Thomas Duguet
email: thomas.duguet@ensiacet.fr

Eliane Amin-Chalhoub

Diane Samélor

Alessandro Pugliara

Constantin Vahlas

Eliane Amin -Chalhoub

Black Co oxides coatings for thermosensitive polymer surfaces by low-temperature DLI-MOCVD

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Cobalt oxide based versatile materials with high surface to volume ratios have attracted substantial research effort in the last twenty years because of their potential applications in various key enabling technologies [1]. Their promising optical [2], magnetic [3], (photo-)catalytic [4,5], sensing [6], electrochromic [7], energy storage [8] and capacitance [9] functionalities find their origin in the appropriate combination of their electronic and morphological properties. The latter depend on the technique used for their production and on the corresponding process conditions. By tuning surface reactivity, and nucleation and growth modes, chemical vapor deposition (CVD) allows precise monitoring of the microstructure and the morphology of the processed coatings and films. This advantage has been considered in the production of cobalt oxide films and nanostructures [10][11][12][13][14][15][16]. However, such infatuation for the CVD of cobalt oxides also reveals that these processes require appropriate chemical routes and engineering to provide materials with the targeted functionalities (see Ref [2] and references therein). This situation also prevails in the investigation of low reflectivity, cobalt oxide films that are used in optical instruments with the purpose of noise attenuation, namely reduction of the stray effect and scattered light in different spectral domains [17]. In this perspective, the authors recently reported the successful, low temperature, CVD processing of black CoO films on silicon [2]. Deposition at low temperature was necessary since the targeted application of the process is the surface functionalization of carbon fiber reinforced polyepoxies (CFRP) composite materials. It was made possible by the use of dicobalt octacarbonyl, Co2(CO)8 as precursor. Co2(CO)8, a solid compound humidified in hexane, was transported to the deposition area by sublimation, in the absence of reactive gas, e.g. oxygen. The reflectivity of the obtained films was as low as 2-5 % and this promising value was attributed to the columnar, fractal, cauliflower-type morphology, a specificity offered by CVD as compared to other deposition techniques. Despite this successful demonstration, it was not possible to elaborate a process operating in a parametric window large enough to allow deposition of films with stable physicochemical and microstructural characteristics, thus providing reproducible high performance optical properties. One of the reasons for this was the sublimation technology adopted for the feeding of the reactor with precursor. Indeed, the thermal sensitivity of Co2(CO)8 which is required for low temperature processing on one hand, presents the drawback of limited shelf life of the compound in the sublimation conditions, on the other hand, with subsequent chemical and morphological modifications of the batch stored in the sublimator. Consequently, the precursor feeding rate decreases during the run, from one run to the other and between different runs, though using similar precursor loads [18].

Considering at the same time the demonstrated potential of the cobalt oxide films as black coatings, and the above reported drawbacks, we present in this contribution the results of a new CVD process for the deposition of low reflective cobalt oxide films. We use the direct liquid injection (DLI) technology which allows to overcome problems of uniform precursor delivery by dispensing a solution of precursor into the reactor. Ideally the solvent will evaporate quickly upon entering the vaporization chamber leaving the gas phase precursor to deposit the desired film on the substrate. An advantage of this approach results from the fact that the precursor is maintained at a lower temperature (where it is stable) until it reaches the vaporization chamber. While one remains limited by the equilibrium vapor pressure, this is now higher due to the higher temperature of the vaporization chamber. Nonetheless, problems can develop if supersaturation leads to formation of precursor particles or if the large quantity of solvent used results in film contamination. These aspects are also considered in this work.

Materials and Methods

10x20 mm 2 silicon (0.5 mm thick) and CFRP composite (1 mm thick) coupons are used as substrates. Synthesis and preparation of the composite coupons can be found elsewhere [19]. Si coupons are cut from 4" Si (100) wafers (Sil'tronix). All substrates are cleaned in detergent, rinsed with water, acetone and ethanol, then dried in Ar flow, and finally baked at 60 °C for 20 min before introduction into the reactor. Si substrates are weighted before and after deposition in order to estimate the equivalent film thickness.

The 0.07M precursor solution is prepared by dissolving Co2(CO)8 powder (Alfa Aesar, stabilized in 1-10vol.% hexane) in anhydrous heptane (99%, Sigma Aldrich) in an Ar glovebox. Then, the Schlenk flask is connected to the injection system and purged several times with pressurized N2 (99.9999%, Praxair). Direct liquid injection (DLI) of the solution is carried out with a Kemstream Vapbox® system at a flow rate of 1 mL/min. The solution enters the mixing chamber where 99.9999% pure N2 (Air Products) is flowed at 200 standard cubic centimeters per minute (sccm). In addition, 120 sccm of 99.99 % pure O2 (Air Products) can be introduced at the entry of the deposition zone. The working pressure is regulated at 5 Torr. Deposition is performed in a horizontal, cylindrical cold wall reactor composed of a glass tube of 50 mm in diameter and 800 mm in length. Inside, a stainless steel sample holder is heated by a resistive coil gyred on its bottom surface. The surface deposition temperature (Td) is regulated with a K-type thermocouple attached to a dummy epoxy sample. It is worth noting that in these conditions the surface temperatures of Si or Epoxy are equal ±5°C.

Finally, in an attempt to understand the role of the C content on the reflectivity, calcination experiments are performed at annealing temperatures (Tc) of 400°C, 800°C, and 1000°C, for 1h in a furnace with flowing air.

Samples surface morphology and roughness are observed using scanning electron microscopy (SEM) on a LEO 435VP instrument. Cross section micrographs are prepared and observed with a FEI Helios 600i station composed of a focused ion beam (FIB) and a field emission gun (FEG) SEM operated at 5 kV. Cross sections are obtained by first depositing a 50x3x2 µm 3 platinum protective layer on the surface. Transmission electron microscopy (TEM) imaging is performed using a JEOL JEM 2100F electron microscope operated at 200 kV. Diffraction patterns are recorded using selected area electron diffraction (SAED) mode with a 150 nm aperture. Crystallographic structures are also determined by X-ray diffraction (XRD) on a SEIFERT-3000TT instrument using a Cu Kα (1.5418 Å) X-ray tube operated at 40 kV and 40 mA, a Ni filter and a solid-state Lynxeye detector. Compositions of the surface and the bulk of the films are determined with X-ray photoelectron spectroscopy (XPS) and Transmission Fourier transform infrared spectrometry (FTIR). XPS is performed on a Thermo Scientific K-Alpha instrument using monochromatic Al Kα (1486.6 eV) radiation. Depth profiling is performed by Ar + ion etching (Ar + , 10 mA, 2kV). High resolution scans are obtained at constant pass energy of 30 eV with energy steps of 0.1 eV. FTIR experiments are performed with a Perkin Elmer Frontier-Microscope Spotlight400 Series. Spectra are recorded in the 4000-400 cm -1 wavelength range with a 30 s scan accumulation and 4 cm -1 resolution. The contribution of Si wafers is subtracted from the spectra and the baseline is subsequently corrected from interference fringes. Finally, the UV-Vis reflectance spectra are measured by a Perkin-Elmer Lambda-19 spectrophotometer equipped with an integration sphere. The reflected light is collected in the directional-hemispherical geometry with an incidence angle of 8°.

Results and Discussion

The DLI-MOCVD Process

A first set of experiments with the DLI-MOCVD process is performed without O2 in the input gas mixture. This leads to the formation of metallic Co films. It is worth recalling that the same O-free protocol previously applied along with the sublimation of the humidified (Co2(CO)8 + 1-10% hexane) precursor led to the formation of CoO [2]. From these former sublimation experiments, the atomic compositions of the films were all close to Co(44)O(44)C (12), in the form of stoichiometric CoO with C contamination. The only consistent supply of oxygen came from the decomposition of the precursor carbonyls (CO → Cads + Oads), hence if one tries to balance the post mortem stoichiometry (Co(44)O(44)C( 12)) with elements coming from carbonyls (1 Cads for 1 Oads), the Cads content is too low. Thus, it had to undergo surface reactions to be eliminated. We assumed Fischer-Tropsch-like reactions that could be active in the synthesis of longer chain hydrocarbons. Moreover, there is a linear dependence on the rate of formation of stable hydrocarbons with the amount of available Cads, in excess in these experiments [20]. In such a process, the only source of hydrogen is hexane that has to undergo C-H bond activation to form longer organic chains or to transform into alkene. Finally, the decomposition of Co2(CO)8 also produces an excess of carbonyls (1 Co for 4 (CO)), but they are likely consumed in the gas phase [21] and oxidized on the growing CoO, therefore eliminated in the form of CO2(g) [22].

In the DLI experiments, we do not deposit Co oxide but metallic Co, instead. The only change is the use of a liquid solution of Co2(CO) 8 in heptane (0.1 mol/L) injected at a high flux. The growing Co surface is catalytic and the growth process is controlled by complex mechanisms for which we do not have an insight in the present work. Nonetheless, macroscopically, the surface kinetics is affected by this change in gas phase composition. A good illustration is the formation of an oxide on a sample that was placed downstream away from the metallic Co deposition zone (not shown). A large part of Co2(CO)8 has already been decomposed upstream in the deposition zone and consequently at that position its concentration in the gas phase is reduced, and may be close to the partial pressure of precursor prevailing in the sublimation process that leads to Co oxide deposition. Overall, we tentatively propose the occurrence of three global reactions to explain the deposition of metallic Co:

(1) the catalyzed decomposition of CO → CO2 + Cads [23,24], (2) the consumption of O in the catalyzed oxidation CO + 1/2O2 → CO2, and (3) a Fischer-Tropsch-like mechanism which partially consumes Cads.

In an attempt to stabilize the Co oxide, we added water vapor to the gas mixture (50 sccm of N2 bubbling in 0.1°C water). We observed a doubling of the growth rate, in conjunction with a decrease of the Co film resistivity (from 22 µOhm.cm without water to 6 µOhm.cm with water). We conclude that water enhances the deposition kinetics and cleans the film from contaminant by-products. The water gas shift reaction (CO + H2O  CO2 + H2) is likely responsible for this effect. Conversely, this confirms that CO and its by-products are responsible for the poor resistivity obtained without water.

Finally, we added O2 to the gas mixture, and again we could form black Co oxides-based films, similar to those obtained in the sublimation process, although with a different chemical scheme and kinetics. The growth rate now reaches two maxima: 1.7 µm/h at 50 °C, and 0.8 µm/h at 125 °C. These films are presented in the next sections below.

