L Lilensten

Y Danard

R Poulain

R Guillou

J M Joubert

L Perrière

P Vermaut

D Thiaudière

F Prima

From single phase

Keywords: Titanium alloy, Microstructural design, Dual phase, Mechanical properties, TRIP-TWIP

HAL is a

Introduction

In recent years, a threshold has been overcome in β-titanium alloys with the quick emergence of a new generation of alloys displaying combined deformation mechanisms such as transformation induced plasticity (TRIP) and twinning induced plasticity (TWIP) effects.

Thanks to these two effects; unprecedented ductility and superior work-hardening are obtained together for these alloys, opening the way for a broader field of applications [START_REF] Marteleur | On the design of new βmetastable titanium alloys with improved work hardening rate thanks to simultaneous TRIP and TWIP effects[END_REF][START_REF] Sun | A new titanium alloy with a combination of high strength, high strain hardening and improved ductility[END_REF][START_REF] Sun | The Role of Stress Induced Martensite in Ductile Metastable Beta Ti-alloys Showing Combined TRIP/TWIP Effects[END_REF][START_REF] Brozek | A βtitanium alloy with extra high strain-hardening rate: Design and mechanical properties[END_REF].

As dislocation glide controls the deformation in stable β (bcc) Ti-alloys, the two alternative deformation mechanisms may be obtained by destabilizing the bcc phase in a controlled way [START_REF] Banerjee | Perspectives on Titanium Science and Technology[END_REF]. The addition of α-stabilizing alloying elements, or the decrease in the β-stabilizers concentration allows to bring the alloy to a metastable region, where its Ms temperature is located just below room temperature [START_REF] Wang | Phase stability and tensile behavior of metastable β Ti-V-Fe and Ti-V-Fe-Al alloys[END_REF][START_REF] Paradkar | Effect of Al and Nb on the trigger stress for stress-induced martensitic transformation during tensile loading in Ti-Al-Nb alloys[END_REF][START_REF] Paradkar | Various stages in stress-strain curve of Ti-Al-Nb alloys undergoing SIMT[END_REF][START_REF] Banumathy | Structure of orthorhombic martensitic phase in binary Ti-Nb alloys[END_REF][START_REF] Hida | Stress induced products and ductility due to lattice instability of β phase single crystal of Ti-Mo alloys[END_REF]. Therefore, upon quenching from the β-region, a 100% metastable β structure can be retained. Depending on its level of metastability, the later will display the TWIP mechanism, or the TRIP one, or a combination of both. Several strategies have been proposed to predict the deformation mechanisms obtained for a nominal composition. They go from very specific DFT calculations to describe the systems [START_REF] Huang | From electronic structure to phase diagrams: A bottom-up approach to understand the stability of titanium-transition metal alloys[END_REF], to more "ready to use" techniques [START_REF] Wang | Structural Stabilities of β-Ti Alloys Studied Using a New Mo Equivalent Derived from [β/(α + β)] Phase-Boundary Slopes[END_REF], such as the Bo-Md d-electron alloy design which will be described in more details later on [START_REF] Marteleur | On the design of new βmetastable titanium alloys with improved work hardening rate thanks to simultaneous TRIP and TWIP effects[END_REF][START_REF] Abdel-Hady | General approach to phase stability and elastic properties of β-type Ti-alloys using electronic parameters[END_REF]. Thanks to all the available design techniques, an increasing number of TRIP/TWIP β-metastable alloys have been proposed and studied recently [START_REF] Marteleur | On the design of new βmetastable titanium alloys with improved work hardening rate thanks to simultaneous TRIP and TWIP effects[END_REF][START_REF] Sun | A new titanium alloy with a combination of high strength, high strain hardening and improved ductility[END_REF][START_REF] Lilensten | On the heterogeneous nature of deformation in a strain-transformable beta metastable Ti-V-Cr-Al alloy[END_REF][START_REF] Castany | Design of strain-transformable titanium alloys[END_REF][START_REF] Sadeghpour | On the compressive deformation behavior of new beta titanium alloys designed by d-electron method[END_REF]. The understanding of the relationship between the mechanical properties and the deformation mechanisms is thus getting better, even though the interplay between twinning, phase transformation and dislocation glide is highly complex, and makes the hypothesis of a single framework applicable to every TRIP/TWIP alloy unlikely [START_REF] Lilensten | On the heterogeneous nature of deformation in a strain-transformable beta metastable Ti-V-Cr-Al alloy[END_REF][START_REF] Lai | On the mechanism of 332 twinning in metastable β titanium alloys[END_REF].

