A Smith

Y El Hafiane

Y El Khessaimi

A Faure

Some examples of mineral eco-materials

Keywords: Eco-materials, cements, dam sediments, plaster, recycling, tape casting, clinker

Eco-materials are materials which can minimize environmental impacts, can be manufactured from recycled materials, or are beneficial for human wellbeing. This paper will illustrate these three aspects on mineralbased eco-materials through research that has been developed in our ceramic institute: preparation of dense tapes of cementitious materials at room temperature by tape casting, valorization of dam sediments as raw materials for sintering of Portland clinker, monitoring the microstructure of plaster boards for improving the acoustical insulation in houses.

Introduction

If one looks for 'ecomaterials', the Mostar Bridge in Central Europe is a superb example. In order to replace a wooden bridge, Suleiman the Magnificent in the 16 th Century commissioned a new bridge [1]. Hayruddin, the architect and builder who supervised its construction, committed suicide on the day the scaffolding was removed from the complete structure. He was scared that the bridge would collapse, stimulating the anger of Suleiman. The truth is, despite the fact the bridge was constructed on a seismic zone, it resisted many centuries until it was bombed in 1993 during the Croatia-Bosnia war. Thanks to a project lead by UNESCO, the bridge was reconstructed by replicating the original materials composition and construction, and was inaugurated in 2004. Beyond the symbolic meaning of reconstructing this bridge, there were some very interesting findings on the materials themselves. The engineers in charge of the bridge reconstruction found that the remains of the original bridge contained various mineral materials and a mortar with horse hair, egg shell. These components gave the bridge a remarkable durability. This is to point out that recycling or valorization of by-products to make new materials, which could be qualified nowadays as 'eco-materials', is quite an old concept.

In the late 90's and at the beginning of the 21 st century, eco-materials were defined as materials that could enhance environmental improvement through the whole life cycle, while maintaining accountable performances [START_REF] Halada | The current status of research and development on ecomaterials around the world[END_REF]. These materials should respect the principles of sustainable development. In this short paper, the presented examples, which correspond to research carried out in our laboratory, will illustrate three points: (1) an eco-material can present similar performances to a 'classical' ceramic material, [START_REF] Halada | The current status of research and development on ecomaterials around the world[END_REF] it should contribute to decrease the environmental impact through recycling and/or valorization of waste minerals, (3) it should permit to improve the home comfort of people in their house. *

Tapes of dense calcium aluminate cement obtained without any firing step

For those who are not familiar with cementitious materials, figure 1 The idea of our research was to see whether it was possible to make a dense aluminous cementitious material that could have some properties of 'classical' alumina but without a firing step. We prepared a slurry that could be used in tape casting or surface coating. The first requirement was to make a well dispersed suspension. Figure 2 presents the rheology of a slurry prepared from a calcium aluminate cement (Secar 71, table 1. The grain size ranges between 1 to 100 µm with d50 around 10 µm) [START_REF]Secar 71[END_REF] mixed with water (water over cement weight ratio, wwater/wcement = 0.3) and containing increasing percentages of additive [START_REF] Hafiane | Role of dispersant and humidity on the setting of millimetric films of aluminous cement prepared by tape casting[END_REF].

The best results, in terms of lowest viscosity, were obtained with the addition of acetic acid. The content which gives the smallest viscosity is wadditive/wcement = 0.005. The next step was to examine the setting time when increasing quantities of acetic acid were added in the water (Figure 3). For a cement paste with no addition of acetic acid, the setting time was quite variable since it ranged between 2 h or 3 h up to 24 h. We found that this variability was due to external parameters such as storage conditions, and ageing of the cement when exposed to the ambient atmosphere. The corresponding pH is alkaline which is quite normal for a cement paste. For a percentage less than 1%, the setting time was very long. However, beyond this value, the setting time drops to 2 h and is very regular. The pH is also acid during the first 15 min. These variations were explained as follows: acetic acid can adsorb at the surface of cement grains, forming surface complex molecules thus delaying the dissolution of anhydrous calcium aluminate grains and subsequently the formation of hydrates. When the quantity of acetic acid is increased, calcium acetates and gibbsite precipitate and the setting time is very regular. In a sense, addition of acetic acid permitted to get rid of the variability of setting time of the cement.

The second set of data concerns the density and the open porous volume as a function of acetic acid content after 4 days or 6 months ageing (Figure 4). First of all, the additive helps the densification of the hydrated cement since the density goes from 2.3 to 2.5 g/cm 3 . Secondly, the open porous volume goes down from 35

to 25 vol% at 4 days and drops to less than 8 vol% after 6 months. The presence of the additive leads to a decrease in pore size and pore volume (Figure 5). In a 6 months old cement, with no additive, there are 2 populations of pores. With acetic acid, the micron size pores progressively disappear and the remaining pore are nanometric pores. The explanation is the following: the presence of acetic acid leads to a good dispersion of cement particles and contribute to a better densification compared to pastes with no addition.

Moreover, calcium acetates and gibbsite can fill in the pores and thus contribute to the disappearance of micrometric pores.

Tape casting was used to prepare calcium aluminate cement tapes and the steps are described in figure 6.

Table 2 gives the composition of the cement slurry and the role of each component. The principle of the process is the following: a slurry with adequate rheology is poured into a container which moves on a support and a tape is deposited. It is interesting to compare the process with the case of tape casting of alumina (Figure 6). The main differences between tape casting of a technical ceramic, such as alumina, and a calcium aluminate cement (which contains 72 wt% of alumina) is the removal of organic additives and the firing step: (i) with the cement, the removal of organic additive (also called 'debinding') is obtained by leaving the set tape in water for 24 h while it requires a heat treatment in the case of alumina, (ii) no sintering is necessary to obtain a dense material in the case of cement. The final materials (4 days old samples) presented similar dielectric constants (of the order of 10) and thermal expansion coefficients (of the order of 7.10 -6 °C-1 between 25 and 1200 °C) [START_REF] Hafiane | Calcium aluminate cement tapes -Part II: physical properties[END_REF]. The last interesting feature about our cement tapes was their surface roughness (of the order of 400 nm), lower than the surface roughness of an alumina substrate prepared by tape casting (of the order of 600 nm). Such a low roughness leads to a very smooth and glossy surface (Figure 7) which contains a mixture of calcium acetate and gibbsite. Michael Eden [6] coated his artworks with our slurries colored with a dye because our slurries gave the glossy aspect he was looking for (Figure 8).

This first example on a cementitious material shows it is possible to obtain at room temperature a fairly dense material which can find applications for making substrates or coating surfaces.

Valorization of dam sediments as raw materials for synthesis of Portland cement clinker

This second example will illustrate how dam sediments, instead of being disposed waste, could be valorized in the cement industry. Hydroelectric power generation represents 71% of renewable electricity all around the world [7]. Reaching 1,064 GW of installed capacity in 2016, it generated 16.4% of the world's electricity output. Some countries account for a significant percentage of the global installed capacities (in 2015, China, USA, Brazil and Canada represented respectively 26%, 8.4%, 7.6% and 6.5%). The immediate problem linked with the dams is the accumulation of sediments that get trapped in the reservoirs behind the dams instead of flowing into the river downstream. This can affect the efficiency of hydroelectric plant. Figure 9 gives a schematic representation on how sediments tend to accumulate in the reservoirs.

Due to watershed erosion, solid particles are transported by the watercourse. The biggest particles can deposit in the highest part of the reservoir while the finest particles (silts and clays) are transported in suspension and can accumulate with time. These accumulation of fine particles can have several potential impacts: for the operator, a reduction of the reservoir capacity and on the recreational activities. To avoid these impacts, it is necessary to have an adequate and sustainable management of these sediments.

Apart from the downstream transfer of the sediments, which is usually managed by the dam operator, there is an incentive to find alternative solutions because these sediments have to be considered as waste when they get extracted from a reservoir and are on-land-managed. Among the possible uses, a specific route we explored in a circular economy approach was the use of these sediments in the cement industry either as alternative materials for the manufacture of clinker (which is the raw material for cement production) or as pozzolanic addition to ordinary Portland cement. Using waste material in Portland cement clinker production is a relevant eco-friendly solution: it limits the mineral extraction in quarries (clays and marls mainly) and, at the same time, it alleviates the tonnages of landfilled waste [START_REF] Taylor | Cement Chemistry[END_REF]. Blending Portland cement with an alternative pozzolanic material has an additional advantage which is a limitation of the carbon dioxide emissions by ton of manufactured cement. Using life-cycle analysis, the CO2 emission reduction using blended cement was found to be around 22 % compared to Ordinary Portland Cement [START_REF]NF EN 197-1. Cement Part 1: Composition, specification and conformity criteria for common cements[END_REF] since less clinker is required in the formulation.

In this respect, we studied sediments extracted from dams operated by Electricité de France (EDF). They contain different minerals, typically quartz, carbonates, clay minerals and feldspars (Figure 10). Table 3 presents the range of chemical composition of the sediments located in the French Alps. Their silica, alumina, iron oxide and calcium oxide are compatible with their use as raw materials to prepare a Portland clinker. In order to obtain a suitable clinker, the mix composition was adjusted so that three parameters, the lime saturation factor (LSF), the silica-ratio (SR) and the alumina ratio (AR) were within appropriate range as shown in table 4 [START_REF] Taylor | Cement Chemistry[END_REF][START_REF] Moir | Cements. In: Advanced concrete technology[END_REF][START_REF] Locher | Cement: principles of production and uses[END_REF][START_REF] Gani | Cement and concrete[END_REF]. These three factors are defined below.