A significant advantage of the DLI-MOCVD process is the control of the precursor flow rate in a wide range, and the efficient use of the precursor, since the solution reservoir is stored and operated at room temperature. To the opposite, the flow rate of precursor in a sublimation process is limited and not well controllable, it varies with time, and it requires to heat the precursor over the whole experiment duration, hence accelerating degradation and agglomeration. Consecutively, films deposited from sublimed precursor were limited to a surface area of approximately 1 cm 2 [2]. In contrast, due to the high precursor flux available, the deposition zone of the present DLI process was extended to approximately 2000 cm 2 . This is an important advantage of the DLI technology since it allows tailoring a coating process to treat large parts over a wide surface area.

Deposition on Si

Films processed on Si in the presence of O2 at Td = 50°C, 125°C, and 160°C, are visually matt and black. At Td = 75°C and 100°C, they are matt and gray, and they delaminate spontaneously. Except for XRD and EPMA results which present qualitative interest, these delaminated coatings are discarded for other characterizations.

The atomic composition of the coatings are determined by EPMA measurements on their surface. The average composition is 30 ±3 at.% Co, 47 ±6 at.% O, 23 ±6 at.% C and it is independent on Td. This is consistent with the results of Ye et al. who identified various carbonaceous species by X-ray photoelectron spectroscopy in Co-based CVD films processed in the same Td range from sublimated Co2(CO)8 [25]. Nonetheless, it is questioning that EPMA results suggest such a high amount of O. Hence, we double check the composition of the films by XPS depth profiling. Results are shown in Error! Reference source not found., where the atomic composition is plotted versus Ar + etching duration.

INSERT FIGURE 1 The as-deposited film surface, which has been exposed to the atmosphere during its transfer from the DLI-MOCVD reactor to the XPS chamber, exhibits a high amount of C contamination (56 at.%). Ar + sputtering is able able to remove this contamination layer entirely after about 700s. There, the atomic composition stabilizes and corresponds to the bulk composition of the film: Co(45)O(45)C (10). The god correspondence with the atomic composition of the films obtained by sublimation is noticeable.

We now analyze the different species within the contamination layer and within the film. Results are shown in Error! Reference source not found., with the high resolution spectra of the C1s, O1s, and Co2p1/2 core levels.

INSERT FIGURE 2

Before etching, two contributions of the O1s peaks (contamination 1 and 2) correspond to the C-O and C=O contributions of the C1s peak; both in terms of chemical shifts and of atomic percentage (10% total). After etching, there are 10% of C remaining which are composed of C-C, C=O, and Co carbides. And the O1s peak is divided into two components, O -I and O -II . O -II belongs to the CoO and Co3O4 oxides structures, whereas O -I arises from the reduction of O -II by the Ar+ ions during sputtering. Finally, based on Refs [26][27][28][29], we propose 7 contributions to decompose the Co2p1/2 peak in Fig. 2e andf: metallic and/or carbidic Co at 793.3 eV (1), Co +III in octahedral sites at 795.3 eV (2) and its satellite at 803 eV (sat.2), Co +II in octahedral sites at 796.5 eV (3) and its satellite at 803.0 eV (sat.3), and Co +II in tetrahedral sites) at 798.0 eV (4) and its satellite at 804.0 eV (sat.4). By etching, the increase of the metallic/carbidic component and the joint decrease of the Co +III component only suggest the reduction of the oxide and the implantation of C because of Ar + sputtering.

The crystallographic structures of the films are determined from the XRD diffractograms shown in Error! Reference source not found. for the different Td. The X-ray bands centered at 36.5°, 42.4°, 61.5°, 73.6°, and 77.5° correspond to (111) (200), ( 220), (331) and (222) planes of the cubic cobalt oxide (II) CoO phase. The XRD pattern of the film deposited at 50°C is composed of poorly defined peaks, both in position and intensity revealing poor crystallinity. Films deposited at 75°C and 100°C, show large X-ray reflections that mainly correspond to small CoO crystallites. Films deposited at 125°C and 160 °C show new X-ray reflections centered at 32.2°, 36.9°, 44.8°, 59.3°, and 65.2° that are attributed to small crystallites of the spinel oxide Co3O4 structure. Schmid et al. deposited cobalt oxide films by MOCVD from cyclopentadienylcobalt dicarbonyl CoCp(CO)2 in the range 200 °C -600 °C. Films were composed of CoO for Td = 200 °C to 500 °C, and of Co3O4 for Td = 600 °C [30]. We assume that the difference with the present work in terms of temperature range, is mainly due to the difference of the initial Co oxidation degrees between the two precursors: 0 in Co2(CO)8, and +1 in CoCp(CO)2. Finally, the strong Si reflection at 55° has been removed of the 50°C and 75°C spectra for the sake of clarity.

INSERT FIGURE 3

To further explore the composition of the coatings we perform FTIR spectroscopy on the black coatings deposited at Td = 50°C, 125°C, and 160°C. Results are summarized in the FTIR spectra in Figure 4. The broad vibration bands centered at 3400 cm -1 show that hydroxyl groups are present at all Td. Several peaks from C-H (2960, 2925, 2860 cm -1 ) and C=C (1490, 1380 cm -1 ) confirm that residual by-products of Co2(CO)8 are present in the films. The films deposited at 125°C and 160°C exhibit two absorption bands at 570 and 660 cm -1 that make us unambiguously identify the spinel Co3O4 structure [31]. The band at 570 cm -1 corresponds to the BOB3 vibration in the spinel lattice, where B denotes the Co3 + cations in the octahedral positions. The band at 660 cm -1 corresponds to the ABO3 vibration, where A denotes Co2 + in the tetrahedral sites. At Td = 50°C, a large band is centered at 500 cm -1 that could be attributed to Co-O vibrations in CoO and/or to defects in the Co oxides structures [31,32]. Overall, the FTIR measurements corroborate the phase transition from CoO at low Td to Co3O4 at higher Td observed in XRD, and the presence of a high content of C.

INSERT FIGURE 4

A selection of reflectivity measurements performed on the black coatings obtained at Td = 50°C, 125°C and 160°C, is presented in Figure a. We consistently observe that the best reflectivity (R) in the visible range is obtained for coatings deposited at 125°C; where R remains below 2% over the 400-800 nm range. This is twice lower than the coatings we obtained previously with the sublimation MOCVD process [2]. But this low reflectivity cannot be solely explained by the intrinsic optical properties of CoO and Co3O4 [33,34]. We suspect a role of the C content, and decide to perform calcinations in air of the 125°C-coatings, at different temperatures (Tc). Figure shows the reflectivity (a), and the evolution of FTIR spectra (b) after annealing at Tc = 400°C, 800°C, and 1000°C. After 1h at 400°C, the reflectivity in the visible range remains unaffected and the reflectivity in the infrared range has significantly decreased. In the corresponding FTIR spectrum, the relative intensity of the Co3O4 absorption bands (570 and 660 cm -1 ) increased, to the expense of the C-H, C=C, and OH bands (3400 cm -1 , 2960-2860 cm -1 , and 1490, 1380 cm -1 , respectively). Noticeable differences are observed after the 800°C annealing. The typical structure of the pure Co3O4 reflectivity spectra developed [33] and the reflectivity increased in the visible range (2-3%). The corresponding FTIR spectrum exhibits intense Co3O4 bands, and very low C contamination bands. Therefore, post deposition thermal treatment at 800°C for 1 h is efficient in the calcination of C and also likely in the complete oxidation of CoO to Co3O4. After annealing at 1000°C, R increases up to 6% in the visible range, and this rise is accompanied by strong structural and morphological changes (see Figure 6. SEM images of the surface of a coating processed at 125°C, as-processed (a) and after calcination at 1000°C (b). A crosssection prepared by FIB of the as-processed film is also presented in (c). b and details in the section 3.3). The FTIR spectrum shows many additional peaks as compared to the one obtained at Tc = 800°C, but it is unclear whether these absorption bands belong to CoO; e.g. the band centered on 500 cm -1 [32]. Anyway, XRD spectra (not shown) after the 1000°C calcination indeed exhibit reflections of the CoO phase, in good agreement with previous works showing that the spinel Co3O4 phase decomposes to CoO above 900°C [35][36][37].

INSERT FIGURE 5

Overall, this indicates that the low reflectivity in the visible range, of the black coatings obtained at 125°C, is due to the intrinsic absorption properties of the Co oxides in combination with the peculiar cauliflower morphology of the coatings, described elsewhere [2] and illustrated below. Figure 6. SEM images of the surface of a coating processed at 125°C, as-processed (a) and after calcination at 1000°C (b). A cross-section prepared by FIB of the as-processed film is also presented in (c). presents SEM surface micrographs of films processed at 125 °C, before (a) and after (b) calcination at 1000°C for 1h in air. The as-processed microstructure is composed of nodules that exceed 1 µm in diameter. Each nodule is composed of nanometric branches. The nodules distribution is dense enough to cover the entire surface, resulting in a rougher microstructure than that obtained at the two other temperatures 50°C and 160°C (not shown). The annealing at high temperature strongly modifies the microstructure, as shown in Figure 6. SEM images of the surface of a coating processed at 125°C, as-processed (a) and after calcination at 1000°C (b). A cross-section prepared by FIB of the as-processed film is also presented in (c). b: the surface is still very rough, with a large amount of open porosity, but it is now composed of facetted grains; an indication for crystallization. Finally, the image in Figure 6. SEM images of the surface of a coating processed at 125°C, as-processed (a) and after calcination at 1000°C (b). A crosssection prepared by FIB of the as-processed film is also presented in (c). c presents a cross section of the as-processed film, still at 125 °C, prepared by FIB. The film is composed of two parts: a compact layer of submicron thickness by the interface with Si, and an external layer whose thickness varies between 1 and 5 µm. The latter is composed of growth patterns which are oriented either perpendicular or inclined with regard to the surface, probably due to steric effects during growth. Their development results in a significant open porosity, at least at the external part of the film. At some particular points of the internal sublayer, the nucleation and development of such patterns results in the elongated, conical, cauliflower-type nodular branches, typical of these Co oxide films [2,38], and which participate to the low reflectivity.

INSERT FIGURE 6

Finally, in order to have a complete overview of the structure and composition of the coatings, we perform TEM on a thin section obtained by focused ion beam milling on a 125°C coating. Results are presented in Figure 6. SEM images of the surface of a coating processed at 125°C, as-processed (a) and after calcination at 1000°C (b). A cross-section prepared by FIB of the as-processed film is also presented in (c).