Balancing the numerous qualities of this family of alloys, their ductility and work hardening, but also their low density for example, the yield strength remains a potential issue for some applications, such as the aerospace related ones. Indeed, rather low yield strength values are generally obtained in most of the developed systems, often below 600 MPa [START_REF] Marteleur | On the design of new βmetastable titanium alloys with improved work hardening rate thanks to simultaneous TRIP and TWIP effects[END_REF][START_REF] Sun | A new titanium alloy with a combination of high strength, high strain hardening and improved ductility[END_REF][START_REF] Sun | The Role of Stress Induced Martensite in Ductile Metastable Beta Ti-alloys Showing Combined TRIP/TWIP Effects[END_REF][START_REF] Lilensten | On the heterogeneous nature of deformation in a strain-transformable beta metastable Ti-V-Cr-Al alloy[END_REF]. Some strategies have been suggested, aiming at increasing the yield strength. Following the Hall-Petch principle [START_REF] Hansen | Hall-Petch relation and boundary strengthening[END_REF], reducing the grain size decreases the dislocation mean free path, and therefore increases the yield strength. However, studies have shown that if grain size reduction does increase the YS in TWIP alloys, it also makes twinning more difficult, resulting in a subsequent decrease of the work hardening rate and the ductility [START_REF] Wang | Role of grain size in tensile behavior in twinninginduced plasticity β Ti-20V-2Nb-2Zr alloy[END_REF]. Similar results have been obtained for TRIP alloys, and this deformation mechanism may even be suppressed [START_REF] Sadeghpour | Effect of cold rolling and subsequent annealing on grain refinement of a beta titanium alloy showing stressinduced martensitic transformation[END_REF]. Another possible route is to use the solid solution effect to harden the material. This has been investigated in a few studies, but, up to now, without tremendous increase of the yield strength, or with a strong decrease of the work-hardening rate and/or the ductility [START_REF] Wang | Phase stability and tensile behavior of metastable β Ti-V-Fe and Ti-V-Fe-Al alloys[END_REF][START_REF] Sadeghpour | A new multi-element beta titanium alloy with a high yield strength exhibiting transformation and twinning induced plasticity effects[END_REF][START_REF] Ren | Development of a high strength and high ductility near β-Ti alloy with twinning induced plasticity effect[END_REF][START_REF] Zhang | Fabrication and characterization of a novel β metastable Ti-Mo-Zr alloy with large ductility and improved yield strength[END_REF][START_REF] Lilensten | Design and tensile properties of a bcc Ti-rich high-entropy alloy with transformation-induced plasticity[END_REF]. Finally, some studies focused on increasing the yield strength by using precipitates to reinforce the matrix. Controlled ω nano-precipitation has proven to be rather efficient and this strategy has been receiving increasing attention recently, though the relationship between precipitates, work hardening and ductility is not straightforward [START_REF] Sun | Strengthening strategy for a ductile metastable β-titanium alloy using low-temperature aging[END_REF][START_REF] Gao | ω phase strengthened 1.2GPa metastable β titanium alloy with high ductility[END_REF]. Following this precipitation strengthening strategy, we propose to design a dualphase TRIP-TWIP alloy, with a β-metastable matrix reinforced by α precipitates. Indeed, αprecipitation has been shown to help increase the yield strength in several studies [START_REF] Sadeghpour | Correlation between alpha phase morphology and tensile properties of a new beta titanium alloy[END_REF][START_REF] Ivasishin | A comparative study of the mechanical properties of high-strength β-titanium alloys[END_REF].