LSF = % CaO 2.80 × % SiO2 + 1.20 × % Al2O3 + 0.65 × % Fe2O3 (1) SR = % SiO2 % Al2O3 + % Fe2O3 (2) AR = % Al2O3 % Fe2O3 (3)
Therefore, we mixed the sediments with limestone in order to synthesize a clinker at 1450 °C, which is the typical temperature for Portland clinker manufacture [START_REF] Faure | Ability of Two Dam Fine-Grained Sediments to be Used in Cement Industry as Raw Material for Clinker Production and as Pozzolanic Additional Constituent of Portland-Composite Cement[END_REF]. By assuming the stoichiometric composition of synthesized phases, the mineral clinker phase fractions, namely alite (C3S), belite (C2S), aluminate (C3A) and ferrite (C4AF) † were calculated using Bogue's empirical formula [START_REF] Stutzman | Uncertainty in Bogue-calculated phase composition of hydraulic cements[END_REF]:

The average percentages of each of these phases -determined by a Rietveld analysis -for a suitable Portland clinker should be 60-65 wt%, 20-25 wt%, 8-12 wt% and 8-10 wt% for C3S, C2S, C3A and C4AF, respectively, which is what we obtained in our mixtures [START_REF] Faure | Capacité d'un sédiment à se substituer à la fraction argileuse de la matière première de l'industrie des liants hydrauliques[END_REF]. Figure 11 shows a typical micrograph of a clinker we prepared by mixing adequate amounts of one sediment coming from the French Alps (27 wt%)

with limestone (73 wt%) and synthesized at 1450°C. The porosity represents about a third of the total clinker grain volume, which can be explained by the granulation step of the powder mixture prior to synthesis. The observations show that the typical phases of a Portland clinker are present: calcium silicates (C3S and C2S), interstitial calcium aluminate phases (C3A, C4AF) [START_REF] Faure | Capacité d'un sédiment à se substituer à la fraction argileuse de la matière première de l'industrie des liants hydrauliques[END_REF].

This second example of eco-materials shows that the valorization of mineral waste is technically feasible and avoid the exploitation of new quarries. Nevertheless, for a large scale implementation, the sediment source should not be far from a cement industrial kiln since transport in this industry should be minimal.

Plaster to improve acoustic insulation in houses

According to a survey, a fair number of persons are bothered by noise in their housing [START_REF] Pedroso | Characterization of eco-efficient acoustic insulation materials (traditional and innovative)[END_REF]. The reduction of noise is a public health issue and it concerns different sector, especially new construction or rehabilitation of dwellings. In order to understand acoustic insulation, two parameters should be considered. The first one is the sound transmission loss, STL; it corresponds to the loss of noise when it goes from one room to another room (Figure 12). An efficient acoustical insulation implies that STL, which corresponds to the difference in noise level, should be as high as possible. In general, STL increases with the frequency [START_REF] Fahy | Sound and structural vibration : radiation, transmission and response[END_REF].

The second parameter is the critical frequency. For a specific frequency of noise called the critical frequency (fc), the sound transmission loss curve shows a dip (Figure 13). This phenomenon can lead to a notably reduced acoustic insulation, especially if the dip of STL affects the midrange frequencies (The human hearing range is commonly given as 20 Hz to 20 kHz but the human ear is most sensitive in the midrange frequencies from 350Hz to 1500 Hz). Amongst the different possible materials for acoustic insulation, there is gypsum, CaSO4.2H2O (commercially called 'plaster'). In fact, gypsum boards either alone or in a multilayer configuration are increasingly used as facing materials for interior walls and ceilings because of their acoustic properties, their good fire endurance, and thermal insulation characteristics. Our contribution was to examine to which extent the acoustic characteristics could be improved or not and how the microstructure could influence on the acoustic properties of gypsum.

In this respect, we prepared gypsum specimens with additive to modify the microstructure. The gypsum boards (thickness: 14 to 18 mm, width: 60 mm and length: 610 mm) were prepared from a mixture of calcium sulfate hemihydrate (CaSO4.½H2O), water (water over CaSO4.½H2O weight ratio, W/P = 0.6) and in some cases addition of citric acid (0.1 to 4 wt% with respect to water) [START_REF] Khessaimi | Effects of microstructure on acoustical insulation of gypsum boards[END_REF]. The sound transmission loss as a function of frequency was measured with the mechanical impedance method (MIM) described in the International Standard ISO 1694 [START_REF]Glass in building --Glazing and airborne sound insulation --Measurement of the mechanical impedance of laminated glass[END_REF]. Figure 14 presents these variations and the microstructure of the corresponding gypsum boards.

The effect of citric acid is remarkable since it shifts the critical frequency towards high values, further away from the human ear sensitive range. The microstructures are also different: gypsum with no additive presents the usual needle like crystals, while citric acid leads to platelets like crystals. Citric acid molecules present interatomic distances between the oxygen atoms of 4 Å, which is the interatomic distance between calcium in (120) and (111) planes in gypsum. Finot et al. [START_REF] Finot | Correlating surface forces with surface reactivity of gypsum crystals by atomic forcemicroscopy. Comparison with rheological properties of plaster[END_REF] confirmed that these faces exhibit the highest surface reactivity for adsorbing organic molecules. Therefore, citric acid molecules behave like a surfactant and can adsorb on these faces and slow down their growth, thus leading to a different morphology.

The consequence on the shift of the critical frequency can be explained as follows: the critical frequency, fc is given by equation [START_REF] Taylor | Cement Chemistry[END_REF].

f = × × × (8)
c0 is the speed of sound in air (340 m s -1 at 20 °C), e is the thickness of the board, Ms is the mass per unit area of the gypsum board and E Young's modulus, which we determined by the pulse echo technique [START_REF] Tessier-Doyen | Experimental and numerical study of elastic behavior of heterogeneous model materials with spherical inclusions[END_REF].

Table 5 presents the data for increasing percentages of citric acid. The parameter which influences on the fc value is Young's modulus: the higher the citric acid content, the lower E is. Values of Young's modulus E are divided by 6 when the citric acid percentage goes from 0 to 4 wt%. A calculation of the number of contacts between the crystals [START_REF] Meille | Etude du comportement mécanique du plâtre pris en relation avec sa microstructure[END_REF] shows that it also decreases by a factor 6. Less contacts induce less rigidity of the material.

To conclude on this example, it shows that monitoring the microstructure enables to obtain gypsum boards with improved acoustic insulation capabilities.

Conclusion

These three examples show that the concept of 'eco-materials' offers interesting technical solutions to manufacture products. The example of calcium aluminate cement tape shows it is possible to obtain at room temperature a material close to sintered alumina tapes. Dam wastes, whose composition is close to clay minerals, could be valorized as raw materials in the cement industry, thus lowering the carbon footprint of cement manufacture. The third example shows that the presence of an additive modifies the microstructure of gypsum crystals and gives a material with improved acoustical insulation compared to gypsum with no addition. These three examples illustrate that these materials could fulfill some needs, like energy savings, waste management, and comfort in housing. Nevertheless, to go further into their wide implementation, there is a necessity to go beyond these scientific solutions and to build network between different actors: academic laboratories, industries, governmental agencies and above all the citizens who will be the consumers of these products [START_REF] Patala | Towards a broader perspective on the forms of ecoindustrial networks[END_REF]. The development of these eco-industrial networks will help to address environmental issues and participate in the social acceptability of different solutions.

C Q KFNaF C D C Q C Q Q D Q C Q CC C Q Q D C D Q Q C C C Q C C Q C QQQ C C C

  is a simplified composition diagram showing two families of cements: Portland cements containing calcia and silica, and calcium aluminate cements (CAC), which are alumina rich.

C3S = 4 ,

 4 0710 CaO -7,6024 SiO2 -6,7187 Al2O3 -1

Figure 1 :Figure 2 :

 12 Figure 1: composition range of calcium aluminate cements compared to Portland cements

Figure 3 :Figure 4 :

 34 Figure 3: setting time and pH after 15 min mixing water with cement (wwater/wcement = 0.3 and wacetic acid/wcement ranges between 0 and 0.05).

Figure 5 :

 5 Figure 5: pore size and pore volume distribution in a 6 months old cement paste (wwater/wcement = 0.3) containing 0, 0.1 and 5 % of acetic acid with respect to water.

Figure 6 :

 6 Figure 6: main steps of tape casting in the case of alumina and a calcium aluminate cement.

Figure 7 :

 7 Figure 7: micrograph of the surface of the cement tape showing a dense and smooth surface.

Figure 8 :

 8 Figure 8: The Wedgwoodn't Tureen 2010 Michael Eden. Collection of Museé des Beaux-Arts, Montreál, Canada. Photo courtesy of Adrian Sassoon, London.

Figure 9 :

 9 Figure 9: schematic representation of sediment deposition in hydroelectric reservoirs.

Figure 10 :

 10 Figure 10: Typical X-Ray Diffraction analysis of a carbonated sediment from French Alps.

Figure 11 :

 11 Figure11: polished surface of a clinker prepared with an alpine sediment and etched with a NaOH solution followed by an ethanol-HNO3 solution (nital), in order to successively reveal and color the interstitial aluminates and the silicate phases[START_REF] Locher | Cement: principles of production and uses[END_REF]. C, S, A and F stand for CaO, SiO2, Al2O3 and Fe2O3 (from[START_REF] Faure | Capacité d'un sédiment à se substituer à la fraction argileuse de la matière première de l'industrie des liants hydrauliques[END_REF]).

Figure 12 :

 12 Figure 12: schematic representation of the sound transmission loss, R, through a partition wall.

Figure 13 :

 13 Figure 13: typical transmission loss as a function of frequency. At a critical frequency, fc, the transmission loss decreases sharply and the acoustic insulation as well. 350 Hz to 1500 Hz is the range where the human ear is most sensitive.

Figure 14 :

 14 Figure 14: sound transmission loss as a function of frequency for gypsum boards (water over CaSO4.½H2O weight ratio, W/P = 0.6) prepared with no additive in water or with the addition of citric acid (1.5 wt% with respect to water).

Table 1 :

 1 mineralogical and oxide composition of Secar 71. C and A correspond to CaO and Al2O3 in cementitious notations.

	Constituent	Function	Weight percentage (%)
	Cement (Secar 71)	Powder	-
	Water	Solvent and binder	33 -35
	Bevaloid 35L	Dispersant	0.05 -2
	PEG 300	Plasticizer	4 -8
	Acetic acid	Dispersant	0.3 -2

Table 2 :

 2 composition of the calcium aluminate cement slurry. The percentages are relative to the weight of cement.