Figure . INSERT FIGURE 7

The micrograph in Figure 7a is obtained in bright-field mode. It reveals porosity around columnar grains that extend from the substrate (bottom) to the free surface (top). SAED patterns are collected in each areas numbered 1 to 15. Figure 7b andc show a selection of two representative diffraction patterns obtained in area 12 and 15, respectively. Diffraction rings are formed from the addition of many crystallites in many different orientations. this is a first indication of nanocrystallinity. The indexation of the diffraction patterns is shown in Table 1. Experimental measurements performed in reciprocal space correspond to the interplanar distances presented in the second column. The correspondence is excellent with the spinel Co3O4 d111 and d 022 distances, and with the cubic CoO d111, d002, d022, d131, and d222 distances. Same rings are observed over the 15 SAED patterns studied. We note that SAED patterns 1 and 15 at the bottom of the section are pure CoO, whereas all other patterns are diphasic with both Co3O4 and CoO. Nevertheless, further investigation is requested to assess if this is random or a real spatial distribution of the phases. In order to check whether some electronic contrast is due to amorphous regions or not, we construct a dark-field image in Figure 7d by adding the 3 dark-field images obtained with the diffracted arcs A, B, and C shown in Figure 7c. Diffracting crystallites fill the majority of the image confirming that the coating are entirely nanocrystalline and that no amorphous Co oxide is formed.

Deposition on Poly-epoxy

Figure 8 presents X-ray diffractograms of the films processed at various Td. The X-ray peaks at 2θ equal 36.5°, 42.4°, 61.5°, 73.6° and 77.5° correspond to (1 1 1), (2 0 0), (2 2 0), (3 3 1) and (2 2 2) planes of the cubic cobalt oxide (II) CoO phase. The diffractograms of all films processed at Td up to 100 °C, present X-ray peaks which exclusively correspond to CoO. The diffractogram of the film processed at 50 °C presents undulations rather than clearly defined peaks, revealing poor crystallinity. Films processed at 160 °C present additional X-ray peaks at 2θ equal to 32.2°, 36.9°, 44.8° and 59.3°, and 65.2° which are attributed to the spinel oxide Co3O4. The film processed at Td 125 °C is composed of CoO with traces of Co3O4. Films present no preferential orientation.

Overall, the results obtained on Si wafers are qualitatively reproduced on CFRP; i.e. CoO is detected for 50°C ≤ Td < 125°C whereas (Co3O4+CoO) grow for Td ≥ 125°C.

INSERT FIGURE 8

We now perform a last experiment with a Si substrate and a CFRP substrate placed at the same position on the substrate holder for MOCVD deposition. Hence, except for the transitional regime where the substrate reactivity has an impact, the two black coatings are deposited in the same conditions. Figure 99 shows the reflectivity of these samples (thickness = 0.7 µm), along with the reflectivity of a commercial reference (Vacuum Black TM , Acktar), a bare CFRP substrate and a bare Si substrate. The inset shows an optical photograph of the black coating obtained on the central part of a 20x10 cm 2 CFRP sample that has been masked on each side with adhesive tape.

INSERT FIGURE 9

The reflectivities of the black coatings obtained on Si or CFRP are similar; i.e. they are constant from 400 to 700 nm and increase beyond. The reflectivity stems from 0.7 to 1.1% on the Si substrate, whereas it ranges from 1.0 to 1.4% on the CFRP substrate. The commercial reference has a little higher reflectivity but it remains constant at about 1.3% over the entire range. The reflectivity of of several black coatings processed in the same conditions with various thickness in the range 0.5-1.5 µm remains below 2%. Nonetheless, it is not possible to extract any correlation between thickness and reflectivity. It is interesting to note that the DLI-MOCVD Co oxide coating performs well on both Si and CFRP substrates despite the fact that the initial reflectivity of the two substrates is not in the same order of magnitude (30.6 -45.5% and 5.8 -7.6%, respectively).

Conclusions

The present work aimed at introducing a new MOCVD process for the controlled deposition of black coatings on thermosensitive carbon fiber reinforced poly-epoxy parts. We implemented the direct liquid injection technology which enables the use of large quantities of liquid precursors maintained at (or below) room temperature before they are flash-evaporated at the reactor entry. Storage degradation is limited and high fluxes can be achieved to treat large surface areas. As compared to previous experiments where the precursor was sublimed and transported by a vector gas, we could multiply the coated surface area by 2000. Nevertheless, the transfer of the process has not been straightforward: by sublimation of the humidified Co2(CO) 8 powder, black Co oxides readily grow, whereas by the injection of the (Co2(CO)8 + heptane) solution, metallic Co grows. Therefore, the adapted DLI-MOCVD process for the deposition of black Co oxide films includes the introduction of O2 in the gas mixture at the entry of the reactor. Black coatings are obtained at 50°C, 125°C, and 160°C. The lowest reflectivity within the optical range (<2%) is found for the deposition temperature of 125°C. Based on the low counts rates in XRD and on TEM experiments, we demonstrate that the films are entirely nanocrystalline. Diffracting phases are CoO for films deposited at low temperatures (50°C, 75°C, and 100°C) and (CoO + Co3O4) for films deposited at higher temperatures (125°C and 160°C). Calcination experiments of the 125°C-films show that (i) carbon plays an important role in the light absorption, and that (ii) the structures are stable for temperatures at least up to 400°C, i.e. well above the admissible temperatures of poly-epoxies. Interestingly, although the chemical scheme and kinetics are different than in the sublimation study, the morphologies are the same, with the typical micrometric "cauliflower" grains that developed on a denser layer. The composition, the crystallographic structures, and the morphology are reproduced on CFRP poly-epoxy substrates. The reflectivity is low, and comparable to commercial references used in the aerospace optics. Hence, we demonstrate that the DLI-MOCVD process is well-suited for the deposition of black coatings on thermosensitive substrates. A next step may be the evaluation of the ability of the process to treat large 3D parts.

Figure 7 .

 7 Bright field TEM micrograph along a cauliflower column (a), and SAED patterns obtained in circled regions 12 (b) and 15 (c). An dark-field image (d) is constructed from the addition of 3 darkfield images obtained with the diffracted beams circled in A, B, and C in (c).

Figure 7 .

 7 Figure 6. SEM images of the surface of a coating processed at 125°C, as-processed (a) and after calcination at 1000°C (b). A cross-section prepared by FIB of the as-processed film is also presented in (c). Figure 7. Bright field TEM micrograph along a cauliflower column (a), and SAED patterns obtained in circled regions 12 (b) and 15 (c). An dark-field image (d) is constructed from the addition of 3 darkfield images obtained with the diffracted beams circled in A, B, and C in (c).

Figure 7 .

 7 Bright field TEM micrograph along a cauliflower column (a), and SAED patterns obtained in circled regions 12 (b) and 15 (c). An dark-field image (d) is constructed from the addition of 3 darkfield images obtained with the diffracted beams circled in A, B, and C in (c).

Figure 7 .

 7 Bright field TEM micrograph along a cauliflower column (a), and SAED patterns obtained in circled regions 12 (b) and 15 (c). An dark-field image (d) is constructed from the addition of 3 darkfield images obtained with the diffracted beams circled in A, B, and C in (c).

Figure 1 .

 1 Figure 1. Elemental depth profile on a 160°C sample.

Figure 2 .

 2 Figure 2. C1s, O1s, and Co2p1/2 XPS core level spectra before and after etching of the 160°C sample.

Figure 3 .

 3 Figure 3. XRD diffractograms of the films deposited on Si coupons at various Td. JCPDS cards of CoO (00-009-0402), and of Co3O4 (00-009-0418) are depicted in gray, and red, respectively. Stars indicate reflections from the Si wafer.

Figure 4 .

 4 Figure 4. FTIR spectra of the black coatings deposited on Si coupons at Td = 50°C, 125°C, and 160°C.

Figure 5 .

 5 Figure 5. (a) UV-vis reflectivity spectra of the black coatings deposited on Si wafers at 50°C, 125°C, and 160°C. Samples obtained at Td = 125°C are annealed at calcination temperatures Tc = 400°C (empty squares), 800°C (empty triangles), and 1000°C (empty circles). (b) FTIR spectroscopy spectra of the 125°C-sample before and after calcination.

Figure 6 .

 6 Figure 6. SEM images of the surface of a coating processed at 125°C, as-processed (a) and after calcination at 1000°C (b). A cross-section prepared by FIB of the as-processed film is also presented in (c).

Figure 7 .

 7 Figure 7. Bright field TEM micrograph along a cauliflower column (a), and SAED patterns obtained in circled regions 12 (b) and 15 (c). An dark-field image (d) is constructed from the addition of 3 darkfield images obtained with the diffracted beams circled in A, B, and C in (c).

Figure 1 .

 1 Figure 1. X-ray diffractograms of the films processed at Td 50, 75, 100, 125, and 160 °C on CFRP substrates. Relative intensities vs. positions for the JCPDS cards #00-009-0402 of CoO (bright, grey) and #00-009-0418 of Co3O4 (dark, red) are also provided.

Figure 2 .

 2 Figure 2. Reflectivity in the visible range of coatings deposited on Si and CFRP substrates, along with references.

Table 1 .

 1 Diffraction rings measurements and corresponding interplanar distances in the Co3O4 and CoO structures.

	Ring ID#	Exp. measurements (Å)	Spinel Co3O4 -ICSD#63165	CoO -ICSD #9865
	1	4,73 ± 0,24	4.668	d111	--	--
	2	2,88 ± 0,15	2.859	d022	--	--
	3	2,49 ± 0,13	--	--	2.461	d111
	4	2,15 ± 0,11	--	--	2.132	d002
	5	1,53 ± 0,08	--	--	1.507	d022
	6	1,31 ± 0,07	--	--	1.285	d131
	7	1,25 ± 0,09	--	--	1.231	d222

Acknowledgements

The DIRECCTE Midi-Pyrénées is acknowledged for financial support in the framework of the AEROSAT 2012 program, under contract n°43186. We are indebted to Sophie Gouy and Claudie Josse, UMS Castaing (Toulouse, FR) for EPMA and FIB-SEM analyses, respectively.