Deformation by these two mechanisms have been observed in dual phase systems [START_REF] Ahmed | The influence of β phase stability on deformation mode and compressive mechanical properties of Ti-10V-3Fe-3Al alloy[END_REF], and a recent study indeed showed that precipitation of 4% of α could help increase the yield strength without loss of ductility or work-hardening, thanks to the TRIP and TWIP mechanisms [START_REF] Lee | Development of sub-grained α+β Ti alloy with high yield strength showing twinning-and transformation-induced plasticity[END_REF]. Their great mechanical properties are however explained by the compositional change of the matrix, the small grain size and the large number of low-angle grain boundaries; the influence of α is rather confined to the prevention of the β grain growth, as well as the formation of a dense network of low angle grain boundaries. Other dual-phase β-metastable Ti-alloys have been claimed before, but to the authors best knowledge, they mostly concern β-metastable alloys in which α is precipitated, and where the TRIP and TWIP properties of the β-matrix are lost [START_REF] Ahmed | The influence of β phase stability on deformation mode and compressive mechanical properties of Ti-10V-3Fe-3Al alloy[END_REF][START_REF] Xue | Mechanical properties and deformation mechanisms of Ti-3Al-5Mo-4.5V alloy with varied β phase stability[END_REF]. In this work, the two phases are co-designed to bring specific properties to the alloy, i.e. the ductility and work-hardening for the β-matrix, and the yield strength from the α-precipitates. The targeted material should thus have an enhanced yield strength, which is the main goal of this study, while maintaining some ductility and a large work-hardening, thanks to the TRIP/TWIP β-matrix. This proof of concept aims at opening the way to a new generation of tunable TRIP/TWIP Ti alloys, beyond single phase materials. The design strategy, combining thermodynamical calculations using the Calphad method, and the Bo-Md alloy design tool, is first described.

The mechanical properties and deformation mechanisms of the alloy are then studied.

Materials and methods

Calphad calculations were performed using the Thermo-Calc software and the Cr-Sn-Ti database published by Gao et al [START_REF] Gao | Thermodynamic description of the Cr-Sn-Ti system[END_REF]. Buttons of 200g were prepared by arc melting, and subsequently forged and hot rolled, to obtain square bars. They were solution treated at 1173 K for 900 s (in the β field) under air, followed by water quenching. Specimen were then cold rolled to a final thickness of about 0.65 mm, corresponding to a thickness reduction around 80%. The cold-rolled specimens were heat treated under high purity Ar at 1103 K for 1800 s (heating rate of 20 K/min) and water quenched to reach a fully recrystallized β structure. The α-phase was then precipitated by a thermal treatment at 1018 K for 1800 s (heating rate of 20 K/min) under high purity Ar, followed by water quenching.

Tensile specimen with dog bone shape and gauge dimensions of 50 x 4 x 0.65 mm 3 were prepared by wire Electro Discharge Machining. Tensile tests were performed at room temperature, at a strain rate of 1.7 x 10 -3 s -1 on an Instron machine equipped with a 10 kN load cell and with an extensometer with a gauge length of 10mm. Samples for microscopy were grinded on SiC papers and finally polished with an OPS-H2O2 mixture.

Back scatter electron (BSE) imaging, to characterize the initial microstructure, was done using a Zeiss Leo1530 field emission gun scanning electron microscope (SEM) operated at 20 kV. Images were treated for quantification of the α-phase using the Image J software. Specimens for electron back-scattered diffraction (EBSD) analyses were electropolished at 278 K with a solution of 2-butoxyethanol, methanol, perchloric acid and hydrochloric acid.

EBSD analyses were performed on a Zeiss-Crossbeam XB 1540 Focus Ion Beam FIB-SEM instrument equipped with a field emission gun operated at 15 kV. The step size was comprised between 30 and 50 nm. EBSD data collection was done by an EDAX/TSL acquisition system, and the results were then analyzed using the TSL/OIM analysis package.

Energy dispersive X ray diffraction (EDX) line profiles were measured across precipitates using a Zeiss Merlin FEG-SEM operated at 20kV.