	Oxide	Composition range (wt%)
	SiO2	42.70 -49.60
	Al2O3	7.50 -13.70
	Fe2O3	3.00 -5.80
	CaO	12.10 -15.60
	TiO2	0.40 -0.70
	MnO	0.05 -0.16
	P2O5	0.13 -0.29
	SrO	0.04 -0.08
	Na2O	0.65 -0.90
	K2O	1.60 -2.70
	SO3	0.10 -0.35
	Ignition loss	< 20

Table 3 :

 3 range of chemical compositions of the dam sediments located in the French Alps.

	Ratio	Taylor [8]	Moir [10]	Locher [11]	Gani [12]
	LSF	0.92 -0.98	0.95 -0.97	0.90 -1.04	0.92 -0.96
	SR	2.0 -3.0	2,4 -2,6	1.6 -4.1	2.3 -2.5
	AR	1.0 -4.0	1.5 -1.8	1.4 -3.7	~ 2.0

Table 4 :

 4 typical ranges of lime saturation factor (LSF), silica ratio (SR) and alumina ratio (AR) for a

	Portland clinker.

Table 5 :

 5 physical parameters of gypsum boards and critical frequency (calculated from (8)) as a function of the quantity of citric acid added in the water + plaster mix.

† In cementitious notations, A, C, F and S correspond respectively to Al2O3, CaO, Fe2O3 and SiO2.

L. Pressouyre : 'Le pont de Mostar, aléas et limites d'une reconstruction à l'identique', Bulletin archéologique du Comité des Travaux Historiques et Scientifiques / Archéologie, histoire de l'art, époques médiévale et moderne (Paris : Ed. du CTHS). N.S. 34 (2008), 187-198. (ISSN 1286-0999).

Acknowledgments

The authors would like to thank all the contributors to these three topics and who are cited in the corresponding references.


Christophe Celindano
email: christophe.celindano@univ-lorraine.fr

Emile Haye

Stéphanie Bruyère

Pascal Boulet

Alexis Boileau

Sylvie Migot

Sandrine Mathieu

Patrice Miska

Silvère Barrat

Fabien Capon

Probing the growth window of LaVO3 perovskites thin films elaborated using magnetron co-sputtering

Keywords: Oxide, Thin film, Sputtering, Crystallization, Vanadate, Perovskite

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The conventional solar cells have a limited energy conversion efficiency because photons with energies lower than the band gap are not absorbed and those with higher energies mainly produce phonons. One of the possible ways to circumvent this problem is the use of multi-junctions with several layers that cover a larger part of the solar spectrum. In this way, Mott insulators as solarabsorbers using carrier multiplication could be considered [START_REF] Manousakis | Photovoltaic effect for narrow-gap Mott insulators[END_REF]. Indeed, lanthanum vanadate LaVO 3 is a promising candidate with an indirect forbidden band gap close to 1.1 eV [START_REF] Arima | Variation of optical gaps in perovskite-type 3d transitionmetal oxides[END_REF], i.e. close to the ShockleyQueisser limit [START_REF] Shockley | Detailed Balance Limit of Efficiency of p-n Junction Solar Cells[END_REF] and a good absorption coefficient [START_REF] Assmann | Oxide Heterostructures for Efficient Solar Cells[END_REF]. This perovskite material and more especially the off-stoichiometry La 1+x VO 3 films have already been studied for photovoltaic solar applications [START_REF] Zhang | Self-regulated growth of LaVO 3 thin films by hybrid molecular beam epitaxy[END_REF][START_REF] Zhang | High-Quality LaVO 3 Films as Solar Energy Conversion Material[END_REF] but mainly by a hybrid molecular beam epitaxy growth method. In such an approach, the authors investigated the off-stoichiometry LaVO 3 samples by varying La/V ratio during the synthesis. However, most of the studies deal with lanthanum over-stoichiometry, in a small range of concentration. The effect of La/V ratio variation over a wide range in LaVO 3 remains an open question.

Usually, lanthanum vanadate films are deposited by pulsed laser deposition, molecular beam epitaxy or are synthesized through powder reactions of lanthanum and vanadium oxides [START_REF] Rotella | Octahedral tilting in strained LaVO 3 thin films[END_REF][START_REF] Choi | Epitaxial growth of semiconducting LaVO 3 thin films[END_REF][START_REF] Wang | Combinatorial pulsed laser deposition and thermoelectricity of (la 1x ca x )VO 3 composition-spread films[END_REF][START_REF] Kourkoutis | Asymmetric interface profiles in LaVO 3 SrTiO 3 heterostructures grown by pulsed laser deposition[END_REF][START_REF] Sheets | Effect of charge modulation in (LaVO 3 ) m (SrVO 3 ) n superlattices on the insulator-metal transition[END_REF][START_REF] Jellite | Investigation of LaVO 3 based compounds as a photovoltaic absorber[END_REF]. These growth processes are limited in terms of upscaling concerns. Therefore, by focusing here on the sputtering technique, we use an alternative way for the synthesis of crystalline LaVO 3 .

The synthesis is based on a two-step process with 1) deposition of a LaVO thin-film by magnetron co-sputtering and 2) ex-situ annealing under a reducing atmosphere. Such a process is easy to up-scale for large surfaces treatments as required for many industrial applications.

For possible use in photovoltaics, the goal of the present paper is to extend the present knowledge about the growth of LaVO3 using magnetron sputtering, particularly by determining LaVO 3 growth window where no formation of additional phases is observed. In this view, thin films with different La/V atomic ratios (from 0.52 to 1.68) are obtained adapting the sputtering electrical parameters and analyzed using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscope (TEM) and infrared spectroscopy.

Experimental section

La x V y O 3 thin films deposited on double side polished silicon substrates are grown using a 30 L sputtering chamber connected to a turbomolecular secondary pump backed by a rotary pump to get a vacuum close to 5 • 10 -5 Pa before each deposition process. Two inches diameter lanthanum and vanadium targets with atomic purity of 99.9% are used for the co-sputtering. The targets are surrounded by plasma suppressors and separated by a distance of 80 mm, long enough to avoid any observable plasma interaction between the two targets during the process. Substrates are mounted on a rotating holder and located at 55 mm and 70 mm away from respectively the lanthanum and vanadium targets. Discharge parameters have been optimized from previous works using similar elements [START_REF] Mercs | Innovative Smart Selective Coating to Avoid Overheating in Highly Efficient Thermal Solar Collectors[END_REF][START_REF] Haye | Nitrogen redistribution in annealed LaFeO x N y thin films investigated by FTIR spectroscopy and EELS mapping[END_REF][START_REF] Haye | LaFeO x N y perovskite thin films: Nitrogen location and its effect on morphological, optical and structural properties[END_REF][START_REF] Haye | Phenomenological study of iron and lanthanum magnetron co-sputtering using two reactive gases[END_REF][START_REF] Haye | Properties of rareearth orthoferrites perovskite driven by steric hindrance[END_REF][START_REF] Capon | Cation size effect on the thermochromic properties of rare earth cobaltites RECoO 3 (RE: La, Nd, Sm)[END_REF]. The lanthanum target is powered by an Advanced Energy Pinnacle 5kW pulsed-DC generator under a power regulation of 75 W, a frequency of 100 kHz and an off-time of 4 µs while vanadium target is powered by an Advanced Energy MDX 500W DC generator with a pre-chosen and fixed power ranging from 200 to 300 W. The pressure control is established by using an MKS Baratron 627 capacitive gage with a 1.33 hPa range and the gas are flown (at 21 sccm for argon and varying flow for the oxygen) using Alphagaz RDM 280 flowmeters. Substrates temperature is evaluated by using a thermocouple, located inside the substrates holder.

Prior to the deposition, the substrates used are cleaned with acetone and ethanol, followed by insitu by reactive-ion etching (RIE) with an Advanced Energy Cesar radiofrequency (RF) generator inside the sputtering chamber for 5 minutes under gas flows close to the growth conditions (i.e. under 21 sccm of Ar and 7 sccm of O 2 ). Two types of annealing treatments have been tested to crystallize the perovskite, namely an air annealing and a reducing annealing. The first annealing is carried out in the air and performed with a Barnstead type 47900 furnace at 900 • C for 5 min. For the second annealing, a homemade furnace is used for the post-growth annealing step, composed of a quartz cylinder receiving a sample-holder where a primary vacuum is done before the heating step. The annealing temperature is regulated with an Eurotherm temperature regulator while the gas flows are controlled via a MKS Type 647C controller. The sample-holder is a quartz semi-cylinder previously cleaned with acetone and ethanol before each use. in a gaseous mixture of 90% Ar and 10% H 2 working pressure is fixed at 700 torr and a temperature ramp has been fixed, with a temperature of 900 • C, reached in 90 min and kept constant for 60 min. When the cycle ends, gas flows are set to zero and the quartz chamber is isolated, with a static pressure imposed by the amount of remaining gas. Samples are then cooled to ambient temperature in almost six or seven hours. FEI/Philips XL-30 field emission environmental scanning electron microscope (ESEM) working at 15 kV provides X-ray energy dispersive spectroscopy (EDS) measurements, useful for the La/V atomic ratio estimation of the different samples with a thickness varying between 200 and 300 nm.

Analyzing thin films, the composition estimation is reliable for studying an atomic ratio evolution, not in absolute value.

The chemical composition of the La x V y O 3 thin films have been investigated by XPS (K-Alpha Thermo Scientific spectrometer) using a monochromatic Al K alpha radiation (1486.68 eV). The X-rays spot size was 250 µm in diameter. Spectra (La 3d, O 1s and V 2p) have been recorded with an energy resolution of 0.5 eV and a pass energy of 20 eV. The number of scans was adjusted (between 5 and 30 scans) for each element to get similar signal-to-noise ratios. A flood gun was used for the charge compensation and spectra were calibrated by fixing the La 3d 5/2 main peak at 833.6 eV, as the intensity of the adventitious carbon peak was too weak, and the La 3d 5/2 peak does not exhibit significant shift [START_REF] Sunding | XPS characterisation of in situ treated lanthanum oxide and hydroxide using tailored charge referencing and peak fitting procedures[END_REF]. Peak-fitting was performed with the Avantage software (Thermo Scientific).