Diana Zindel

Claire Vol

Odile Lecha

Isabelle Bourquignon

Merve Bilgic

Marion Vereecke

Fabienne Charrier-Savournin

Maité Romier

Eric Trinquet

Jean-Philippe Pin

Laurent Prézeau
email: laurent.prezeau@igf.cnrs.fr

Isabelle Bequignon

Maïté Romier

Julie Pannequin

Thomas Roux

Elodie Dupuis

HTRF® total and phospho-YAP(Ser127) cellular assays

Keywords: YAP phosphorylation, YAP-S127-Phosphorylation-directed antibodies, Antibody-based assay, Resonance energy transfer, HTRF®

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

The Hippo-YAP-TAZ pathway is a potent regulator of physiological processes controlling organ size as well as cellular proliferation and differentiation. Deregulation of this pathway is frequently associated with anomalous cellular growth leading to cancer [START_REF] Cordenonsi | The hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells[END_REF]. Thus, accurate assessing of the activity of this pathway is crucial to better understanding of its function and to perform screening of compounds and setup strategies to control it. The Hippo pathway comprises a kinase module and a transcriptional module. The kinase module is the major regulator of YAP/TAZ transcriptional module activity [START_REF] Dupont | Role of YAP/TAZ in mechanotransduction[END_REF][START_REF] Azzolin | YAP / TAZ Incorporation in the b -Catenin Destruction Complex Orchestrates the Wnt Response[END_REF][START_REF] Hansen | YAP and TAZ: a nexus for Hippo signaling and beyond[END_REF]. By phosphorylation mostly through LATS 1/2 kinases YAP and TAZ co-transcription factors are retained in the cytosol and degraded and thereby the transcriptional module is inactivated (Figure 1). Thus, there is a crucial need to identify inputs and screen bio-active molecules that could modulate the Hippo-YAP/TAZ pathway in pathological processes like cancer, leading to generating innovative therapeutic anti-tumoral strategies. In the present chapter we present an innovative HTSapplicable HTRF® approach to determine YAP expression and YAP activity by its phosphorylation status of the residues S127 (Figure 1) [START_REF] Zhao | Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control[END_REF], generally assessed in cellular models, native tissues, and organs, as a marker of YAP activity, location, and regulation by numerous partners.

Resonance energy transfer (RET)-based biophysical methods like FRET and BRET (for Förster-and Bioluminescence Resonance Energy Transfer, respectively) have been used by many laboratories to generate signaling assays. The physical principle states that an excited donor fluorophore that displays compatible spectra with another fluorophore (called acceptor) can transfer its energy to the latter in a non-radiative manner by dipole-dipole interaction. FRET requires (I) that the integral of the excited donor´s emission spectrum overlaps with the integral of the acceptors absorption spectrum, (II) that both fluorophore are in close distance to one another in the range of a few nm, and (III) that the transition dipole orientation of the donor and the acceptor are approximately parallel to each other [START_REF] Shrestha | Understanding FRET as a Research Tool for Cellular Studies[END_REF].

Thus, signaling assays are built on variation of distance or orientation of two fluorophore attached to the signaling proteins of interest (for example, G protein BRET assays, EPAC cAMP FRET assays, etc., [START_REF] Jares-Erijman | FRET imaging[END_REF][START_REF] Comps-Agrar | Cell-Surface Protein--Protein Interaction Analysis with Time-Resolved FRET and Snap-Tag Technologies: Application to G Protein-Coupled Receptor Oligomerization[END_REF][START_REF] Zindel | Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling[END_REF]. Although well adapted for specific experimental conditions, mostly in recombinant systems, these methods display several limitations. Indeed, the BRET or FRET donor and acceptor fluorophore are generally luminescent and fluorescent proteins (Luciferase, Green Fluorescent Protein (GFP) variants, etc.) that need to be fused to the proteins of interest and then expressed by the cells after transfection of the encoding plasmids. Second, the absorption and emission spectra of the donor and acceptor fluorophore are not very well separated, meaning that excitation of the donor will lead to a background excitation of the acceptor (cross excitation). Moreover, the emission spectrum of the acceptor is contaminated by the emission of the donor (bleed through).

To overcome these issues, we used the Homogenous Time Resolved FRET (HTRF®) (Figure 2), based on RET between a rare earth Europium [START_REF] Lehn | Synthesis and Properties of Sodium and Europium(III) Cryptates Incorporating the 2,2′-Bipyridine 1,1′-Dioxide and 3,3′-Biisoquinoline 2,2′-Dioxide Units[END_REF] or Terbium cryptate as a donor fluorophore and a Cy5-like dye (D2) as an acceptor fluorophore [START_REF] Bazin | Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions[END_REF][START_REF] Maurel | Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology[END_REF][START_REF] Mathis | Probing Molecular Interactions with Homogeneous Techniques Based on Rare Earth Cryptates and Fluorescence Energy Transfer[END_REF] . As an advantage, this type of donor displays a long fluorescence emission lifetime in the millisecond range (in comparison to the nanosecond range of standard fluorophores) (Figure 2), allowing a recording of FRET signal in a time-gated manner (Time-Resolved FRET or TR-FRET). Indeed, recording the TR-FRET signal 50µsec after the excitation of the donor allows the removal of the background arising from both the auto-fluorescence of the cells and the fluorescence of the acceptor, as their fluorescence is rapidly negligible. As such, TR-FRET signal can be measured without washing out the reagents, then in a homogenous way (HTRF®). In addition, considering that this pair of donor-acceptor displays optimal compatible spectral properties, that the D2 acceptor emits light at wave lengths where the donor emission if barely detectable, TR-FRET provides a good signal-to-noise ratio compared to most other RET methods.

Thus, HTRF® is used to generate signaling assays suitable for HTS (https://www.cisbio.com/drugdiscovery/htrf-technology) [START_REF] Ayoub | Homogeneous time-resolved fluorescencebased assay to monitor extracellular signal-regulated kinase signaling in a high-throughput format[END_REF][START_REF] Scholler | Chapter Seven -Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development[END_REF]. Four main advantages can be pointed out: first HTRF® displays a good signal to noise ratio due to its properties explained above; second, HTRF® assays are easy-torun assays, avoiding purification or washing steps, allowing miniaturizing, and thus are ideal for HTS; third, for most of the assays, great specificity is given by a coincidence detector principle; and fourth, such assays are ideally suited to record endogenous signaling events in native systems. Furthermore, classical FRET assays are limited by the relative orientation of the fluorophores, whereas there is minimal orientation constraint as terbium has multiple electronic transitions [START_REF] Selvin | Luminescence energy transfer using a terbium chelate : Improvements on fluorescence energy transfer[END_REF]. For many assays, the coincidence detection is due to the use of two different antibodies, one recognizing a general state of the signaling protein of interest, and the other one recognizing an active state (e.g. phosphorylated state) of the signaling protein of interest. By using these antibodies being labelled by either donor or acceptor TR-FRET fluorophore, the TR-FRET signal is recorded only when both antibodies are bound to the signaling protein of interest thereby highly reducing any non-specific signal due to binding to non-specific targets of each antibody. This is typically the case for the S127-P-YAP assay. Note that, for T-YAP assay, the coincidence detector is due to the binding of both antibodies to the same protein whatever its active-inactive state (see below). In such sandwich assays [START_REF] Scholler | Chapter Seven -Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development[END_REF], normalized TR-FRET signal is generated through energy transfer between two labelled antibodies, and the larger the amount of target produced, the higher the normalized FRET signal. The TR-FRET signal measured ratiometrically is then proportional to the quantity of target produced.

In the present chapter, we describe the HTRF® protocol for two assays, one detecting the total amount of YAP protein (T-YAP assay), and one detecting the Serine127 phosphorylation state of the protein YAP (S127-P-YAP assay) (Figure 3). T-TAP assay will then be used 1-to determine the relative amount of YAP in different samples and its regulation over time, and 2-to help normalizing the level of phosphorylation of YAP in samples, especially when regulated by drugs or other experimental conditions.

Materials

Some of the material is common for both T-YAP or S127-P-YAP assays except the antibodies.

Sample preparation (HEK293 cells)

1-Phosphate-Buffered Saline (PBS) (Lonza, Basel, CH).

2-96 wells black plate with black bottom (CellStar®; Greiner, Kremsmünster, AUT). Note that 384well plates could also be used depending on the type of cells and the level of YAP expression (Note

1).

3-HEK293 cells. In the present chapter/section, the protocol will be described for HEK293 and can be adapted to any other cell types, heterologous or primary cells (cultured or freshly isolated), and even pieces of tissue (Note 1).

4-Culture medium for HEK293 cells: Complete Dulbecco's Modified Eagle's Medium (DMEM)

medium: DMEM supplemented with 10% foetal bovine serum, 1% penicillin/streptomycin and 1% non-essential amino acids. All the products used for cell culture are purchased from Gibco/BRL-Life Technologies/Thermo Fisher Scientific (Waltham, MA, USA).

YAP detection

1-Stimulation agents, agonists and inhibitors as required by the scientific project 2-Antibodies raised against YAP and labelled with donor or acceptor HTRF® fluorophore (Cisbio, Codolet, FR). Two antibodies recognizing all forms of YAP but with non-overlapping epitopes for the T-YAP assay. For the S127-P-YAP assay, one antibody recognizing all forms of YAP and one antibody recognizing the S127-phosphorylated form of YAP.

Methods

In this chapter, we will focus on the two-plate protocol for adherent cells (Figure 4). Few steps of the protocol for HTRF® YAP assays will differ when cells are grown in suspension, as only one plate will be used (One-plate protocol), and they will be briefly described at the end of the methods Part 3.7.

For accurate total YAP and phosphorylated YAP detection, it is crucial to determine the optimal amount of sample to use (Note 1 & 2), and the process to follow will be described in Part 3.6. below, after the description of the different steps to run the protocol for the assays. T-YAP and S127-P-YAP assays can be run separately. Indeed, T-YAP is dedicated to the assessment of the relative amount of total YAP in samples and its regulation over time or upon treatment, on a time-scale different than that of YAP phosphorylation (Note 3). On the other side, S127-P-YAP assay by itself detects the phosphorylation state of YAP and its modulations by drugs and experimental conditions. But both T-YAP and S127-P-YAP assays can also be run in parallel, and this will be used to normalize the data obtained with the S127-P-YAP assay with that obtained with the T-YAP assay (Note 4). The protocols are the same for both assays except for the required antibodies.

Preparing and dissolving the reagents

Allow all reagents to thaw before use and centrifuge the vials gently before pipetting the stock solutions. Prepare the working solutions from stock solutions by following the instructions below.

1-Dilute the stock solution of Phospho-YAP-d2-antibody/ phospho-YAP-Europium cryptate antibody (20-fold concentrated antibody stock solution) 1:20 with detection buffer: e.g. dilute 0.05 ml of d2antibody or phospho-YAP-cryptate antibody solution in 0.95 ml of detection buffer.

2-Dilute the 4X-concentrated lysis buffer 1:4 in deionized water: e.g. add 1.25 ml of 4X lysis buffer to 3.75 ml deionized water, mix gently.

3-The blocking reagent is provided as 100X concentrated. Dilute the 100X blocking reagent 1:100 in 1X lysis buffer: e.g. add 0.05 ml of blocking reagent to 4.95 ml of 1X lysis buffer. Mix gently.