In situ SXRD (synchrotron X ray diffraction) analyses were carried out at the DiffAbs (diffraction and absorption) beamline at Soleil, the French synchrotron facility. XRD measurements were performed in reflection mode with an energy of the X-ray beam fixed to 12.475 keV (λ=0.994 Å). Using this energy combined with an incident angle of 9°, the penetration depth was approximately 20 µm. A 600 x 300 µm² x-ray beam size (HxV fwhm) at the center of the 6-circle diffractometer was used in order to obtain good statistics of grains during measurements. The diffraction patterns were recorded thanks to a bi-dimensional XPAD-S140 detector (hybrid pixel technology, 560 x 240 pixels² image size) [START_REF] Basolo | A 20kpixels CdTe photon-counting imager using XPAD chip[END_REF][START_REF] Bourlot | Synchrotron Xray diffraction experiments with a prototype hybrid pixel detector[END_REF]. The detector was mounted on the 2θ goniometer arm with a detector sample distance of 580 mm (granting an angular resolution of 0.013°). After recording, each diffraction pattern was converted to diffraction diagram (radial integration) with a typical [32°-50°] 2θ angular range. A Proxima-Micromecha tensile machine with a 3kN load cell was used for in situ tensile tests with specimen's dimensions of 50 x 2.95 x 0.49 mm and a gauge length of 20 mm.

Results

Alloy design approach

The approach proposed here is to reinforce a TRIP/TWIP matrix with hardening precipitates.

Care must be taken to consider the precipitation of the second phase in the determination of the alloy's nominal composition, and therefore anticipate the compositional change of the matrix. For this study, the precipitation of α particles inside the β-grains is proposed and will induce chemical partition of the elements. The determination of the composition was done based on the following reasoning: the microstructure prior deformation should be made of about 20% of α-precipitates, in a β-matrix, in order to keep β as the main phase that controls the deformation mechanisms, and have enough α precipitates to harden the microstructure. This content of α-precipitates is, at this point, arbitrary. It only aims at challenging a design approach and provide a proof of concept. Thus, after precipitation of 20% of α, the β matrix should have an optimum composition with adequate Bo-Md parameters for TRIP/TWIP.

Establishing the alloy's nominal composition is done by coupling two techniques: Calphad is used to calculate the composition of the α and the β phases for a given nominal composition and microstructure, and the Bo-Md technique to assess the TRIP/TWIP character of the β phase. The Bo-Md tool was first developed by Morinaga et al. for superelastic Ti alloy design [START_REF] Abdel-Hady | General approach to phase stability and elastic properties of β-type Ti-alloys using electronic parameters[END_REF][START_REF] Morinaga | Alloy Design Based on Molecular Orbital Method[END_REF]. In this method, the average bond order (Bo) and average energy of the d-orbital (Md) of an alloy's composition are calculated. They reflect the cohesion strength and the chemical stability of the alloy, respectively. These theoretical data, extrapolated from molecular orbital calculations, are coupled with a stability map based on experimental results [START_REF] Abdel-Hady | General approach to phase stability and elastic properties of β-type Ti-alloys using electronic parameters[END_REF]. That allows to draw the limits of stability for the β-phase, and then to target the instability region for the TRIP/TWIP alloy design. This method has been proven to be efficient and easy to use [START_REF] Marteleur | On the design of new βmetastable titanium alloys with improved work hardening rate thanks to simultaneous TRIP and TWIP effects[END_REF][START_REF] Sun | A new titanium alloy with a combination of high strength, high strain hardening and improved ductility[END_REF][START_REF] Lilensten | On the heterogeneous nature of deformation in a strain-transformable beta metastable Ti-V-Cr-Al alloy[END_REF][START_REF] Castany | Design of strain-transformable titanium alloys[END_REF][START_REF] Sadeghpour | On the compressive deformation behavior of new beta titanium alloys designed by d-electron method[END_REF].