The structure of the sample is investigated by XRD with a Brucker D8 Advance diffractometer (λ CuKα1 = 1.54056 Å) using the Bragg-Brentano θ -2θ configuration.

A doubly corrected JEOL ACCEL ARM Cold FEG 200F TEM coupled with a GATAN GIF Quantum ER was used in scanning transmission electronic microscopy (STEM) mode for Electron Energy Loss Spectroscopy (EELS) measurements (energy dispersion of 0.1 eV/channel, a full width at half maximum (FWHM) of 0.6 eV, a 2 nm pixelsize and a pixeltime of 0.5 s/pixel to avoid beam damage). Fast Fourier transform (FFT) of high resolution transmission electron microscopy (HRTEM) images were recorded to obtain the crystallographic structure. Thin foils of selected samples were prepared by the focused ion beam (FIB) technique using a FEI HELIOS NanoLab 600i FIB/SEM.

Results and discussion

Discharge characteristic

The sputtering of lanthanum and vanadium in the presence of a reactive atmosphere has been investigated using hysteresis experiments, by sputtering each target separately or simultaneously.

The parameters used are the further deposition parameters given in the table 1 except that for the hysteresis study we use a fixed power of 250 W applied to the vanadium target and a variable O 2 flow rate. Before the hysteresis experiments, targets are sputtered in pure Ar gas for half an hour using the previous given parameters in order to avoid residual oxides and start the hysteresis experiments with metallic elements. The oxygen partial pressure is obtained by making the difference of total pressures measured with or without oxygen. In the absence of oxygen, pure metallic atoms are sputtered from the targets, which is often called the elemental sputtering mode (ESM) [START_REF] Capon | Strontium-doped lanthanum manganite coatings crystallised after air annealing of amorphous co-sputtered films[END_REF]. When oxygen is introduced in the chamber, this leads to a target poisoning caused by both chemisorption and ion implantation of oxygen molecules [START_REF] Depla | Discharge voltage measurements during magnetron sputtering[END_REF]. In the case of the lanthanum we could consider that the target surface is fully covered by an oxide layer when the oxygen pressure starts to increase linearly with the O 2 flow rate i.e. around 2 sccm O 2 flow rate (see point A in Figure 1) leading to a regime where only compounds are sputtered and called reactive sputtering mode (RSM). For the vanadium target the transition occurs for an O 2 flow rate higher than 4 sccm (see point B in Figure 1.b) then, taken separately, the lanthanum is more reactive in an oxygen atmosphere than the vanadium.

When targets are sputtered together, the hysteresis curve (see point C Figure 1.c) is very similar to the vanadium one indicating that the simultaneous sputtering is driven by the vanadium element because even more reactive, the lanthanum target is still sputtered in ESM until the vanadium target becomes fully oxidized on the surface and then both targets are sputtered in RSM. At 10 sccm, the O 2 flow rate is slowly decreased to zero (backward direction of the hysteresis) but the RSM is still effective for 0 2 flow rates lower than before for simple and co-sputtering (see points D, E and F in Figure 1). This could be explained by the fact that despite the reduction of oxygen content, targets are still oxidized and become metallic only for smaller O 2 flow rates.

Further, this result implies that changing the deposition parameters is more efficient by modifying the vanadium parameters than the lanthanum ones. Next, the choice is made to change the discharge power applied to the vanadium target.

Elaboration and annealing of La

x V y O 3 thin films
The deposition parameters are given in table 1. The discharge power applied to the vanadium target is changed from 200 to 300 W inducing the modification of the La/V ratio.

As-deposited thin films are amorphous as observed by XRD in Figure 2. An annealing step is 120 necessary to crystallize the perovskite-phase [START_REF] Boileau | Mechanisms of Oxidation of NdNiO 3-δ Thermochromic Thin Films Synthesized by a Two-Step Method in Soft Conditions[END_REF]. We first used an annealing made under air at 900 • C for 5 min inducing the formation of LaVO 4 structure (Figure 2), where the vanadium has a +V oxidation state. Consequently, reaching the +III oxidation state for the vanadium in LaVO 3 The air annealing is then replaced by a reducing annealing process, performed under pumping while injecting Ar/H 2 gas mixture. For all samples, SEM cross-sections performed on both preand post-annealed steps (see Figure 3) show a morphological modification, from dense (for an asdeposited sample in

Growth window of LaVO 3

La/V atomic ratio, evaluated by EDS measurement and which is not varying with the reducing annealing step, exhibits a linear evolution as a function of the discharge power applied to the vanadium target (see Figure 4).

135

Figure 5 shows the diffractograms from XRD measurements after reducing annealing. All the films present LaVO 3 orthorhombic structure [START_REF] Seim | Non-Stoichiometric LaVO 3 . II. Powder Neutron Diffraction Study of Crystal and Magnetic Structure for La 1 -xVO 3 , 0.00 <= X <= 0.10[END_REF] with the space group Pnma (62). Beyond the La/V value of 1.22, a crystallized La 2 O 3 cubic phase [START_REF] Gobichon | Potassium Lanthanum Nitrate System: Phase Equilibria and Thermal and Structural Properties[END_REF] appears in addition to the orthorhombic LaVO 3 with two peaks at 28. To obtain more information about the chemical composition, XPS Spectra were recorded between 510 and 535 eV and are reported in Figure 6.a in order to obtain both O 1s and V 2p core levels for the different La/V ratios. As the O 1s and V 2p levels are near, they have been recorded

and fitted with a unique background, as suggested by Biesinger [START_REF] Biesinger | Resolving surface chemical states in XPS analysis of first row transition metals, oxides and hydroxides: Sc, Ti, V, Cu and Zn[END_REF]. The peak fitting is shown in Figure 6.c: O 1s signal presents two contributions, centered at 528.8 and 530.7 eV, corresponding to O-La and O-V binding energies respectively. The V 2p signal exhibits two peaks corresponding to 3/2 and 1/2 spin-orbit splitting, with an energy difference of 7.5 eV. It appears that several contributions have to be used to fit these peaks, indicating different oxidation states of vanadium as previously reported [START_REF] Hotta | Electronic structure of the Mott insulator LaVO 3 in a quantum well geometry[END_REF][START_REF] Wang | Device Performance of the Mott Insulator LaVO 3 as a Photovoltaic Material[END_REF]. Three contributions have been used for the V 2p 3/2 peak fitting, centered at 514.1, 515.6 and 516.2 eV, that corresponds to +II, +III and +IV vanadium oxidation states. As La/V ratio increases, two major effects occur. First, the O-V binding contribution extinguishes (see Figure 6.a) due to the lower concentration of vanadium at the surface. Contrary to previous works dealing with LaVO 3 elaborated by PLD [START_REF] Wang | Device Performance of the Mott Insulator LaVO 3 as a Photovoltaic Material[END_REF][START_REF] Jana | Mott-Hubbard type insulating nature of epitaxial LaV O 3 thin films[END_REF][START_REF] Wadati | Characterization of LaVOx thin films by photoemission spectroscopy[END_REF], the O-V binding contribution to the O 1s level compared to the O-La one seems to be higher, which could be explained by the possible presence of vanadium oxides VO x at least at the surface of the V-richer samples. The effect is predominant from a ratio of 1.22. Secondly, a shift from 528.8 to 529.4 eV of the O-La contribution is observed for ratios higher than 1.22 (Figure 6.a and 6.b). This difference is attributed to the presence of La not associated with a variation of the La-O coordination among the LaVO 3 perovskite phase. The signal corresponding to the perovskite phase is shifted around 0.6 eV compared to La 2 O 3 .

160

The evolution of the La 3d signal for the different La/V ratios is reported in Figure 7.a. The signal exhibits a doublet due to the splitting of La 3d into 5/2 and 3/2 contributions. Then, a second multiplet splitting is observed in each contribution, that is attributed to the interatomic charge transfer of the O 2p to La 4f level, by the shake-up process, that ends in 3d 9 f 1 final state [30,[START_REF] Bolwin | Influence of valence band states on the core hole screening in lanthanide perovskite compounds[END_REF]. The delta between these two contributions (main peak and satellite) is widely dependent 165 on the species, and may vary in a wide range (from 3.37 to 4.27 eV, see [START_REF] Mickeviius | Investigation of epitaxial LaNiO 3-x thin films by high-energy XPS[END_REF]), while the main peak position is only slightly affected [START_REF] Han | Electronic structures of the CMR perovskites R 1-x A x MnO 3 (R = La, Pr; A = Ca, Sr, Ce) using photoelectron spectroscopy[END_REF]. The plot of the energy difference ∆E between the main contribution of La 3d 5/2 , centered at 833.6eV, and its satellite is shown in confirms the previous observation, with a drop of ∆E from 3.71 to 3.57 eV, when the atomic ratio reach 1.22 or higher values. As previously, this drop is attributed to the apparition of La 2 O 3 .

In order to see the effect of the La/V ratio variation, cell parameters refinements were performed (Fullprof Suite). Figure 8 presents the cell-volume evolution with the La/V ratio obtained after the refinement (varying from 233 to 241 Å3 with an error of almost 4 Å3 ). The refined volume does not evolve with this two-step synthesis in opposition to films obtained by molecular beam epitaxy [START_REF] Zhang | High-Quality LaVO 3 Films as Solar Energy Conversion Material[END_REF] where the out-of-place parameter decreases when La/V ratio differs from one. In our 0 close to the 242 Å3 value calculated with the cell-parameters found by Bordet et al. [START_REF] Bordet | Structural Aspects of the Crystallographic-Magnetic Transition in LaVO 3 around 140 K[END_REF].