4-

The control lysate is ready to use. It is provided as an internal positive control to determine the assay quality.

Cell preparation in 96w plates

Cells are plated in 96w plates (but could also be plated in 384w plates, see Part 3.7. on non-adherent cells), but the detection and reading of the HTRF® signal will be performed in low volume 384w plates, to minimize the volume of reagents to be used (donor-and acceptor-fluorophore bearing antibodies). 2-Depending on the signaling input you intend to study, a starvation step with serum free medium can be included (Note 5). Wash the cells 1-2 times carefully with serum free DMEM and starve them several hours or overnight in serum free DMEM, 50 µl/well.

Cell stimulation and lysis

Steps 1 & 2 are required when one wants to assess the effect of exogenous added compounds acting on receptors, enzymes, etc., on YAP phosphorylation and level of expression. Otherwise, directly start at

Step 4.

1-Add 50 µl/ well containing or not compounds to be tested, diluted at the desired concentration in serum free DMEM.

2-Incubate at 37 °C and 5 % CO2. A time course study to determine the optimal stimulation time is recommended for each compound or target stimulated. Note that the stimulation incubation time is different when running the T-YAP assay (4h to OVN) separately from the S127-P-YAP assay (usually 30min, but ranges from 10 min to 2 h) (Note 3, Figure 5).

3-Remove the stimulation buffer from the cells, and immediately add 50 µl of lysis buffer/ well. Lysis buffer volume can be decreased down to 25µl if higher volume of lysate is required.

Conjugate-Antibody addition and signal detection

1-For T-YAP detection, mix 2 µl of diluted anti total YAP-d2 antibody with 2 µl of diluted anti-total YAP-Europium cryptate antibody. Add 4 µl of the mix to the 16 µl of lysate for a final volume of 20 µl / well for the detection of the total endogenous YAP proteins in the test condition or in the positive/negative control condition, respectively. 2-For S127-P-YAP detection, mix 2 µl of anti-phospho-YAP-d2 antibody with 2 µl anti-total YAP-Europium cryptate antibody. Add 4 µl of the mix to the 16 µl of lysate for a final volume of 20 µl /well to detect the fraction of phosphorylated YAP proteins in the test condition or in the positive/negative control condition respectively.

2-Incubate at least 2h (up to overnight) at room temperature in the dark.

4-Read the fluorescence emission at 665 nm and 620 nm with an appropriate plate reader displaying an HTRF® detection module (e.g. PHERAstar FS, BMG Labtech, Ortenberg, GER).

Data evaluation and normalization

HTRF® ratio must be calculated for each individual well. The mean and standard deviation can then be worked out from ratio replicates. Normalization of the data obtained with S127-P-YAP vs T-YAP assays is most of the time required for proper analysis (Note 4, Figure 6).

1-Calculate the ratio between the emission at 665 nm and the emission at 620 nm from raw data obtained with the plate reader:

HTRF® ratio = [(Signal 665nm) / (Signal 620nm)] x 10 4
2-Normalize the HTRF ® signal for phospho YAP to the HTRF ® signal of total YAP (Note 4).

The normalization value can be used to monitor changes in YAP expression versus changes in YAP phosphorylation. This relative value represents the proportion of phosphorylated YAP compared to the total YAP present within the sample, and is then calculated for each well as follows:

Normalization value (P/T) = [(Phospho YAP HTRF® ratio)/(Total YAP HTRF® ratio)] x 100

Determination of the amount of lysate required for optimal detection with T-YAP and S127-P-YAP (Note 1 & 2)

When starting with new cellular model, samples, conditions, etc., it is recommended to determine the amount of lysate necessary to obtain a HTRF ratio lying within the linear range of the assay (Note 2).

1-Transfer a range of 1 to 16 µl of cell lysate (out of the 50 µl in 96w plate) into a 384w plate, add the respective amount of lysis buffer to a volume of 16µl lysate/well. Add the antibody conjugates (Part 1) to a total volume of 20 µl/ well. 2-Determine the HTRF values for the negative control by pipetting 16 µl of supplemented lysis buffer (1x) per well, add 2 µl of d2 antibody and 2 µl of europium cryptate antibody to the final volume of 20 µl per well (Table 1).

and Table

3-Determine the HTRF values for the positive control by pipetting 16 µl of control lysate, add 2 µl of d2 antibody and 2 µl of europium cryptate antibody to the final volume of 20 µl per well (Table 1).

The ratio of positive control lysate signal/ non-specific signal from the negative control should be ≥ 2 (see step 3.5 for data evaluation).

One-plate protocol for cells grown in suspension

1-Transfer 8 µl of cells in small volume white 384w plates in appropriate medium (culture medium, for example, depending on the cells) 3.3x10 5 are generally sufficient but optimal density needs to be determined experimentally (Note 1).

2-Dispense 4 µl of compound (3X) in appropriate medium (culture medium, for example, depending of the cells) and incubate between 10 and 30 min at 37°C. I order to determine the optimal stimulation time, perform a time course study.

3-Add 4 µl of supplemented lysis buffer (4X) and incubate for 30 min at room temperature. The optimal lysis time may vary depending on the cell line (Note 6).

4-Add 4 µl of premixed antibodies prepared in detection buffer. Cover the plate with a sealer and incubate overnight at room temperature. 5-Read the fluorescence emission on a compatible HTRF reader as described in Part 3.4. and 3.5.

Notes

Note 1: It is generally required to probe different cellular densities to determine the amount or volume of cell lysate to use. Indeed, cell lines express various level of YAP proteins, and then will generate various level of HTRF® signal. Moreover, the confluency greatly influences the expression and activity of Hippo-YAP/TAZ pathway. High cell density induces YAP phosphorylation in position S127 [START_REF] Meng | MAP4K family kinases act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway[END_REF] via contact inhibition [START_REF] Zhao | Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control[END_REF], resulting in cytosolic sequestration of YAP whereas low cellular density results in nuclear YAP accumulation [START_REF] Juan | Targeting the Hippo Signaling Pathway for Tissue Regeneration and Cancer Therapy[END_REF]. This is correlated to the fact that YAP/ TAZ inhibition is the key mechanism of contact inhibition through Hippo kinase activation, that in turn inhibits and induces degradation of YAP [START_REF] Zhao | Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control[END_REF][START_REF] Aragona | A Mechanical Checkpoint Controls Multicellular Growth through YAP/TAZ Regulation by Actin-Processing Factors[END_REF]. Thus, there is more YAP protein in proliferating cells.

That is the reason why the cells types, the conditions of culture and treatment, the confluence, meaning the cell seeding density, have to be taken into account for optimal conditions of detection.

The determination of the amount or volume of cell lysate to be used is described in Part 3.6. Note 2: It is crucial to obtained values in the linear range of each of both assays to be able to compare data. This is crucial to precisely detect any regulation of YAP expression or YAP phosphorylation upon stimulation by drugs, upon conditions of culture, etc., as the variation of signal has to stay in the optimal linear range detection of the assay. The linear range values vary depending the biological models, the amount of endogenous YAP, the way the cells or samples are treated, etc. Thus, in order to work properly and get data in the linear range of detection, it is recommended to check the linearity of the data obtained with a range of diluted samples, in addition to the negative controls. This will give the range of variation of signal optimally detected for a given sample model and help adjusting the volume of samples to use for detection. an appropriate stimulus, lysed and incubated with Eu 3+ Cr -donor labeled and d2-acceptor labeled antibodies raised against total YAP and S127 phosphorylated YAP. After 2 (up to 24 h) incubation with the antibodies, HTRF signal is detected using a plate reader.

3 -

 3 HTRF® detection buffer (Cisbio, Codolet, FR) 4-Lysis buffer 4X concentrated (Cisbio, Codolet, FR) 5-Deionized water 6-Blocking reagent (Cisbio, Codolet, FR) 7-Control cell lysate (Cisbio, Codolet, FR) 8-Low volume 384w plates 2.3. FRET measurements 1-HTRF® compatible reader, e.g., PHERAStarFS® plate reader (BMG Labtechnologies, Ortenberg, GER)

2 - 2 and

 22 Seed 5-8 x 10 4 HEK293 cells/well in appropriate (usually black plate with black bottom) 96w plates and incubate them in DMEM supplied with 10 % FCS at 37 °C and 5 % CO2 for 6-8 h until they are well attached. For each cell line, optimization of cell seeding densities is recommended (Note 1 & Part 3.6.).

4 - 5 - 7 -

 457 Incubate for 30 min at room temperature under gentle shaking (Note 6). Homogenize the lysate by gently pipetting it up and down, avoiding bubbles. 6-Transfer 16 µl of each test condition well from the 96w cell-culture plates, onto a low volume 384 w white plate. Depending on the cell line used, it may be necessary to dilute the cell lysate to ensure samples are within the assay linear detection range (Note 1 & 2 and Part 3.6.). Transfer 16 µl of the control lysate for S127-P-YAP or total T-YAP assays, for positive control conditions.

Note 3 :Note 4 :

 34 Modulation of total YAP amount and of S127 phosphorylated YAP display a different timecourse as illustrated in Figure5. Indeed, T-YAP is mostly dedicated to assess the relative expression of YAP itself, in different samples, in different conditions, or upon drug actions. In the case of a modulation of YAP expression, the time-course needed to record this variation is in the range of 4h to OVN. By comparison, the time-course for S127 phosphorylation of YAP is in the 10min-2h range.Time-course has to be taken into account when assessing the dynamics of either the YAP protein amount or the YAP phosphorylation regulation. YAP phosphorylation in position S127 and S381 has been shown to activate a phosphodegron resulting in poly-ubiquitination and degradation[20]. Compounds inducing YAP phosphorylation consequently can induce YAP degradation. Hence, measuring S127-P-YAP before and after compound incubation may possible result in an apparent decrease in phosphorylation, due to a reduction of the total protein level. In order to avoid false negative results for detection of S127-P-YAP it is advantageous to normalize the HTRF ® result of S127-P-YAP to the HTRF ® result determined for T-YAP.

Note 5 :Note 6 :

 56 Serum-derived factors such as the lipids S1P or LPA are known to be potent small molecule activators of YAP [21]. It has been demonstrated that serum deprivation for several hours induces YAP cytoplasmic retention accompanied by an increase of YAP phosphorylation in position S127. The addition of serum to starved cells leads to a concentration dependent decrease in pS127 YAP phosphorylation resulting in nuclear YAP localization and target gene expression [21,22]. It is therefore recommended to test different serum concentrations or to apply a serum starvation step before probing for YAP activity in cellular systems. Optimal lysis time may be cell line dependent and it is recommended to carefully determine it experimentally by performing a time-course experiment.