According to recent results, the single phase Ti -8.5Cr -1.5Sn (wt%) alloy, designed by the Bo-Md method, displays a remarkable work-hardening (with an ultimate tensile strength above 1200 MPa), a uniform deformation of 0.36, and a yield strength of 550 MPa [START_REF] Brozek | A βtitanium alloy with extra high strain-hardening rate: Design and mechanical properties[END_REF], based on intense TRIP/TWIP combined effects. Furthermore, the thermodynamical properties of the Ti-Cr-Sn system have been published and could be used in this study for the Calphad calculations [START_REF] Gao | Thermodynamic description of the Cr-Sn-Ti system[END_REF]. The Ti-Cr-Sn system is therefore an excellent candidate for this study, and Ti-8.5Cr-1.5Sn (wt%) will be considered as the "reference alloy" in the following, in order to design a "dual-phase" grade based on this system.

The design results are illustrated in Figure 1. Considering a nominal composition Ti-7Cr-1.5Sn (wt.%) (point 1 in Figure 1a), Calphad predicts that, for a microstructure containing 20% α (points 2 in Figure 1a), the β-phase has the composition Ti-8.6Cr-1.4Sn (wt.%), very close to the targeted composition of the reference alloy. As for the α precipitates, they would have the composition Ti-0.5Cr-2.1Sn (wt.%): with no surprise, Cr, which is a β stabilizer, is rejected from the precipitates to the matrix. One can note that the C15 phase is predicted by the calculation, however it has not been observed experimentally. Constrained calculations omitting C15 do not show any difference for the bcc and the hcp phases compositions and temperatures, in the α+β domain. The stability and expected deformation mechanisms following the Bo-Md design are illustrated in Figure 1b. The alloy with nominal composition Ti-7Cr-1.5Sn (wt.%) (point 1 in Figure 1b) sits in the blue area, that deforms preferably by formation of stress induced martensite (TRIP) if the alloy is loaded in its fully β microstructural state. The Bo and Md parameters of the β-phase after precipitation of α are indicated by a light blue dot (see also point 2 in Figure 1b). One can see that it is very close to the targeted Ti-8.5Cr-1.5Sn (wt.%) reference alloy, in dark blue in Figure 1b. 1) Ti-7Cr-1.5Sn (wt.%) and its β-phase (2) after precipitation of 20% of α, compared to the reference alloy Ti-8.5Cr-1.5Sn (wt%) [START_REF] Brozek | A βtitanium alloy with extra high strain-hardening rate: Design and mechanical properties[END_REF].

Based on these design results, the dual phase Ti-7Cr-1.5Sn (wt.%) alloy involving a α volume fraction of 20% is expected to have the desired properties. The alloy is thus prepared and studied in the following.

Initial microstructure

The alloy with nominal composition Ti-7Cr-1.5Sn (wt.%) is prepared, based on the design results. After thermomechanical treatments, a recrystallized β microstructure is obtained by a heat treatment in the high temperature domain (point (1) in Figure 1a), and the α phase is precipitated in the β grains by a second thermal treatment in the α domain (points (2) in Figure 1a). The adequate experimental temperatures to obtain the desired microstructure were slightly off compared to the Calphad predictions, thus were used after experimental optimization as they allow to reach the desired microstructure. The latter is represented in Figure 2. On this scanning electron microscopy (SEM) image taken with BSE contrast, the α-precipitates appear dark, which indicates that they are depleted in heavy elements. As Sn is expected to partition only slightly between the two phases, and Cr is expected to be massively rejected towards the β-matrix, the observed contrast is consistent with the expected Cr trend. Qualitative EDX analyses given in supplementary material confirm the depletion of Cr and enrichment of Ti at the α precipitates.

One can see that α is precipitating mostly as platelets inside the grains, with a homogeneous repartition. This phase is also found at the grain boundaries of the β grains, which is a classical observation in β-Ti alloys, where it forms αGB (elongated precipitate between the two grains) and αWGB, characterized by precipitates aligned rather perpendicular to the grain boundary.

An average amount of 20% of α-phase is quantified by image analyses, by averaging the amount calculated for several images. This value is in perfect agreement with the design strategy.