In addition to chemical and structural investigation, a study of infrared properties has been carried out (Figure 9). Three major peaks are visible at 17.9 µm( 559 cm -1 ), 27.9 µm (358 cm -1 ) 180 and 59.9 µm (166 cm -1 ) attributed to the three vibration modes of the perovskite, namely V-O stretching, V-O-V bending and La translation respectively [START_REF] Haye | Experimental and Theoretical Infrared Signatures of REMO 3 (RE = La, Pr, Nd, Sm, and M = Co, Fe) Perovskites[END_REF]. These vibration modes are not affected by stoichiometry variations. However, the La/V ratio modification leads to the appearance of a peak at 11.13 µm (885 cm -1 ) attributed to La-O-Si vibration mode [START_REF] Kwon | In Situ Infrared Characterization during Atomic Layer Deposition of Lanthanum Oxide[END_REF] and suggests a different structure at the interface with the silicon substrate. A slight contribution at 20.5 µm (487cm -1 ) 185 appears for La/V ratio of 0.52 and may be attributed to a vanadium oxide. FTIR spectra thus confirm the XPS and XRD analyses with LaVO 3 -type formation without additional phases for La/V ratio ranging from somewhere between 0.52 and 0.83 up to a ratio of 1.22. XRD diffractograms in Figure 5 reveal the presence of La 2 O 3 cubic structure and the XPS spectra shown in Figure 6 reveal the 2p 3/2 vanadium contribution extinguishment with increasing La/V 190 atomic ratio. Those facts are motivating a TEM investigation in order to locate both structures and element positions using respectively fast Fourier transforms of HRTEM images or EELS analyses on three FIB lamellae and both cut using the FIB technique; the first for the V-richer sample with a La/V ratio of 0.52, the second for the La/V = 0.99 sample closest to the stoichiometry and the third for the lanthanum-richer La/V = 1.68 sample.

Several STEM images have been taken with the high angle annular dark field (HAADF) detector in order to select regions for EELS spectrum acquisitions. Figure 10 exhibits the presence of carbon and platinum elements, both used for lamella preparation. Between the lanthanum-vanadiumoxygen film and the silicon substrate, we detect a thin silica film corresponding to native silicon oxide. In order to study the crystallographic structure, Fast Fourier Transforms (FFT) on HRTEM images or diffraction patterns (not shown) were recorded on different crystallites. As expected, for the La/V = 0.99, only the LaVO 3 phase was found. For the lanthanum-deficient sample with the La/V ratio of 0.52, we have only observed the LaVO 3 phase, despite that the regions with less lanthanum were revealed by EELS for this V-richer sample (see Figure 10.a) and even more, that a signature of vanadium oxides was visible when using on the infrared spectrum (see Figure 9). This fact could be explained by small crystallite sizes of the potential vanadium oxides. On the other hand, for the La/V = 1.68 sample, at the surface (interface between the layer and the carbon, Figure 10.b) we found La 2 O 3 cubic crystallites (using lattice parameters given in [START_REF] Gobichon | Potassium Lanthanum Nitrate System: Phase Equilibria and Thermal and Structural Properties[END_REF]) for almost three nanometers i.e. approximately the thickness without vanadium found using EELS. At the center of the layer only LaVO 3 orthorhombic crystallites were identified, surrounded by amorphous domains. The transition between La 2 O 3 and LaVO 3 regions is not abrupt since a mixture of La 2 O 3 and a very few LaVO 3 orthorhombic crystallites (identified with lattice parameters of [START_REF] Seim | Non-Stoichiometric LaVO 3 . II. Powder Neutron Diffraction Study of Crystal and Magnetic Structure for La 1 -xVO 3 , 0.00 <= X <= 0.10[END_REF]) is present on a thickness of three nanometers despite the low vanadium EELS signal (EELS signal in Figure 10.b). Finally, close to the native silica between the film and the substrate we only detected the presence of the La 2 O 3 cubic structure too. Another surprising fact is the presence of silicon from the substrate at the interfaces but only for this sample, with a large lanthanum excess which is not occurring before annealing. This fact may be attributed to the possible insertion of the silicon into the La 2 O 3 structure that is confirmed by a La-O-Si infrared vibration mode (Figure 9). The La 2 O 3 /LaVO 3 non negligible thickness ratio close to 20 % justifies the La 2 O 3 phase present at the surface and the interface revealed by XRD, XPS and FTIR.

To summarize, the growth window of LaVO 3 structure ranges from La/V ratio around 0.5 up to 1.7. The increase of the lanthanum content induces the formation of La 2 O 3 phase containing silicon at each perovskite film boundaries whereas when increasing the vanadium concentration, vanadium oxides seem to be present in volume, where a lake of lanthanum was observed.

Conclusion

The LaVO 3 growing window has been investigated by the means of La x V y O 3 thin films synthesis, by using three complementary characterization methods. The thin films have been elaborated using a two-step method based on the magnetron co-sputtering followed by an ex-situ reducing thermal treatment (under Ar/H 2 mixture). Surprisingly, the structure presents a high tolerance, even using an annealing step. FTIR, XPS and XRD analyses clearly demonstrate structural modifications at La/V ratios higher than 1.2 whereas for the La/V ratio of 0.52 the FTIR highlights a new vibration mode, related to the presence of vanadium oxide(s). According to TEM and FTIR observations, vanadium oxide crystallites are present in volume contrary to the La 2 O 3 lanthanum oxide observed at the interface meaning that in the case of a vanadium excess no oxide layer is preferentially formed at the interfaces. This is an interesting fact for photovoltaics applications because stabilizing an unique crystallographic structure means a constant and stable 1.1 eV indirect optical gap in such materials and also since the presence of the La 2 O 3 oxide at the interfaces (out of the determined growth window) could affect the physical properties of a solar cell [START_REF] Shaikh | La 2 O 3 interface modification of mesoporous TiO 2 nanostructures enabling highly efficient perovskite solar cells[END_REF]. Furthermore, the use of magnetron sputtering makes up-scalable process over a large surface possible for example in the solar panels realization. For the selected area an EELS spectrum was taken each 2 nanometers and elemental maps of both lanthanum and vanadium were then constructed with colored intensities; the more bright it is the more the element is present. The "spatial drift" is used for the correction of any sample displacement due to vibration during the measure.

Figure 1

 1 Figure 1 shows the oxygen partial pressure behavior as a function of the O 2 flow rate using single sputtering (targets sputtered separately, Figure 1.a and 1.b) and the co-sputtering (Figures 1.c).

Figure 1 :

 1 Figure 1: Oxygen partial pressure vs the oxygen flow rate of the hysteresis experiment for the sputtering of (a) the lanthanum target, (b) the vanadium target and (c) both targets together.

Figure 2 :

 2 Figure 2: Diffractograms for a sample with a La/v ratio close to 1. As deposited film is amorphous whereas an ex-situ annealing leads to LaVO 4 or LaVO 3 depending on the annealing atmosphere (air or Ar/H 2 mixture).

Figure 3 .

 3 a) to porous (after annealing in Figure 3.b) reducing the thickness of almost twenty percents. This density drop i.e. porosity increase can be explained by the oxygen 130 losses during the annealing.

Figure 3 :

 3 Figure 3: Layer morphology observed with the SEM high-resolution mode in cross-section at a working distance of 5 mm, for a 3 kV acceleration voltage and a 200k magnification. The La/V ratio is around 1.68 in this case. Micrograph (a) shows cross-section of the as-deposited film and (b) after the reducing annealing.

Figure 5 :

 5 Figure 5: X-ray diffractogram in 2θ geometry of samples elaborated for different La/V atomic ratio. Two new peaks are appearing with the lanthanum richer samples (see the two orange triangles) which can be attributed to the La 2 O 3 cubic structure.

Figure 7 Figure 6 :

 76 Figure 6: O1s and V2p core level spectra for sample with different La/V ratio (a). Evolution of the O-La binding energy as a function of the different La/V ratio (b). Example of peak fitting with the six contributions of V2p signal and the two contribution of O1s signal (c).

Figure 7 :

 7 Figure 7: Evolution of the XPS La 3d spectra with the different La/V atomic ratio (a), and evolution of the energy gap between the two contributions of La 3d 5/2 (b).

3 Figure 8 :Figure 9 :

 389 Figure 8: Evolution of the orthorhombic unit cell-volume with the La/V atomic ratio. The red dashed-line distinguishes the vanadium or lanthanum-richer samples while the blue and green boxes give the structures in presence.

Figure 10

 10 Figure10shows two HAADF cross-section images and for selected regions, the corresponding elemental maps of lanthanum M 4,5 edges and vanadium L 2,3 edges. Colored intensities depend on the area of the selected characteristic energy-loss peaks and respectively correspond to lanthanum M 4 or vanadium L 3 rays among the different EELS spectra acquired for each pixel of the maps.Then intensities are proportional to the elemental contents. The right color bar representing the total signal of the lanthanum and vanadium EELS peak intensities (in cyan color in the Figure10.a, 10.b and 10.c) seems to have the same brightness on the whole thickness of the sample with a La/V ratio close to 0.52 (Figure10.a) meaning that both vanadium and lanthanum are present whatever the depth in the film. Nevertheless, the EELS spectra shown at the right of the figure clearly brings out a region where there is less lanthanum than the others. In the case of the sample with a La/V ratio close to 1 (see Figure10.b) the right color bar has an almost constant brightness except for the film-substrate border where a lake of vanadium which is confirmed by the lower EELS spectrum at the right of the same figure is observed on a thickness smaller than 3 nm (where the total film thickness is close to 260 nm). However, for the La-rich sample with a ratio near 1.68 and a total film thickness close to 192 nm, the total signal (shown in Figure10.b) highlights a lake of vanadium close to the film borders, namely up to approximately 6 nm in depth at the surface and 35 nm at the layer-substrate interface, which represents almost 20 percents of the total film thickness could be a potential localization for the formation of La 2 O 3 .

Figure 10 :

 10 Figure 10: HAADF images of the (a) La/V = 0.52, (b) La/V = 0.99 and (c) La/V = 1.68 sample cross-sections.

Table 1 :

 1 Experimental parameters for the elaboration of the samples by magnetron sputtering

	Parameter	value	unit
	Ar flow	21	sccm
	O flow	7	sccm
	Working pressure	1.5	Pa
	P V (DC)	variable	W
	V target distance	7	cm
	P La (DC-pulsed)	75	W
	f La	100	kHz
	Toff La	4	µs
	La target distance	5.5	cm
	Substrates temperature	≈ 60

• C requires a post-growth annealing treatment under an Ar/H 2 (100/10 sccm respectively ) reducing atmosphere.

