C. At the present stage of our investigations, the microscopic driving force at the origin of the observed phase separation is not yet clearly identified. Further theoretical work using kinetic Monte Carlo simulations and/or even molecular dynamics or nano-thermodynamic simulations would be needed to clarify this important issue. This work brings a comprehensive view of the optical properties of Ce-doped Si-based layers in relation to their structure and then represents an important step towards the realization of blue light emitting devices fully compatible with the well-known Si technology.

Notes and references

Fig. 1

 1 Fig. 1 (a) Steady state PL spectrum of a 3% Ce-doped SiO 1.5 thin film measured at room temperature (red solid line). The corresponding PLE spectrum is shown as a black solid line. The inset shows a photograph of the sample showing the blue luminescence visible with the naked eyes. (b) Integrated Ce-related PL intensity as a function of annealing temperature for three different Ce contents.

Fig. 2 X

 2 Fig. 2 X-ray photoelectron spectra of the Ce 3d core level measured on a 3% Ce-doped SiO 1.5 thin film after annealing at 500 1C (a) and 900 1C (b). The result of the fit (solid lines) is superimposed on the experimental data (crosses).

Fig. 3 Fig. 4

 34 Fig. 3 HAADF image of a 3% Ce-doped SiO 1.5 thin film annealed at 900 1C showing the presence of Ce-rich clusters (a) and the corresponding bright field image (b). HAADF image (c) showing the areas (red square for the matrix and blue square for a particle) used for the EDS analysis (d)

Fig. 5

 5 Fig. 5 Raman spectra measured on a 3% Ce-doped SiO 1.5 thin film after annealing at various temperatures between 900 1C and 1150 1C. All spectra are vertically offset for clarity. The vertical dashed-dotted line indicates the position of the crystalline Si-TO peak at 520 cm À1 . The inset shows the evolution of the integrated Raman peak intensity at 520 cm À1 as a function of annealing temperature.

Fig. 6

 6 Fig. 6 Atom probe tomography analysis of the 3% Ce-doped SiO 1.5 thin film annealed at 980 1C. (a) Plane view of a 5 nm thick slice taken from the reconstructed volume. The Si atoms are shown as red dots, providing evidence of the presence of Si-rich clusters, (b) analogous plane view showing Ce atoms as blue dots, evidencing the presence of Ce-rich clusters, (c) superposition of the maps shown in (a) and (b) demonstrating that both Si-and Ce-rich clusters no longer have the same locations (also indicated by dashed circles). All the views have the size of 33 Â 33 Â 5 nm 3 .

2 and Ce 2

 2 Si 2 O 7 or Ce 4.667 (SiO 4 ) 3 O.

Fig. 7

 7 Fig. 7 Atom probe tomography analysis of the 3% Ce-doped SiO 1.5 thin film annealed at 1100 1C. (a) Plane view (33 Â 33 Â 5 nm 3 ) of a 5 nm thick slice taken from the reconstructed volume showing both Si atoms (red dots) and Ce atoms (blue dots). The phase separation between pure Si nanocrystals and Ce-rich clusters is evident. (b) High resolution cross sectional TEM image showing the presence of a pure Si nanocrystal (circled in red) in the immediate vicinity of a Ce-rich cluster (circled in blue).

Fig. 8

 8 Fig.8FTIR spectra of Ce-doped SiO 1.5 thin films annealed at 1200 1C. The Ce content is varied between 0.3% and 4%. All spectra are vertically offset for clarity. The dashed area is enlarged in the inset. The vertical dashed lines in the inset indicate the wavenumber range in which features corresponding to the formation of a cerium silicate are expected.

J. Mater. Chem. C, 2015, 3, 12499--12506 | 12503

J. Mater. Chem. C, 2015, 3, 12499--12506 | 12505


Pierre Trinsoutrot

Hugues Vergnes

Brigitte Caussat
email: brigitte.caussat@ensiacet.fr

Three dimensional graphene synthesis on nickel foam by chemical vapor deposition from ethylene

Keywords:

3D multi-layers graphene networks were synthesized on nickel foam from ethylene between 700 and 1000 • C by chemical vapor deposition. Large nickel foam substrates were used allowing the accurate measurement of graphene masses. The weight of graphene increased with run duration and when decreasing temperature. Graphene was also present inside the hollow branches of the foam. We demonstrated that the weights of graphene formed largely exceed the masses corresponding to carbon solubility into nickel. Indeed weight percentages of graphene as high as 15% were obtained, corresponding to graphene layers of 500 nm to 1 m thick. This means that graphene formation could not be due only to carbon dissolution into nickel and then precipitation during the cooling step. Another mechanism probably coexists, involving continuous graphene formation in presence of ethylene either by segregation from the dissolved carbon into nickel or by surface CVD growth.