Mechanical properties

The true stresstrue strain tensile curve and the work-hardening rate curve of the new alloy, given in Figure 3, are compared with the reference alloy Ti-8.5Cr-1.5Sn (wt.%) (100% β TRIP/TWIP alloy) [START_REF] Brozek | A βtitanium alloy with extra high strain-hardening rate: Design and mechanical properties[END_REF]. The dual phase alloy displays an elastic modulus of 95 GPa, a very high yield strength of MPa, as well as a uniform elongation strain of 0.29 and an ultimate tensile strength of MPa. In spite of a small loss of ductility and work-hardening, compared to the reference alloy, an increase of the yield strength larger than 200 MPa is obtained. This result itself validates the strategy consisting in precipitating a secondary phase to reinforce the matrix.

The dual-phase alloy displays a marked increase of the work-hardening rate around 0.15 strain, following a noticeable stress-plateau, that is commonly observed in TRIP alloys [START_REF] Castany | In situ synchrotron Xray diffraction study of the martensitic transformation in superelastic Ti-24Nb-0.5N and Ti-24Nb-0.5O alloys[END_REF],

suggesting the occurrence of this deformation mechanism in the new alloy. Comparing the intensity of the hump in the work-hardening rate curves, it can be suggested that the TRIP effect is stronger in the dual-phase alloy (bigger hump) than in the reference alloy.

Deformation mechanisms

Strain induced martensite formation was characterized by in-situ synchrotron X ray diffraction. The intensity in the X ray diffractograms corresponds to the integration of the intensities measured on the whole length of the XPAD-S140. Figure 4 shows the evolution of the diffraction patterns as the strain increases, from the initial state up to 16%. At 16%, the sample was unloaded and a diffractogram was recorded in the unloaded condition.

Diffractograms recorded at larger deformation levels are not displayed here, as they do not bring additional information, and furthermore become less defined due to the very large strains accommodated in the microstructure. The diffractogram at 0% strain shows the presence of the  phase and the  phase only, with lattice parameters a=2.933 Å and c=4.669 Å for  and a=3.231 Å for . Due to the very large grain size of the β grains (>100 μm) in the initial state compared to the beam size, artifacts are observed on the 0% strain diffractogram, taking the shape of double peaks for the plan (002) at ~36°. This effect disappears as soon as the deformation starts, with subdivision of the grains into smaller domains linked to the deformation features (see supplementary materials for more details). At 4% strain, peaks that can be attributed to the stress induced martensite are already seen, such as the ones corresponding to the (112) and the (022) planes. This confirms that the TRIP mechanism is effective in this alloy, as was suggested already by the shape of the tensile and work-hardening rate curves. As the deformation increases, the intensity of the '' phase increases as well, such as the (130) one. However, the larger strains accumulated in the structure broaden the peaks that become less resolved. When unloading, some of the martensite seems to reverse back, as would indicate the decrease in intensity of its (130) peak, and a new peak corresponding to the (200)'' reflection appears.

One can also note the appearance of a peak around 41.5°. This does not match any of the possible reflections for the considered , '' and  phases, but it is consistent with the (002) reflection of the  phase, considering that ω originates from β, and fulfills the following relationships: 𝑎(𝜔) = √2𝑎(𝛽) and 𝑐(𝜔) = √3 2 𝑎(𝛽) [START_REF] Lai | Origin of shear induced β to ω transition in Ti-Nb-based alloys[END_REF][START_REF] Silcock | An X-ray examination of the to phase in TiV, TiMo and TiCr alloys[END_REF]. Investigations of the deformation mechanisms was further completed using the EBSD technique ("post-mortem" observations). Due to the proximity in the diffraction patterns of the α hcp phase and the α'' martensite (orthorhombic), the indexing of the patterns is done only with the α-phase and the β-phase. Therefore, identification of the martensite with this technique is not possible.

Orientation profile along the black line labelled 2 in (b). The point to point misorientation is plotted in red and the point to origin misorientation is plotted in blue. (e) Zoom into the zone 1 inset of (b). (f) Zoom into the zone 2 inset of (b).