  7 • and 31.2 • . Figure 4: Evolution of the La/V atomic ratio (estimated using EDS) varying the power applied on the vanadium target. Power on the lanthanum target is kept constant and other parameters are given in table 1.

		1.8
		1.6	La-richer samples	V-richer samples
	La/V atomic ratio	0.8 1.0 1.2 1.4
		0.6
		0.4
		200	220	240	260	280	300
		Power applied on the vanadium target (W)
				8

  2 O 3 which appears with this high ratio as confirmed by XRD measurements meaning that the O-La contribution shift could be attributed to the La 2 O 3 phase apparition and

	(020)	(121)	(220)	(040)	(230)	(042)	(232)	La/V
								1.68
								1.58
	Intensity (a.u)							0.99 1.05 1.11 1.22 1.44
					(222)	(311)		0.83
								0.52
	20	30	40		50			60
			2 (°)
			9

case, this means that all the lanthanum in excess introduced in the lanthanum-richer samples is not incorporated in the LaVO 3 phase but in the La 2 O 3 phase. The mean cell-volume value is 240 Å3

Acknowledgements

The SIAM platform in UNamur (for the XPS measurements) is acknowledged. The authors thank Régis Peignier from the Institut Jean Lamour of Nancy for his precious technical expertise and Jérôme Grausem from the LCPME laboratory (Nancy, France) for the FTIR spectrometer used. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


Marie Reynier°

Sophie Allart

Dominique Goudounèche

Alain Moga #

Guy Serre°

Michel Simon°

Corinne Leprince°^

Corinne Leprince
email: corinne.leprince@inserm.fr

THE ACTIN-BASED MOTOR MYOSIN Vb IS CRUCIAL TO MAINTAIN THE EPIDERMAL BARRIER INTEGRITY

Keywords: Cer, ceramide, Ctrl, Control, CDSN, corneodesmosin, DAPI, 4′, TEER, trans-epithelial electric resistance, TEM: transmission electron microscopy, TEWL, trans-epidermal water loss, TJ, Tight Junction, TGN, Trans Golgi

Myosin Vb (Myo5b) is an unconventional myosin involved in the actin-dependent transport and tethering of intracellular organelles. In the epidermis, granular keratinocytes accumulate cytoplasmic lamellar bodies (LB), secretory vesicles released at the junction with the stratum corneum which participate actively in the maintenance of the epidermal barrier. We have previously demonstrated that LB biogenesis is controlled by the Rab11a GTPase, known for its ability to recruit the Myo5b motor. In order to better characterize the molecular pathway that controls LB trafficking, we analyzed the role of F-actin and Myo5b in the epidermis. We demonstrated that LB distribution in granular keratinocytes was dependent on a dynamic Factin cytoskeleton. Myo5b was shown to be highly expressed in granular keratinocytes and associated with corneodesmosin-loaded LB. In reconstructed human epidermis, Myo5b silencing led to epidermal barrier defects associated with structural alterations of the stratum corneum and a reduced pool of LB showing signs of disordered maturation. Myo5b depletion also disturbed the expression and distribution of both LB cargoes and junctional components such as claudin-1, which demonstrates its action on both LB trafficking and junctional complex composition. Altogether, our data reveal the essential role of Myo5b in maintaining the epidermal barrier integrity.

INTRODUCTION

In the epidermis, the outermost living cell layer is composed of highly differentiated granular keratinocytes which play a crucial role in the maintenance of the epidermal barrier [START_REF] Elias | The secretory granular cell: the outermost granular cell as a specialized secretory cell[END_REF][START_REF] Madison | Barrier function of the skin: "la raison d'être" of the epidermis[END_REF]. First, granular keratinocytes have the capacity to assemble tight junction (TJ) complexes, essential for regulating the transit of small molecules across the epithelium [START_REF] Brandner | Importance of tight Junctions in Relation to Skin Barrier Function[END_REF]. Second, they are secretory cells containing cytoplasmic vesicles called lamellar bodies (LB) [START_REF] Feingold | The role of epidermal lipids in cutaneous permeability barrier homeostasis[END_REF]Schmitz and Muller, 1991). LB contain lipid precursors and lipid-modifying enzymes, structural proteins such as corneodesmosin (CDSN), proteases such as kallikreins (KLK), anti-bacterial peptides, etc. All these components are intended to be released by exocytosis in order to fill the intercorneocyte spaces of the stratum corneum (SC).

In a previous report, we demonstrated that the biogenesis of LB is regulated by Rab11a, which belongs to the guanosine triphosphate hydrolase (GTPase) Rab family [START_REF] Reynier | Rab11a is essential for lamellar body biogenesis in the human epidermis[END_REF]. In three-dimensional in vitro reconstructed human epidermis (RHE), Rab11a silencing generated epidermal barrier defects due to an impaired LB pool and drastic changes in the composition of the SC. In skin granular keratinocytes where it is particularly abundant and detected on the surface of LB, Rab11a is believed to control the biogenesis of pre-LB emerging from the Trans Golgi Network (TGN) and maturing along a secretory pathway.

However, the molecular cascade that leads to LB exocytosis at the apical plasma membrane of granular keratinocytes remains to be fully characterized.

Rab11a effectors have been clearly identified in various cell types and tissues: most of them are elements of the trafficking machinery [START_REF] Hales | Rab11 family interacting protein 2 associates with myosin Vb and regulates plasma membrane recycling[END_REF][START_REF] Cullinane | Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization[END_REF][START_REF] Roland | Rab GTPase-Myo5B complexes control membrane recycling and epithelial polarization[END_REF][START_REF] Welz | Orchestration of cell surface proteins by Rab11[END_REF][START_REF] Gruber | Autosomal recessive keratoderma ichthyosis-deafness (ARKID) syndrome is caused by VPS33b mutations affecting Rab protein interaction and collagen modification[END_REF]. In polarized epithelial cells such as small intestine enterocytes [START_REF] Sobajima | Rab11a is required for apical protein localization in the intestine[END_REF] or bladder epithelium umbrella cells [START_REF] Wankel | Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusioform vesicles in urothelial umbrella cells[END_REF], the partnership between Rab11a and the actin-based motor myosin 5b (Myo5b) plays a crucial role in the regulation of apical trafficking. Mutations in the MYO5B gene cause a very severe intestinal disorder with life threatening diarrhea [START_REF] Müller | MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity[END_REF][START_REF] Ruemmele | Loss-offunction of MYO5B is the main cause of microvillus inclusion disease: 15 novel mutations and a CaCo-2 RNAi cell model[END_REF] called Microvillus Inclusion Disease (MVID; OMIM #251850). Furthermore, a number of studies have revealed that exocytosis of secretory vesicles requires an active Factin cytoskeleton in multiple subcellular compartments, beyond the TGN exit sites (Guttierez and Villanueva, 2018). First, F-actin fibers may supplement microtubules as long distance tracks, facilitating vesicle motility towards the cortical sites [START_REF] Schuh | An actin-dependent mechanism for long-range vesicle transport[END_REF][START_REF] Pylypenko | Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes[END_REF]. Second, cortical actin at the cell periphery plays a scaffolding role for the exocytic machinery and its regulators. Third, F-actin acts as a driving force during the exocytic process at the plasma membrane.

In this study, in order to decipher the molecular basis of trafficking pathways that control the epidermal barrier, we analyzed the importance of the F-actin cytoskeleton and the molecular motor Myo5b, in the epidermis. Disruption of the F-actin cytoskeleton by drug treatment was shown to modify LB distribution in the cytoplasm of granular keratinocytes. In normal epidermis, Myo5b could associate with corneodesmosin loaded structures which are likely to be LB. In RHE, Myo5b silencing induced severe defects of the SC, decreased LB density and modified the expression of LB cargoes and junctional components. From these results, we hypothesize that the molecular motor Myo5b regulates F-actin-dependent vesicular trafficking which ultimately controls the epidermal barrier.

RESULTS

LB distribution is dependent on the F-actin cytoskeleton in granular keratinocytes

In order to study the molecular basis of LB trafficking in the epidermis, we first investigated the importance of the F-actin cytoskeleton through latrunculin treatment, a drug that affects actin polymerization. On tissue sections from drug-treated RHE, the LB cargoes CDSN and KLK7 were less concentrated on the apical side of granular keratinocytes and showed a more dispersed distribution in the cytoplasm compared to control RHE (Figure 1a). These data were strengthened by whole-mount imaging of RHE which substantially improved the detection and 3D analysis of fluorescent signals, i.e. actin and CDSN. The images presented in Figure 1b are Z sections of flattened granular keratinocytes [START_REF] Yokouchi | Epidermal cell turnover across tight junctions based on Kelvin's tetrakaidecahedron cell shape[END_REF] and corresponding videos are shown in supplementary material. The CDSN LB marker was present in the whole cell body including the cell edges in control conditions, when it was more localized in the cell center, on larger punctate structures, after latrunculin treatment (highlighted in boxes 1-4, Figure 1c). This suggests that a dynamic F-actin cytoskeleton is important for LB positioning in granular keratinocytes. A possibility is that an actin-based motor might control LB trafficking.

Actin-based Myo5b is associated with LB markers in granular keratinocytes

It has been previously shown that after TGN exit, LB carry the Rab11a GTPase known for its regulatory role in vesicular trafficking [START_REF] Reynier | Rab11a is essential for lamellar body biogenesis in the human epidermis[END_REF]Ishida-Yamamoto et al., 2018).