Introduction

Graphene is a two-dimensional mono-layer of sp 2 -hybridized carbon atoms. These last years, it has attracted huge attention because of its unique physical properties [1,2], in particular large surface area, exceptional electron mobility, high thermal conductivity, extraordinary mechanical robustness, . . . To exploit these properties for industrial applications, both large-scale synthesis and integration of individual graphene sheets into multifunctional structures are required. One promising application concerns lithium ion batteries, whose electrochemical performances could be enhanced by developing new electrode materials of higher electronic conductivity than the existing ones [3,4]. Several approaches have been developed for the large-scale synthesis of graphene for this application, such as chemical exfoliation or reduction of graphite oxide [5,6]. But these processes lead to poorly conductive graphene due to the presence of important structural defects and of oxygen containing groups. Chemical vapor deposition (CVD) is an alternative technology able to synthesize large-area high-quality graphene on planar metal substrates [7].

Recently, some research groups have succeeded in forming three dimensional (3D) foam-like graphene macro-structures by template-directed CVD [1,3,4,8,9]. They have used nickel foams as scaffold and a reactive gaseous mixture composed of methane, hydrogen and argon at 1000 • C. They have obtained 3D networks of few-layers and high-quality graphene exhibiting high conductivity (600 S/cm), high reversible capacity and good rate performance in lithium ion batteries [3].

The mechanism of graphene formation on nickel involves first methane decomposition on the catalytic surface, then carbon dissolution into nickel and finally carbon precipitation during the cooling step due to the decrease of carbon solubility [10]. Few studies are available concerning the use of acetylene, ethylene or ethanol to form graphene on nickel substrates. Jiang et al. [11] have synthesized graphene based carbon nano-fibers on nickel fibers from ethanol at 700 • C. Xiao et al. [12] have used acetylene on nickel foam at 1000 • C to form 3D 10 nm-thick graphene with large amounts of defects. Chae et al. [13] have shown that a decrease of temperature from 1000 to 700 • C using acetylene on planar polynickel substrates at 10 -3 Torr led to an increase of the number of graphene layers and of the amount of defects. Addou et al. [14] have formed monolayer graphene from ethylene in ultrahigh vacuum conditions (10 -6 Torr) on Ni (1 1 1) planar substrates between 500 and 600 • C. The higher reactivity of ethylene in comparison with methane allows to decrease the deposition temperature [15]. To the best of our knowledge, ethylene has never been used to form graphene on nickel foams.

In this paper, we report the synthesis of 3D graphene networks on nickel foam from ethylene between 700 and 1000 • C at ambient pressure. Large catalytic substrates were used allowing the accurate measurement of graphene masses.

Experimental

The CVD synthesis of graphene was carried out in a 100 mm quartz tube of 1400 mm in length. A three zones furnace of 760 mm length surrounds the reactor. The 1.8 mm-thick nickel foam samples (99.9% Alantum, 40 mm × 60 mm) were placed in the central isothermal zone of the reactor, using a substrate holder which maintains them vertically during the process. They were heated between 700 and 1000 • C in a H 2 and Ar flow and annealed for 5 to 40 min. Then 15 sccm of ethylene were introduced into the reactor during 5-40 min with the same Ar and H 2 flow rates. The samples were then cooled to room temperature under the same Ar/H 2 flow rates without carbon precursor. All experiments were performed at 700 Torr.

The samples were systematically weighed before and after graphene synthesis on a Mettler Toledo (AX105 Delta Range) balance (accuracy of ±0.0003 g). Raman spectroscopy (confocal Raman microscope Labram-Horiba Yvon Jobin) with a laser excitation wavelength of 532 nm and optical microscopy were used to evaluate the thickness, crystalline quality and uniformity of the graphene at room temperature. Each spectrum was obtained by 5 acquisitions of 30 s accumulation time. Nine measurements were systematically performed for each sample on pre-definite positions covering the whole foam surface. The morphology of graphene films was analyzed in cross-section using Scanning Electron Microscopes (Jeol JSM 6700F and SEM Hitachi 5500) after having cut the foam at the center of the samples.