Figure 1 :

 1 Figure 1: Multiple upstream inputs regulate the Hippo-YAP-TAZ axis through phosphorylation.

Figure 2 :

 2 Figure 2: HTRF ® (Homogeneous Time Resolved FRET) donor, e.g. Eu 3+ Cryptate, is excited at 337 nm

Figure 3 :

 3 Figure 3: Overview of the HTRF ® assay to detect YAP-S127 phosphorylation in 96w or 384w format

Figure 4 :

 4 Figure 4: Overview of both One-plate and Two-plate assay protocols for non-adherent and adherent

Figure 5

 5 Figure 5

Figure 5 :

 5 Figure 5: Time-course for S127 phosphorylated and total YAP detection.

Figure 6 :

 6 Figure 6: YAP activation monitored by HTRF.

Table 1

 1

		Non-	Treated	Control	Blank	Negative
		treated cell	cell lysate	lysate	control	control
		lysate
	Non-treated cell	16 µl	-	-	16 µl	-
	lysate
	Treated cell	-	16 µl	-	-	-
	lysate
	Control lysate	-		16 µl	-	-
	Supplemented	-	-	-	-	16 µl
	lysis buffer (1X)
	Detection buffer	-	-	-	2µl	-
	S127-P-YAP-d2	2µl	2µl	2µl		2µl
	antibody
	S127-P-YAP	2µl	2µl	2µl	2µl	2µl
	Cryptate
	antibody
	Total volume	20µl	20µl	20µl	20µl	20µl

Zhao, B., Li, L., Lei, Q. and Guan, K. (2010) The Hippo -YAP pathway in organ size control

Acknowledgements

The work described was made possible thanks to Cisbio and the technological Pharmacology facilities (Plateform ARPEGE Pharmacology Screening Interactom) of Biocampus (IGF). This work has been supported by CisBio (Cooperative research team Eidos, CNRS N°039293 /CBB DRD-09-04 avenant 4), by the Labex EpiGenMed, an « Investissements d'avenir » program (ANR-10-LABX-12-01), the Fondation pour la Recherche Médicale (DEQ20170336747), CNRS, INSERM, and the University of Montpellier.


Jean-Philippe Pin

Julie Kniazeff

Laurent Prézeau

Jiang-Feng Liu

Philippe Rondard

GPCR interaction as a possible way for allosteric control between receptors

For more than twenty years now, GPCR dimers and larger oligomers have been the subject of intense debate in the field. Evidence for a role of such complexes in receptor trafficking to and from the plasma membrane have been provided, but one of the main issue is of course to determine whether or not such a phenomenon can be responsible for reciprocal control (allosteric control) of the subunits. Such a possibility would indeed add to the possible ways a cell can integrate various signals targeting GPCRs. Among the large GPCR family, those of the class C that include mGlu and GABAB receptors, represent excellent models to examine such a possibility as they are mandatory dimers. In the present review, we will report on the observed allosteric interaction between the subunits of class C GPCRs, both mGluRs and GABABRs, and on the structural bases of these interactions. We will then discuss these findings for other GPCR types such as the rhodopsin-like class A receptors. We will show that many of the observations made with class C receptors have also been reported with class A receptors, providing interesting evidence that such allosteric interactions can be of physiological relevance.

Introduction

G protein-coupled receptors are essential elements involved in intercellular communication as well as communication with the environment making them ideal targets for drug development (Overington, Al-Lazikani andHopkins, 2006,Santos, Ursu, Gaulton et al., 2017). These receptors encoded by 3% of our genes (more than 800 genes in humans) are expressed in every cell, and each cell express ten's of different types of receptors (Moore-Morris, Varrault, [START_REF] Moore-Morris | Identification of potential pharmacological targets by analysis of the comprehensive G protein-coupled receptor repertoire in the four cardiac chambers[END_REF]. The GPCR expression pattern in a given cell type is even dependent on its developmental and physiopathological status, then providing a way to target a specific set of cells in a given situation for an optimal therapeutic effect (Moore- [START_REF] Moore-Morris | Identification of potential pharmacological targets by analysis of the comprehensive G protein-coupled receptor repertoire in the four cardiac chambers[END_REF]. These GPCRs are activated by a large variety of signals including external signals, various metabolites, hormones and transmitters as well as proteins involved in cell adhesion. These multiple types of signals are being transduced inside the cell through a limited set of signaling pathways involving or not G proteins of 4 major types, Gs, Gi/o, Gq and G12. An important question is then raised: how can all the GPCR-mediated signals reaching a given cell be integrated? Are there mechanisms that can control one GPCR pathway when another is or as been activated? As for many other receptor-mediated signaling, multiple possible cross-talks exist that allow the cell to respond differently whether one or two pathways are activated simultaneously or consequently. One best example is the synergy reported between the Gq and Gi-mediated responses [START_REF] Prezeau | Functional crosstalk between GPCRs: with or without oligomerization[END_REF]. When both pathways are activated simultaneously, either through one or two different GPCRs, the Ca 2+ signal is largely amplified, due in part to the action of the G protein bg dimer on PLCb3. Such potentiation of the Gq-mediated response is even larger when the Gi pathway is activated first, few minutes ahead [START_REF] Rives | Crosstalk between GABA(B) and mGlu1a receptors reveals new insight into GPCR signal integration[END_REF]. Moreover, the Gi-coupled receptor can generate a Ca 2+ signal if activated few minutes after the Gq cascade is activated [START_REF] Rives | Crosstalk between GABA(B) and mGlu1a receptors reveals new insight into GPCR signal integration[END_REF]. This illustrates a complex signaling synergy between these two pathways that can not only integrate coactivation of both pathways, but also provide specific information when activated sequentially. Cross talks between other G protein signaling pathways have also been reported.

Although such signal integration through signaling crosstalk is of main interest, the observation that GPCR can interact with each other, leading to the formation of dimers and larger oligomers, has generated a lot of interest as this may represent another possible way for a receptor to control the activity of the other [START_REF] Angers | Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function[END_REF]Bouvier, 2002,Bouvier, 2001 , 2007). The initial observation using biophysical methods illustrated the possible interaction between different GPCRs expressed in heterologous cells, likely at higher densities than the physiological ranges [START_REF] Angers | Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function[END_REF][START_REF] Bouvier | Oligomerization of G-protein-coupled transmitter receptors[END_REF]. As such, functional cross-talk between different GPCRs has also been often used as an argument to support the formation of such GPCR heterodimers [START_REF] Gomes | G Protein-Coupled Receptor Heteromers[END_REF][START_REF] Pin | International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the recognition and nomenclature of G protein-coupled receptor heteromultimers[END_REF]. However, most of the reported studies could not bring strong enough data illustrating a crosstalk resulting from a direct interaction between the receptors, or from a functional interaction between the signaling cascades not requiring GPCR interaction [START_REF] Gomes | G Protein-Coupled Receptor Heteromers[END_REF],Pin et al., 2007). This, plus the more recent studies that question the existence of GPCR dimers in native cells due to their low density, and the probable instability of the GPCR dimers [START_REF] Calebiro | Single-molecule analysis of fluorescently labeled Gprotein-coupled receptors reveals complexes with distinct dynamics and organization[END_REF][START_REF] Felce | Single-Molecule Analysis of G Protein-Coupled Receptor Stoichiometry: Approaches and Limitations[END_REF][START_REF] Hern | Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules[END_REF][START_REF] Kasai | Single-molecule imaging revealed dynamic GPCR dimerization[END_REF]) largely limited the enthusiasm in better understanding the possible bases of GPCR crosstalk through dimerization (Bouvier and Hebert, 2014,Bouvier and Hebert, 2014[START_REF] Lambert | CrossTalk opposing view: Weighing the evidence for class A GPCR dimers, the jury is still out[END_REF].

Among the various types of GPCRs, those of the class C, composed of the eight mGlu receptors, the GABAB receptor as well as the CaSR and the sweet and umami taste receptors are mandatory dimers, either homodimers or heterodimers [START_REF] Kniazeff | Activation mechanism and allosteric properties of the GABAB receptor[END_REF][START_REF] Pin | Organization and functions of mGlu and GABAB receptor complexes[END_REF],Rondard and Pin, 2015). In contrast to most other GPCRs in which agonists bind to the seven transmembrane (7TM) domain, class C agonists interact on a venus flytrap (VFT) domain that is part of their large extracellular domain (Pin andBettler, 2016,Kunishima, Shimada, Tsuji et al., 2000). Dimerization has been shown to be mandatory for the agonist to activate the 7TM domain leading to G protein activation (El Moustaine, Granier, [START_REF] El Moustaine | Distinct roles of metabotropic glutamate receptor dimerization in agonist activation and G-protein coupling[END_REF]. Such a process involves clear allosteric interactions between the subunits, at the level of both their VFT and their 7TM domains [START_REF] Pin | Organization and functions of mGlu and GABAB receptor complexes[END_REF].

In the present review, we will report on what it is known on the allosteric interactions between the 7TMs of class C dimers, and the structural bases for these. We will then discuss these observations in relation to what has been observed with class A GPCRs

1-Class C GPCRs as interesting models of other GPCRs

Class C GPCRs diverged from the other GPCRs early during evolution [START_REF] Nordstrom | Independent HHsearch, Needleman--Wunsch-based, and motif analyses reveal the overall hierarchy for most of the G protein-coupled receptor families[END_REF]. Their structure, stoichiometry and activation mechanism is quite different from the other GPCRs [START_REF] Pin | Organization and functions of mGlu and GABAB receptor complexes[END_REF]. These receptors have a large extracellular domain composed of a VFT domain where agonists bind, linked to the 7TM domain via a cysteine-rich rigid segment. These receptors are mandatory dimers associated via a hydrophobic surface on one side of the N-terminal lobe of the VFT. Agonist bind in the cleft that separates both lobes of the VFT, leading to the stabilization of a closed conformation of this domain. This closed conformation allows the VFTs of the class C dimer to not only interact via their N-terminal lobe-I, but also through their lobe-II, an interaction made possible by a 70° rotation of one VFT compared to the other [START_REF] Kunishima | Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor[END_REF][START_REF] Doumazane | Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors[END_REF][START_REF] Muto | Structures of the extracellular regions of the group II/III metabotropic glutamate receptors[END_REF][START_REF] Tsuchiya | Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+[END_REF]. This important movement of the two VFTs during activation, is expected to affect the relative position of both 7TM domains [START_REF] Hlavackova | Sequential inter-and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1[END_REF][START_REF] Tateyama | Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1alpha[END_REF], resulting in the generation of the intracellular signal.