Among Rab11a effectors, one of the most frequently reported is the actin-based molecular motor, Myo5b. The expression of MYO5B mRNA was analyzed in distinct keratinocyte populations separated as previously described by [START_REF] Toulza | Large-scale identification of human genes implicated in epidermal barrier function[END_REF] The ratio between granular and basal cells was 23.63 ± 12.26 indicating an increasing expression gradient along with keratinocyte differentiation. Such a gradient was not found for another member of the Myo5 family displaying Rab11a binding capacities, MYO5A (mRNA ratio of 1.37 ± 0.03). On epidermal sections, the Myo5b protein showed a strong wavy signal at the apical side of granular keratinocytes, a signal which is reminiscent of Rab11a, when Myo5a was homogenously present in the entire epidermis (Figures S1a andS1e). In these cell layers, Myo5b and Rab11a could be found in the same subcellular compartment (Figure S1b). Moreover, Myo5b expression in RHE was reduced after Rab11a silencing, when Myo5a expression was not affected (Figure S1c,d,e). Altogether, these results highlight the possibility of a Rab11a-Myo5b partnership in the cytoplasm of granular keratinocytes.

Thereafter, we examined the distribution of Myo5b along with LB markers in native epidermis. In the outermost granular cell layers, Myo5b partially co-localized with CDSN as well as KLK7 (Figure 2a). By using super-resolution confocal microscopy (STED), Myo5b and CDSN signals were more specifically analyzed and revealed co-localization on punctate structures with a diameter of 100 to 140 nm (see enlarged boxes 1 and 2 in Figures 2b and2c). When the diameter of the co-labelled structures was calculated on a larger picture (Figure 2b, Merge), it was evaluated at 108 ± 58 nm, which is totally consistent with the known size of LB. The Pearson's coefficient of the two fluorochromes was evaluated as r=0.708, which revealed a significant co-localization between Myo5b and CDSN. These data suggest that Myo5b may associate with cytoplasmic structures which on the basis of their size as well as CDSN labelling are likely to be LB. This association may be dependent on a molecular recruitment by the Rab11a GTPase.

Myo5b silencing induces functional and structural defects in the epidermis

In order to analyze the functional relevance of Myo5b in the epidermis, we performed RNA interference in RHE. Two shRNA that specifically target the MYO5B mRNA induced a consistent inhibition of MYO5B expression compared to the non-targeting shRNA control (shCTRL): 76 ± 15% for shMYO5B1 and 48 ± 8% for shMYO5B2 (Figure S2a). In the same RHE, the expression of MYO5A was slightly decreased by shMYO5B2 but almost not affected by shMYO5B1. The silencing effect of the shRNAs was confirmed at the protein level (Figure S2b ans S2c).

After hematoxylin-eosin staining, Myo5b-depleted RHE were reproducibly thinner than the controls with a more compact SC, but maintained a normal structure of stratified epithelium, which indicates that keratinocyte differentiation occurred quite normally (Figure 3a). We then investigated the consequences of Myo5b depletion on the functional properties of RHE. First, as a global indicator of barrier tissue integrity, Myo5b-silenced RHE showed decreased transepithelial electric resistance (TEER) compared to controls (Figure 3b). Second, in a transepidermal water loss (TEWL) assay, Myo5b-depleted RHE demonstrated higher inside-out permeability compared to controls (Figure 3c). Third, in a Lucifer Yellow assay, outside-in permeability was higher after Myo5b silencing and this was most striking with shMYO5B1 (Figure 3d). Altogether, these functional assays suggest that Myo5b depletion impairs the barrier function of the epidermis.

These functional data are consistent with substantial alterations of the SC observed by transmission electron microscopy (TEM), after Myo5b silencing. As shown in Figure 4a, corneocytes were tightly packed, inter-corneocyte spaces had almost disappeared and corneocyte matrices were filled with a heterogeneous material that could be remnants of keratinocyte organelles. We performed a quantitative biochemical analysis of major lipids present in the SC, i.e. cholesterol and various types of ceramides, including the ω-acyl ceramides (EOS and EOP) with long carbon chains that are essential for lamella organization.

All the types of lipids tested were substantially decreased in the SC after Myo5b depletion (Figure 4b). Since most of the SC lipids are metabolized from lipid precursors transported by LB, we analyzed the LB pool after Myo5b silencing. In representative electron micrographs shown in Figure 4c, LB (white arrows) were fewer in the cytoplasm of granular keratinocytes compared to control RHE. This difference was confirmed by quantification of LB in a larger series of images where LB density was 0.71 ± 0.37 LB/µm 2 in Myo5b-depleted granular keratinocytes, versus 1.18 ± 0.48 LB/µm 2 in control cells (Figure 4d, upper panel). When LB quantification was restricted to an apical band in granular keratinocytes 1 and 2, as described in supplementary Figure S3, the difference between the 2 groups was even more significant: LB density was 1.74 ± 0.43 LB/µm 2 after Myo5b depletion, versus 2.88 ± 0.92 LB/µm 2 in control conditions (Figure 4d, lower panel). Moreover, it was frequent to find ovoid structures of the same size as LB but with no lamellar content (Figure 4e). These electron-lucent abnormal vesicles could be LB which had undergone a bad maturation. Altogether, our results indicate that the actin-based molecular motor Myo5b regulates the epidermal barrier by controlling the LB cytoplasmic pool, LB maturation along the secretory pathaway and ultimately, the composition and structure of the SC.

Myo5b depletion affects the expression of LB markers and cell junction components

For further examination of the functional properties of Myo5b in the epidermis, we analyzed the consequences of Myo5b depletion on the expression and distribution of critical proteins:

(i) LB cargoes such as CDSN and KLK7 and (ii) proteins of junctional complexes such as desmoglein (DSG), E-cadherin and claudin 1 as part of desmosomes, adherens junctions and TJ, respectively. Firstly, once Myo5b was silenced, CDSN and KLK7 had a more dispersed distribution in granular keratinocyte cytoplasm (Figure 5a), and the amount of KLK7 protein detected in a total RHE extract was decreased compared to a control RHE (Figure 5b). Therefore, Myo5b depletion reduced the amount of LB cargoes available in the apical area of granular keratinocytes, which suggests an impairment of LB trafficking. Secondly, Myo5b silencing decreased the expression of junctional proteins with the highest impact on the TJ component, claudin 1, as shown by western blotting (Figure 5c) or immunostaining of RHE sections (Figure 5d). Such a decrease could impact the TJ structure and function, which are crucial parameters of the epidermal barrier.

DISCUSSION

This work provides evidence that the F-actin cytoskeleton and Myo5b, an actin-based molecular motor, control the epidermal barrier by supporting stages of vesicular trafficking in keratinocytes. We have demonstrated that (i) the F-actin cytoskeleton affects LB distribution in the cytoplasm of granular keratinocytes, (ii) Myo5b is associated with CDSN loaded LB, controls LB trafficking before exocytosis at the SC junction and finally, (iii) Myo5b regulates membrane expression of junctional proteins such as the tight junction component, claudin 1.

The importance of Myo5b in granular keratinocytes is directly related to the previous demonstration that the Rab11a GTPase is essential for LB biogenesis [START_REF] Reynier | Rab11a is essential for lamellar body biogenesis in the human epidermis[END_REF]. In other tissues, Rab11a has been reported to recruit molecular motors on endosomal vesicles for trafficking regulation along microtubules and actin tracks [START_REF] Welz | Orchestration of cell surface proteins by Rab11[END_REF]. In particular, actin-dependent Myo5b is a major effector of the active Rab11a GTPase [START_REF] Lapierre | Myosin vb is associated with plasma membrane recycling systems[END_REF][START_REF] Hales | Rab11 family interacting protein 2 associates with myosin Vb and regulates plasma membrane recycling[END_REF]. In mouse oocytes, long-range motility dependent on the actin tracks has been described for Rab11a positive vesicles that are able to recruit Myo5b and actinnucleation factors [START_REF] Schuh | An actin-dependent mechanism for long-range vesicle transport[END_REF]. In our RHE model, treatment with an actin-depolymerizing drug tended to maintain larger CDSN-bearing structures in the nuclear periphery. This suggests that actin-dependent transport may apply to LB in granular keratinocytes, just after the TGN exit sites. The flexibility of the actin-based connections between vesicular compartments may facilitate LB maturation through exchange with endosomal compartments.

The detection of electron-lucent organelles lacking lamellar content in TEM micrographs of Myo5b-silenced RHE, suggests that the Myo5b molecular motor is crucial for LB maturation. Such atypical LB have also been described in the epidermis of ARC and ARKID patients presenting mutations in the VPS33B or VIPAS39 gene encoding two elements of membrane tethering complexes [START_REF] Hershkovitz | Defective lamellar granule secretion in arthrogryposis, renal dysfunction, and cholestasis syndrome caused by a mutation in VPS33B[END_REF][START_REF] Rogerson | VPS33b and VIPAR are essential for epidermal lamellar body biogenesis and function[END_REF].

In addition, this work shows that Myo5b controls expression and localization of junctional proteins such as the tight junction-associated claudin 1. This is consistent with the decreased TEER and increased TEWL measured on RHE after Myo5b silencing. In the epidermis, the tight junctional complexes are assembled in the penultimate granular keratinocytes, a dynamic process which is linked to cell differentiation and turnover [START_REF] Rübsam | E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning[END_REF]. Our hypothesis is that Myo5b participates in targeting junctional components through endosomal trafficking. When Myo5b is depleted, the endosomal vesicles may be directed towards the degradation compartment, as previously shown in the Rab11a study [START_REF] Reynier | Rab11a is essential for lamellar body biogenesis in the human epidermis[END_REF].