Results and discussion

Table 1 presents the operating conditions tested and the graphene weights obtained. The nickel foam scaffold used to form graphene network is a 3D porous structure with hollow branches and quite circular pores of 50-200 m in diameter, as shown in Fig. 1a. After graphene synthesis, thanks to the thermal treatment applied, the foam is constituted of polycrystalline nickel grains in the range of 2-20 m (Fig. 1b). Numerous Raman spectra and SEM analyses have been performed after graphene formation. They indicate that the foam is fully and uniformly covered by graphene even at its bulk and even after 5 min of deposition, as found by other studies [8,9]. Graphene is also present inside the branches of the foam, with a thickness equivalent to that outside the foam, as revealed by SEM views (Fig. 1c).

On the Raman spectra, the average number of graphene layers is estimated from the 2D (∼2670 cm -1 )/G (∼1582 cm -1 ) peaks ratios. The graphene crystalline quality can be deduced from the ratio between the disorder-induced D-peak (∼1350 cm -1 ) and the G peak. It is worth noting that when using methane, the 2D/G peak ratios often vary from 0.5 to 3, corresponding to few layers to multi-layers (>3) graphene on a sample, according to the position on the foam [8,9]. This is true also on nickel planar substrates [10,16] and has been attributed to the fact that carbon preferentially precipitates out at the grain boundaries of polycrystalline nickel substrates. When using ethylene, the 2D/G peaks ratios are always comprised between 0.32 and 0.38, indicating that graphene is everywhere multi-layers (Fig. 1d). This could be due to the higher reactivity of ethylene in comparison with methane [15]. So, it could be less sensitive to the catalytic surface state.

The influence of temperature has then been studied for an annealing duration of 40 min and a deposition duration of 20 min, as detailed in Table 1. The main result obtained is that for all runs, the mass of graphene is higher than the mass of carbon corresponding to the C solubility into nickel at the operating temperature (for instance 0.26 wt% at 1000 • C). We verified that this was also true for the works of Bi et al. [8] and Chen et al. [9] on nickel foam from methane. This means that the mechanism of graphene formation on nickel can not be due only to carbon dissolution into nickel and then precipitation during cooling, contrarily to what is generally recognized in the literature [10,17]. Graphene could also be formed by a continuous mechanism existing as long as ethylene is present, either of isothermal carbon segregation from the carbon dissolved into nickel or of CVD surface growth. Some authors [18,19] have already mentioned the possible contribution of CVD surface growth for graphene formation on planar nickel from methane. So, it is likely that such continuous mechanism also exists on any form of nickel substrates from any carbon gaseous precursor. The great advantage of nickel foams compared to nickel planar substrates is that they allow forming measurable weights of graphene.

Another result is that the wt% of graphene increases when decreasing temperature, from 0.75% to a max. value of 9.5% at 700 • C (Fig. 2a). However, the quality of graphene is strongly dependant of the temperature. Characteristic Raman spectra obtained for each temperature (Fig. 2b) indicate that at 700 • C, the 2D and G bands are well-defined, which is characteristic of carbon sp 2 hybridation, then of graphene [20]. The shouldering visible on the G band can be attributed to the crystalline defects revealed by the high D/G peak ratio. According to the 2D/G ratio, graphene is multi-layers. At 750 • C, graphene is also multi-layers but with a less marked D band. At 800 and 850 • C, multi-layers graphene without D band is obtained. At 1000 • C, the 2D band does not appear anymore and the G band is broad and poorly defined, which could be linked to the presence of a great variety of C bonds, with a lot of defects as revealed by the D peak. So for these conditions, a temperature of 1000 • C does not allow the formation of graphene but probably of amorphous carbon, as indicated by the absence of the 2D band [20]. The fact that contrarily to methane, ethylene allows to form more selectively carbon nanomaterial around 750-850 • C rather than at higher temperature is well-known for carbon nanotubes, due to its higher reactivity [21].

The thickness of graphene formed at 750 • C estimated from cross-section SEM analyses ranges between 500 and 900 nm (Fig. 3a). Similar or even higher values were measured at 700 • C (Fig. 3b, see dotted lines). These thicknesses and the weights of graphene formed are clearly much higher than those reported in the literature from methane or acetylene. Indeed, Chen et al. [1] formed mono to few layers graphene for 5 min of deposition. Bi et al. [8] obtained 4 wt% max. of graphene presenting 3-10 layers for 30 min of deposition. Chen et al. [9] formed 1.7 wt% max. of graphene for run durations of 10 min. These much higher values could be due to the higher reactivity of ethylene in comparison with methane, to the presence of graphene inside the foam branches and to the higher precursor flow rate and run duration used in our case. At 850 • C and 1000 • C, the thickness has been estimated close to several tens of nm. Such thick carbon layers could appear as closer to graphite than to graphene. But the 2D band of the Raman spectra obtained for temperatures between 700 and 850 • C (Fig. 2b) is clearly typical of graphene, knowing that for graphite, the 2D peak is formed of two bands [20].