Such a divergent general structure and activation mechanism raised the question whether or not these receptors can bring information useful for our understanding of specific properties of receptors from the other classes.

Despite their low sequence similarity, class C and class A 7TM domains share a number of important similarities. First, their general structure is similar, although the class C 7TM domain is more compact, still the 3D repartition of the seven transmembrane helices is very similar . The ionic interaction between transmembrane segment 3 (TM3) and TM6 that maintains class A GPCRs in an inactive state is also found in class C receptors (Dore, Okrasa, Patel et al., 2014,Wu, Wang, Gregory et al., 2014). Although not in the same environment, this ionic lock seems to play the same role in class C and class A GPCRs, consistent with a movement of TM6 being critical for the activation of either class A and class C receptors [START_REF] Dore | Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain[END_REF][START_REF] Binet | Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors[END_REF]. Indeed, as for class A GPCRs, conformational changes in class C 7TMs is important to control signaling. This is best illustrated by the discovery of a number of negative allosteric modulators that bind in the 7TM domain of class C GPCRs and prevent their activation (Gregory, Noetzel andNiswender, 2013,Urwyler, 2011). This is also supported by the discovery of a number of positive allosteric modulators that also bind in the 7TM domain at a site similar to that occupied by class A agonists, and that stabilize the active conformation of the receptor. In addition, when truncated of their large extra and intracellular domain, class C 7TM domain can be expressed in cells where their conserved their basal activity. Most importantly, these isolated class C 7TM domains can be directly activated by PAMs further demonstrating their structural similarity with class A receptors (Goudet, Gaven, [START_REF] Kniazeff | Closed state of both binding domains of homodimeric mGlu receptors is required for full activity[END_REF].

Taken together, these observations bring confidence that, although they diverged very early during evolution, class C 7TM domain still conserved a similar mode of activation as that of the other GPCRs, consistent with their action on the same effectors including heterotrimeric G proteins.

2-Negative interaction between the 7TM domains of class C GPCRs

The analysis of the allosteric interaction between the subunits in class C GPCRs first concentrated on their VFT domains. It was found that agonist binding in one VFT increases affinity in the associated VFT, then facilitating the double occupancy of the homodimeric receptors which is the most effective state for the mGlu receptors (Kniazeff, Bessis, [START_REF] Kniazeff | Closed state of both binding domains of homodimeric mGlu receptors is required for full activity[END_REF]). In the case of the GABAB receptor composed of two distinct subunits, GABAB1 and GABAB2, agonist affinity in GABAB1 is largely increased by its interaction with GABAB2 VFT due to additional contacts between the two VFTs in the ligand occupied active state of the dimer (Geng, Bush, Mosyak et al., 2013). It was therefore soon considered that such interaction between the VFTs would favor the activation of both 7TM domains likely in a symmetrical way, as proposed for allosteric proteins(Changeux and Christopoulos, 2016,Monod, Wyman and [START_REF] Monod | On the Nature of Allosteric Transitions: A Plausible Model[END_REF].

Thanks to the development of an optimized quality system allowing the perfect control of the subunit composition of an mGlu dimer at the cell surface [START_REF] Brock | Activation of a Dimeric Metabotropic Glutamate Receptor by Inter-Subunit Rearrangement[END_REF], it has been possible to clarify the role of each 7TM domain in the coupling properties of the mGlu homodimeric receptors. By using PAMs and NAMs acting in a single 7TM within the dimer, associated with a point mutation introduced in only one subunit and preventing G protein activation, it was found that only one 7TM domain could be active at a time (Goudet, Kniazeff, Hlavackova et al., 2005,Hlavackova, Goudet, Kniazeff et al., 2005). This is best illustrated by the full inhibition of G protein activation by a mGlu5 dimer in which the mutated subunit unable to activate G proteins is stabilized in an active conformation by a PAM acting exclusively on this subunit [START_REF] Goudet | Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators[END_REF]. Such a conclusion was confirmed using FRET-based sensor technologies [START_REF] Hlavackova | Sequential inter-and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1[END_REF]. This illustrates that, if this subunit is in an active conformation, the associated subunit cannot couple to G proteins [START_REF] Hlavackova | Sequential inter-and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1[END_REF],Goudet et al., 2005). Although surprising, these findings were consistent with the observation that in the heterodimeric GABAB receptor, one subunit only is responsible for G protein activation, the GABAB2 subunit (Galvez, Duthey, [START_REF] Galvez | Allosteric interactions between GB1 and GB2 subunits are required for optimal GABAB receptor function[END_REF]. Similarly, in the heterodimeric taste receptors, data also suggest that T1R3 is the only subunit responsible for G protein activation [START_REF] Xu | Different functional roles of T1R subunits in the heteromeric taste receptors[END_REF].

Whereas mGlu receptors were thought to only form homodimers, we and others brought evidence for the existence of mGlu heterodimers, not only in heterologous cells ( ). We then examined whether both subunits could activate G protein in the mGlu2-4 heterodimer. We found that G protein coupling was assumed by the mGlu4 7TM only in this receptor (Liu, Zhang, Moreno-Delgado et al., 2017), and not mGlu2 even though the mGlu2 7TM is capable of activating G protein when assembled in a homodimeric form.

Taken together, these data indicate that in class C dimers only one 7TM domain activates G protein at a time. It can be either one in a homodimer, but the allosteric interaction is such that only a specific one does it in a heterodimeric entity [START_REF] Galvez | Allosteric interactions between GB1 and GB2 subunits are required for optimal GABAB receptor function[END_REF][START_REF] Xu | Different functional roles of T1R subunits in the heteromeric taste receptors[END_REF][START_REF] Liu | Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer[END_REF]. Such an asymmetry can be explained in two ways. It can be either due to the inability, resulting from steric hindrance for example, of both subunits to activate two G proteins at a time. The second possibility is that the physical interaction of the 7TM domains in the active state is such that only one domain can reach the active conformation as a result of an allosteric interaction. This second possibility is more likely than the first one, as the former cannot explain that a given subunit is responsible for G protein activation in the heterodimeric receptors.

3-Positive interaction between the 7TM domains of class C GPCRs

We have shown above that in class C dimers, one subunit, when in the active form, prevents the associated one from being active. Does this mean the associated, not functional subunit, remains in a basal state? Is the inactive associated subunit able to influence the activity of the other? The analysis of the role of each subunit in heterodimeric receptor function brought important information along these lines. The analysis of the role of the GABAB1 subunit in the GABAB heterodimer early indicated that it was playing an important role in the activation of the GABAB2 7TM, with a positive action (Monnier, Tu, Bourrier et al., 2011). It was also found that a change in conformation in the GABAB1 7TM was likely essential in this effect as introducing a disulfide bridge within this domain prevented activation of the GABAB2 7TM [START_REF] Monnier | Transactivation between 7TM domains: implication in heterodimeric GABAB receptor activation[END_REF]. Such a conclusion was further supported by our recent data using the mGlu2-4 heterodimer. Indeed, stabilizing the mGlu2 7TM in an inactive state with a NAM known to interact within this domain, largely diminished the ability of the mGlu4 7TM to activate G proteins [START_REF] Liu | Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer[END_REF]. This was observed not only with the mGlu2-4 heterodimer, but indeed with any mGlu heterodimeric receptor composed of a group-II (mGlu2 or 3) and a group-III (mGlu4, 7 or 8) subunits, with the group-II 7TM domain facilitating the G protein coupling efficiency of the group-III 7TM domain. These findings then show that, even though only one subunit in a class C dimer is responsible for G protein activation, the other subunit is far from being neutral, and is indeed involved in the process, facilitating the activation of the active subunit through a conformational change that remains to be determined. Such observations further support a clear allosteric interaction between both 7TM domains with class C GPCR dimers.

4-Class C as dimers or larger oligomers ?

If class C receptors are commonly considered as dimers, the GABAB receptor was soon found to associate into larger entities composed of at least two heterodimeric entities associated via the GABAB1 subunit (Maurel, Comps-Agrar, [START_REF] Maurel | Cell surface protein-protein interaction analysis with combined time-resolved FRET and snap-tag technologies: application to GPCR oligomerization[END_REF]. Such complexes were not only observed in transfected cells at physiological densities, but also in brain membranes (Comps-Agrar, Kniazeff, Norskov-Lauritsen et al., 2011,Schwenk, Metz, Zolles et al., 2010). In addition, these complexes appear stable at least in transfected cells as heterodimers are poorly exchangeable between these complexes (Comps-Agrar, [START_REF] Comps-Agrar | Stability of GABAB receptor oligomers revealed by dual TR-FRET and drug-induced cell surface targeting[END_REF]. These data strongly suggest that such complexes are real in neurons and then may be of physiological importance. The obvious next question was to examine whether one GABAB heterodimer could affect the signaling of the other in such complexes. It was soon observed that within a tetrameric entity, only one heterodimer was functional [START_REF] Maurel | Cell surface protein-protein interaction analysis with combined time-resolved FRET and snap-tag technologies: application to GPCR oligomerization[END_REF][START_REF] Comps-Agrar | The oligomeric state sets GABA(B) receptor signalling efficacy[END_REF]. The selective coupling of only one GABAB2 subunit is unlikely due to steric hindrance as the GABAB2 subunits are distant from each other, separated by the GABAB1 subunits [START_REF] Maurel | Cell surface protein-protein interaction analysis with combined time-resolved FRET and snap-tag technologies: application to GPCR oligomerization[END_REF][START_REF] Comps-Agrar | The oligomeric state sets GABA(B) receptor signalling efficacy[END_REF]. These data reveal a negative interaction between the GABAB heterodimers within such tetrameric entities. Although we cannot exclude a direct negative interaction via the 7TM domains within the complex, it was found that a negative interaction already occurs at the level of the VFTs, with a single ligand being able to bind in a tetrameric GABAB receptor [START_REF] Stewart | Allosteric interactions between GABAB1 subunits control orthosteric binding sites occupancy within GABAB oligomers[END_REF].