The MYO5B encoding gene is mutated in MVID, an autosomal recessive disease most frequently characterized by severe diarrhea leading to life-threatening dehydration (Mueller et al., 2008;[START_REF] Ruemmele | Loss-offunction of MYO5B is the main cause of microvillus inclusion disease: 15 novel mutations and a CaCo-2 RNAi cell model[END_REF]. This phenotype has been recapitulated in cell line models [START_REF] Thoeni | Microvillus inclusion disease: loss of myosin Vb disrupts intracellular traffic and cell polarity[END_REF][START_REF] Knowles | Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease[END_REF] as well as in KO mice after targeting of the MYO5B gene [START_REF] Carton-Garcia | Myo5b knockout mice as a model of microvillus inclusion disease[END_REF][START_REF] Schneeberger | An inducible mouse model of microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking[END_REF][START_REF] Weiss | Loss of MYO5B in mice recapitulates microvillus inclusion disease and reveals an apical trafficking pathway distinct to neonatal duodenum[END_REF]. All these data demonstrate that in enterocytes, Myo5b is a key molecule for the control of apical transport that leads to epithelial cell polarity and function. Despite the ubiquitous expression of Myo5b, MYO5B gene mutations have a predominant intestinal impact, with rare cases leading to hepatic cholestasis [START_REF] Gonzales | MYO5B mutations cause cholestasis with normal serum gamma-glutamyl transferase activity in children without microvillous inclusion disease[END_REF][START_REF] Qiu | Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis[END_REF]. A targeted study recently performed on a Navajo population of MVID patients reported lighter defects in stomach, liver, kidney and pancreas [START_REF] Schlegel | Apical membrane alterations in non-intestinal organs in microvillus inclusion disease[END_REF]. These data suggest that MYO5B mutations may impact many epithelial tissue types with varying intensities, possibly due to variable mechanisms of compensation. More specifically, the absence of skin defects in MVID patients could be due to the action of another Myo5 isoform, such as Myo5a which is much more expressed in skin tissue than in the small intestine [START_REF] Roland | Alternative splicing in class V myosins determines association with Rab10[END_REF].

In addition to genetic data, a number of biological studies have documented the importance of Myo5b for apical vesicular trafficking in polarized cells [START_REF] Roland | Rab GTPase-Myo5B complexes control membrane recycling and epithelial polarization[END_REF][START_REF] Sobajima | Rab11a is required for apical protein localization in the intestine[END_REF][START_REF] Thoeni | Microvillus inclusion disease: loss of myosin Vb disrupts intracellular traffic and cell polarity[END_REF][START_REF] Wankel | Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusioform vesicles in urothelial umbrella cells[END_REF]. In particular, in bladder epithelium umbrella cells, Myo5b controls the transport of uroplakin containing vesicles which emerge from the TGN and undergo exocytosis in response to bladder filling [START_REF] Khandelwal | Rab11a-dependent exocytosis of discoidal/fusiform vesicles in bladder umbrella cells[END_REF][START_REF] Khandelwal | A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells[END_REF]. In drosophila photoreceptor cells, Myo5 participates in the transport of rhodopsin-containing vesicles, leading to rhabdomere formation [START_REF] Li | Myosin V, rab11, and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors[END_REF]. Therefore, the control of apical vesicle motility by the Myo5b motor is a preserved evolutionary process in polarized cells.

Our data show that this process also applies to LB apical trafficking in polarized epidermal keratinocytes.

The diversity of the experimental models mentioned above helped to decipher the molecular basis of the Myo5b-dependent apical trafficking pathway between the TGN exit sites and the exocytic sites. From a proximal Rab11a-Myo5b partnership, these studies identified other players, among which are the Rab8 [START_REF] Khandelwal | A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells[END_REF][START_REF] Knowles | Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease[END_REF] and Rab27 GTPases [START_REF] Wankel | Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusioform vesicles in urothelial umbrella cells[END_REF], the Myo5a motor and elements of tethering/fusion complexes [START_REF] Vogel | Cargo-selective apical exocytosis in epithelial cells is conducted by Myo5B, Slp4a, Vamp7, and Syntaxin 3[END_REF][START_REF] Knowles | Rab11a regulates syntaxin 3 localization and microvillus assembly in enterocytes[END_REF]. In the epidermis, further studies are necessary to test the relevance of these other trafficking molecules. Since Myo5b is considered to be an anchoring/tethering molecule as much as a motion molecule [START_REF] Roland | Rab GTPase-Myo5B complexes control membrane recycling and epithelial polarization[END_REF], further work is needed to analyze its importance for LB docking at the actin-rich cell cortex.

In conclusion, F-actin and Myo5b control epidermal barrier function through their coordinated action on LB trafficking and membrane targeting of junctional elements. Therefore, along with other epithelia, the ubiquitously expressed Myo5b is shown to have a noteworthy role in the skin.

MATERIALS AND METHODS

Antibodies and reagents

The following antibodies were used: rabbit anti-Myo5b (Sigma-Aldrich, St Louis, MO), rabbit anti-Myo5a (Novus Bio, Littleton, CO), rabbit anti-Rab11a (USBio, Salem, MA), mouse anti-Rab11a (Invitrogen, Carlsbad, CA), mouse anti-CDSN (G36.19 mAb, [START_REF] Serre | Identification of late differentiation antigens of human cornified epithelia, expressed in re-organized desmosomes and bound to cross-linked envelope[END_REF], goat anti-KLK 6 (R&D systems, Minneapolis, MN), goat anti-KLK 7 (R&D Systems), rabbit anti-claudin 1 (Abcam, Cambridge, UK), mouse anti E-cadherin (BD Biosciences, Franklin Lakes, NJ), mouse anti-desmoglein 1/2 (DG3.10 Progen, Heidelberg, Germany), mouse anti-actin (Millipore, Darmstadt, Germany) and mouse anti-GAPDH (6C5 Santa Cruz Biotech, Dallas, TX). The following secondary antibodies: Alexa Fluor 488 or 555 donkey anti-rabbit IgG, Alexa Fluor 488 or 555 donkey anti-mouse IgG, Alexa Fluor 488 or 555 donkey anti-goat IgG and Alexa Fluor 555 conjugated phalloidin (all from Invitrogen) were used for regular immunofluorescence staining. For STED microscopy, ATTO 594 conjugated anti-mouse IgG and ATTO 647N anti-rabbit IgG were used (Rockland Immunochemicals, Limerick, PA). For western blotting, the following secondary antibodies were used: goat antirabbit IgG conjugated to horseradish peroxidase (HRP), swine anti-goat IgG conjugated to HRP (Southern Biotech, Birmingham, AL) and goat anti-mouse IgG conjugated to HRP (Bethyl Laboratories, Montgomery, TX). All reagents were from Sigma Aldrich unless stated.

shRNA lentiviral particles

For RNA silencing in keratinocytes, we used MISSION pLKO.1-puro vector-based lentiviral particles containing a puromycin resistance gene and a shRNA insert under the human U6 promoter (Sigma Aldrich). The shRNA targeting the RAB11A mRNA sequence was the same as previously used [START_REF] Reynier | Rab11a is essential for lamellar body biogenesis in the human epidermis[END_REF]. Two shRNAs that target the MYO5B mRNA sequence (NCBI reference NM_001080467) were tested (shMYO5B1 and shMYO5B2). In some experiments, only shMYO5B1 was used and referred to as shMYO5B. A shRNA that does not target any known mammalian mRNA sequence was used as a negative control (shCTRL).

Skin samples, keratinocyte populations and RHE

Human abdominal skin samples (from females undergoing plastic surgery, without any history of skin diseases) were obtained from Genoskin (Toulouse, France), following written, informed consent of the donor and as authorized by the French Ministry of Research (#AC-2017-2897). Normal human keratinocyte populations were obtained from epidermal fragments treated with trypsin-EDTA at 4°C and dissociated. For RHE, 350,000 normal human keratinocytes in Epilife medium (Invitrogen) containing 1.5 mmol/l calcium were seeded on polycarbonate culture inserts (area of 0.63 cm 2 with pores of 0.4µm in diameter, Sigma Aldrich). After 48 h of incubation at 37°C in a humidified atmosphere containing 5% CO 2 , cells were exposed to the air-liquid interface, and 50 µg/ml vitamin C (Sigma Aldrich) and 10 ng/ml keratinocyte growth factor (KGF, Sigma Aldrich) were added to the medium in the lower compartment. The medium was renewed every 2 days during the 12-day culture at the air-liquid interface (Franckart et al., 2012;[START_REF] Pendaries | Knockdown of filaggrin in a three-dimensional reconstructed human epidermis impairs keratinocyte differentiation[END_REF]. Experiments were performed with normal human keratinocytes from 5 individuals.

In order to inhibit the dynamics of actin cytoskeleton, RHE were treated for 2h with Latrunculin A at 0.5 µM versus vehicle (DMSO).

Keratinocyte transduction

Normal human keratinocytes were cultured in complete Dermalife medium (Cellsystems, Troisdorf, Germany) in a humidified atmosphere containing 5% CO2 at 37°C. Keratinocytes were transduced with lentiviral particles containing either shCTRL, shRAB11A, shMYO5B1 or shMYO5B2 at a multiplicity of infection of 5 in the presence of 6 µg/ml of protamine sulfate (Sigma Aldrich). After 24h of incubation, the culture medium was replaced with fresh medium containing 2 µg/ml of puromycin (Sigma Aldrich) for selection. After selection, when keratinocytes covered at least 70% of the flask area, they were used to produce RHE as described above.

Statistical analysis

All data are presented as mean ± standard deviation. Statistical differences were determined with student t tests. The threshold for statistical significance was set at p<0.05.

Further details are available in Supplementary Material.

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Actin-based Myo5b is highly expressed in granular keratinocytes and

Figure 3 .

 3 Figure 3. Myo5b silencing alters the epidermal barrier

Figure 4 .

 4 Figure 4. Myo5b silencing alters the structure and composition of the SC, and disrupts

Figure 5 .

 5 Figure 5. MYO5B silencing alters the expression and distribution of LB cargoes and

ACKNOWLEDGEMENTS

This research was supported by the French National Institute of Health and Medical Research (INSERM), the University of Toulouse and the French Society of Dermatology (SFD). We are indebted to C. Pons for her excellent technical assistance, her suggestions and tremendous contribution to this work. We acknowledge P. Descargues (Genoskin, Toulouse, France) for his help in obtaining skin samples, F. Capilla from the histopathology facility, D. Daviaud and A. Canivet from the imaging facility (Toulouse RIO, University of Toulouse, France). We also thank M. Masson-Regnaut, M.C. Méchin and N. Gaudenzio for their help and advices.

CONFLICT OF INTEREST

The authors declare no conflict of interest.