For the thickest layers between 700 and 800 • C, the crosssection morphology appears to be of cream-slice type and graphene presents a high surface roughness due to the formation of numerous cracks. These cracks are certainly due to differences of thermal expansion coefficients between nickel and graphene [13] and their formation is probably favoured when the graphene thickness increases. When the thickness is lower (samples obtained at 850 and 1000 • C, Fig. 3c andd), a smoother morphology close to that reported in the literature from methane [9] is observed. It is worth noting that for these samples, two distinct layers of graphene appear in the cross-section SEM views. They could be related to the co-existence of the two mechanisms of graphene formation previously detailed. This behaviour was not observed for the thicker graphene, but more precise analyses are necessary to verify if two kinds of graphene structure could be distinguished.

The influence of deposition duration has been studied at 750 and 1000 • C (Table 1). In both cases, the graphene weight clearly increases with run duration, reaching 15.5 wt% for 40 min of deposition at 750 • C. Such evolution was also observed by Bi et al. [8] from methane. This confirms that the mechanism does not only involve carbon precipitation during the cooling step, but also a continuous mechanism in presence of the carbon precursor. Such high masses of 3D graphene are very promising, especially for Li-ion battery applications [4].

An increase of graphene weight is also observed at 850 • C when increasing annealing duration (Table 1), but the effect is less marked than that of deposition duration, confirming the relatively low sensitivity of ethylene to the nickel crystalline state.

Conclusions

Weight percentages as high as 15% of 3D graphene networks can be produced by chemical vapour deposition from ethylene on nickel foam, between 750 and 850 • C. Such graphene masses largely exceed the weights corresponding to carbon solubility into nickel at these temperatures. This means that the mechanisms of graphene formation on nickel could involve carbon precipitation during the cooling step, but also continuous graphene formation from either the dissolved carbon brought by the carbon precursor supply or surface CVD growth. This assumption is reinforced by the fact that (i) the weight of graphene increases with deposition duration and (ii) two distinct layers of graphene appear for the thinnest layers, which could be related to two different mechanisms. The thickness of graphene formed at 700 and 750 • C can be as high as 1 m, with cream slice morphology, numerous cracks and high surface roughness. Electrochemical studies are in progress to test this material as anode of Li-ion batteries.

Fig. 1 .

 1 Fig. 1. (a) SEM view of the nickel foam. (b) SEM view of the nickel surface after annealing at 1000 • C. (c) FEG SEM view of a cut branch after run Ni31. (d) Raman spectra of graphene at various points of the foam after run Ni22.

Fig. 2 .

 2 Fig. 2. (a) Characteristic Raman spectra of graphene after runs Ni25, Ni27, Ni29, Ni30, Ni31, (b) Influence of deposition temperature on graphene wt%.

Fig. 3 .

 3 Fig. 3. FEG SEM view of graphene (a) after run Ni31, (b) after run Ni30, (c) after run Ni22, (d) after run Ni28.

Table 1

 1 Experimental conditions tested and results obtained.

	Run	Temperature ( • C)	Annealing	Deposition	Weight of the	Weight of	Wt% of deposited
			duration (min)	duration (min)	nickel foam (g)	graphene (g)	graphene
	Ni26	1000	5	5	1.7026	0.0100	0.59
	Ni28	1000	5	20	1.4559	0.0128	0.88
	Ni27	1000	40	20	1.6891	0.0127	0.75
	Ni22	850	5	20	1.3002	0.0084	0.65
	Ni25	850	40	20	1.6898	0.0149	0.88
	Ni29	800	40	20	1.7002	0.0313	1.8
	Ni30	750	40	20	1.6988	0.1308	7.7
	Ni33	750	40	40	1.5232	0.2361	15.5
	Ni31	700	40	20	1.6022	0.1522	9.5

Acknowledgements

The work was supported by the FP7-NMP GRENADA European project. The authors acknowledge M. Molinier and E. Prevot from LGC/INP de Toulouse, for technical support.