Although GABAB receptors were found to associate into large entities, mGlu receptors were instead soon reported as strict dimers both in cell lines [START_REF] Maurel | Cell surface protein-protein interaction analysis with combined time-resolved FRET and snap-tag technologies: application to GPCR oligomerization[END_REF] and in xenopus oocytes [START_REF] Levitz | Mechanism of Assembly and Cooperativity of Homomeric and Heteromeric Metabotropic Glutamate Receptors[END_REF]. This was confirmed in cultured hippocampal neurons at expression levels close to the native conditions [START_REF] Moller | Oligomerization of a G protein-coupled receptor in neurons controlled by its structural dynamics[END_REF]. Surprisingly, co-diffusion analysis using the number and brightness analysis of labeled snap-tagged mGlu2 receptors reveal that they associate into larger entities, mostly tetramers when their conformation is stabilized either in the inactive state or in the active state with ligands [START_REF] Moller | Oligomerization of a G protein-coupled receptor in neurons controlled by its structural dynamics[END_REF]. Because mGlu2 receptors were found to oscillate between various conformations under their basal state (Olofsson, Felekyan, Doumazane et al., 2014,Vafabakhsh, Levitz and[START_REF] Vafabakhsh | Conformational dynamics of a class C G-protein-coupled receptor[END_REF], we proposed that the stabilization into a given state, either active or inactive, favors the association between receptors through specific interfaces, likely due to the increase in the number of receptors in a conformation compatible with such oligomerization. However, so far, there is no direct indication that allosteric interactions can occur between mGlu dimers within such complexes.

5-Concerted movements between the 7TM domains of class C GPCRs

How can one 7TM domain control the activity of its associated subunit? A clear answer to this question definitively needs the determination of the 3D structure of the dimeric complex in various states, and eventually with the associated G protein. So far, the only structure of a truncated class C GPCR 7TM domain showing a possible dimeric entity is that of mGlu1 [START_REF] Wu | Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator[END_REF]. In that case, the two domains are found in contact via TM1. However, for several reasons, this is unlikely the case in the full-length receptor. First, this dimer interface is unlikely affected upon receptor activation making it very unlikely involved in an allosteric coupling between the two subunits. Moreover, the distance between the TM1s of both subunits is expected to be driven by the distance between the C-terminal ends of the CRDs as the later are directly linked to TM1, and these are not expected to be in close contact. Cys crosslinking experiments were then conducted to identify the possible interface of mGlu 7TM domains. The replacement of many lipid exposed residues of most TM helices in the mGlu2 receptor led to the crosslinking of the two subunits when introduced into TM4, TM5 or TM6 [START_REF] Xue | Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer[END_REF]. It was also found that stabilizing the receptor in its inactive state using competitive antagonists and NAMs increases the proportion of receptors crosslinked through TM4 or TM5, while stabilizing the receptor in its active state favors TM6 crosslinking. Moreover, crosslinking through TM4-5 results in a receptor that can no longer be activated by glutamate, while TM6 crosslinking generate a receptor almost fully active in the absence of agonist [START_REF] Xue | Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer[END_REF]. Accordingly, it was proposed that both 7TM domains may contact each other mainly through TM4-5 under basal conditions, while via TM6 in the active state. Such a proposal nicely fit with the expected distance between the N terminal ends of the TM1 helices deduced from the distances of the C terminal ends of the CRDs in the active and inactive states determined in the 3D structures of the entire extracellular domain [START_REF] Muto | Structures of the extracellular regions of the group II/III metabotropic glutamate receptors[END_REF]. Such a proposal also provides possible explanation for the asymmetric activation of the class C 7TM domains, with one being active only per dimer. Because of the large movement of TM6 needed to fit the C terminal end of the G protein alpha subunit within the 7TM domain, a dimer interface involving TM6 may allow only one subunit to reach the active state, while the other may require to change conformation to allow this TM6 to move. Such a model nicely explains the allosteric interaction observed between class C 7TM domains.

Such a model has been further validated when analyzing the 7TM domain association and movements in a tetrameric GABAB receptor. In that case it was observed that the helices corresponding to TM6 of the GABAB1 subunits are facing each other in the inactive tetramer, while both contact those of the GABAB2 subunits in the active state, allowing the later to reach the active conformation. This concerted movement between the 4 subunits of a possible GABAB tetramer also provides possible explanation for the control of a class C dimer by another GPCR, as observed with the 5HT2a receptor regulation of the mGlu2 signaling possibly involved in the action of anti-psychotics (Fribourg, Moreno, Holloway et al., 2011,Gonzalez-Maeso, Ang, [START_REF] Gonzalez-Maeso | Identification of a serotonin/glutamate receptor complex implicated in psychosis[END_REF]. Indeed, the interaction of the 5HT2a receptor with the TM4-5 of mGlu2 is expected to favor mGlu2 subunit interaction through their TM6, then influencing their ability to be active. Work is however necessary to bring experimental evidence for this model.

6-Comparison with what has been reported for other GPCRs

Although class C GPCRs represent an excellent model to analyze the possible allosteric interaction between 7TM domains, the question remains to know whether the observations made on class C GPCRs can be applied to other GPCRs, especially class A receptors. Interestingly, when possible allosteric interactions between subunits within GPCR dimers were examined, a negative interaction was very often observed, with one subunit active preventing the other from being active. This has been well demonstrated with the D2 and 5HT2C receptor dimers [START_REF] Han | Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation[END_REF][START_REF] Herrick-Davis | Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein[END_REF], as well as with the alpha2A adrenergic -mu opioid receptor heterodimer using FRET-based receptor biosensors [START_REF] Vilardaga | Conformational cross-talk between alpha2A-adrenergic and mu-opioid receptors controls cell signaling[END_REF]. This is also consistent with a number of studies reporting a negative cooperativity between agonist binding on various GPCRs [START_REF] Springael | Allosteric properties of G protein-coupled receptor oligomers[END_REF]. Indeed, if only one subunit can reach an active state stabilized by a G protein, then only one subunit can reach the high agonist affinity state, and not the other. Such a proposal help explain the increased off rate of bound agonist upon addition of high concentration of unlabeled ligands [START_REF] Springael | Allosteric properties of G protein-coupled receptor oligomers[END_REF][START_REF] Urizar | Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity[END_REF]. This was further confirmed with the oxytocin receptor for which both positive and negative cooperativity for ligand binding were reported [START_REF] Albizu | Probing the existence of G protein-coupled receptor dimers by positive and negative ligand-dependent cooperative binding[END_REF], and for which a single high affinity site for agonist per dimer was clearly demonstrated by measuring FRET between ligands [START_REF] Albizu | Time-resolved FRET between GPCR ligands reveals oligomers in native tissues[END_REF]. Moreover, when studied with recombinant purified leukotriene B4 receptor, a dimer was found to interact with a single G protein [START_REF] Baneres | Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein[END_REF], and clear allosteric interaction were observed between the subunits leading to the activation of only one subunit per dimer, consistent with what is proposed for class C GPCRs [START_REF] Damian | Asymmetric conformational changes in a GPCR dimer controlled by G-proteins[END_REF]. Such a property of GPCR dimers does not appear to be general as in some cases it has been proposed that both subunits in a dimer can simultaneously activate G protein [START_REF] Pellissier | G protein activation by Serotonin type 4 receptor dimers: evidence that turning on two protomers is more efficient[END_REF]. Several studies also examined the possible dimer interfaces in class A GPCR dimers. Most studies pointed toward TM4 and TM5 as a possible interface ( , 1996). Of interest, and consistent with the observation that class A GPCR dimers may not be very stable, such a dimer interface appears to be dynamic, with changes of the interface being observed between active and inactive states, as revealed by Cys cross-linking experiments [START_REF] Mancia | Ligand sensitivity in dimeric associations of the serotonin 5HT2c receptor[END_REF][START_REF] Guo | Crosstalk in G protein-coupled receptors: changes at the transmembrane homodimer interface determine activation[END_REF]. Moreover, several crystal structures revealed possible dimer assembly via TM4-5 (adenosine A2A receptor) [START_REF] Liu | Structural basis for allosteric regulation of GPCRs by sodium ions[END_REF], TM5 (CXCR4 receptor) [START_REF] Wu | Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists[END_REF], or via TM5-6 (Mu opioid receptor) [START_REF] Manglik | Crystal structure of the micro-opioid receptor bound to a morphinan antagonist[END_REF]. More recently, the dimer interface of the neurotensin receptor was reported to be dynamic, possibly resulting from a possible change in the interface over time [START_REF] Dijkman | Dynamic tuneable G protein-coupled receptor monomerdimer populations[END_REF], as suggested for the class C GPCRs. Class A GPCR dimers have also been proposed to associate via TM1 and helix 8. In the case of the Mu Opioid receptor, this interface has been proposed to be responsible for a possible oligomerization of the receptor. If this can be the case, because such an interface is not expected to undergo conformational changes upon ligand binding, it is unlikely that this interface be involved in allosteric interaction between the subunits. This may explain why, in some cases, no evidence for cooperativity between subunits of a GPCR dimer has been reported [START_REF] Pellissier | G protein activation by Serotonin type 4 receptor dimers: evidence that turning on two protomers is more efficient[END_REF].

Conclusion

Class C GPCRs represent an interesting model to examine the allosteric interactions between 7TM domains. The recent studies point to important possible interactions, both positive and negative leading to an asymmetric activation of these dimeric receptors, with one subunit facilitating the activation of the other, such that a single subunit activate G proteins. Further studies pointed to a dynamic dimer interface involving TM4-5 in the inactive state, and TM6 in the active conformation of the dimer. Because major conformational changes are expected at this level of a 7TM protein upon G protein activation, such a TM6 interface appear ideal to explain the important allosteric interactions observed. While the debate is still active regarding class A GPCR dimers, it is puzzling to observe that many of the characteristics of the class C GPCR dimers were also reported for many class A dimers. These include the control of one subunit by the other leading to the activation of only one subunit in a dimer, as well as the likely involvement of TM4-5 and 6 as a possible dimer interface. One main issue will be to elucidate when and how such a dimeric process can occur for class A GPCRs that appear not to be stable in contrast to class C receptors. Could such class A dimers be stabilized in some way with scaffolding proteins? Whatever, even if such interactions are transient, this does not exclude a possible physiological relevance. Indeed, GPCR and G protein interaction is also not stable, and everyone won't question its physiological implications.

  ,Ferre, Baler, Bouvier et al., 2009,Ferré, Casadó, Devi et al., 2014,Gomes, Ayoub, Fujita et al., 2016,Smith and Milligan, 2010,White, Grodnitzky, Louis et al.

  Doumazane, Scholler, Zwier et al., 2011,Moreno Delgado, Møller, Ster et al., 2017), but also in native neurons (Moreno Delgado et al., 2017,Yin, Noetzel, Johnson et al., 2014

  Mancia, Assur, Herman et al., 2008,Guo, Shi, Filizola et al., 2005,Liu, Chun, Thompson et al., 2012,Wu, Chien, Mol et al., 2010,Manglik, Kruse, Kobilka et al., 2012), with TM6 being also reported (Baneres and Parello, 2003,Damian et al., 2006,Hebert, Moffett, Morello et al.