EBSD analyses of a 5% deformed microstructure after unloading are displayed in Figure 5 (tensile direction indicated by the white double arrow in Figure 5a). The phase map in Figure 5a confirms a surface fraction of α phase of 20%. The index quality (IQ), over imposed on the phase map, reveals the presence of deformation bands in the grains (lower IQ). The orientation map is given in Figure 5b. The misorientation profiles (indicated by ( 1) and [START_REF] Sun | A new titanium alloy with a combination of high strength, high strain hardening and improved ductility[END_REF] in Figure 5b), across two deformation bands (see arrows) are measured and plotted in Figures 5c and5d respectively. The profile 1 (Figure 5c) highlights a misorientation of 60° corresponding to a β {112}<111> twin. This twin is observed at the interface between αGB and the β grain. Orientation analyses indicate that the BOR is not fulfilled between αGB and the matrix in this case, nor it is between the twin and αGB. The IQ shows that this area is not so well indexed, which can be explained by the local strain. The second deformation profile (Figure 5d) highlights a misorientation of 51°, and this deformation band was identified as a {332}<113> β twin. Several {332}<113> twins with the same orientation are observed in grain 2. Other deformation bands identified as {332}<113> twins are found, such as in the red neighboring grain (blue orientation in Figure 5b). Therefore, it is confirmed that the βmatrix of this dual-phase alloy clearly displays TWIP properties additionally to the TRIP effect observed on SXRD patterns.

Figures 5e and5f are magnified views of the insets labeled Zone 1 and Zone 2 in Figure 5b.

They display the interaction of a twin with α-precipitates of 7 different orientations (P1 to P7) corresponding to different variants. All the precipitates are sheared by the twin: the precipitate Pi transforms to a precipitate Pi' on the sheared zone. The general behavior observed here relates to a simple shearing of the precipitates by the mechanical twins, upon hitting them. It is worth noting that the twins trajectory remains quite straight even after successive interactions with α-precipitates, without visible deviation. The fact that the twins do not seem disturbed by the precipitates, and that this deformation mechanism is not hindered by the high density of precipitates, is remarkable.

Discussion

The alloy Ti -7 Cr -1.5 Sn (wt. %) was designed by coupling the electronic Bo-Md approach and the Calphad method, in order to obtain a dual-phase TRIP/TWIP alloy. Such alloy would benefit from the ductility and the work hardening of the β-matrix, when the precipitates could help increasing the stress, and especially the yield strength. It can be noted that the role of the precipitates is most likely more complex than a simple increase of the alloy's strength, especially considering the alloy's plasticity and the interplay of the various deformation mechanisms of the targeted alloy. After alloy preparation and processing, a microstructure made of 20% of α precipitates in the  matrix is obtained. The precipitates are found at the grain boundaries and inside the grains. They always fulfil the BOR with the matrix in the case of intragranular α, and the grain boundary α always fulfills the BOR with at least one of the two neighboring  grains. The mechanical properties obtained for the alloy reveal that from a mechanical point of view, the design strategy was successful, since a very good elongation and a good work-hardening are obtained, along with an increase in yield strength larger than 200 MPa. Study of the deformation mechanisms by in situ SXRD and postmortem EBSD show that the deformation mechanisms of the dual phase alloy are in good agreement with the Bo-Md predictions. Orthorhombic martensite is forming from the early stages of the deformation. {332}<113> twins are observed in the grains, with several twinning variants activated at a strain of 0.05, and {112}<111> twinning was also observed, indicating that the two twinning modes contribute to the alloy's plasticity. Formation of  phase at large strains observed with SXRD (Figure 4) could be obtained by stress induced transformation, alongside with the formation of {332}<113> twins, as was already evidenced in several works [START_REF] Mantri | Deformation Induced Hierarchical Twinning Coupled with Omega Transformation in a Metastable β-Ti Alloy[END_REF][START_REF] Hanada | Transmission electron microscopic observations of mechanical twinning in metastable beta titanium alloys[END_REF][START_REF] Sun | Investigation of early stage deformation mechanisms in a metastable β titanium alloy showing combined twinning-induced plasticity and transformation-induced plasticity effects[END_REF]. It could also be correlated to the formation of the {112}<111> twins, where  has already been observed to form at the interface between such twins and the matrix [START_REF] Lai | Origin of shear induced β to ω transition in Ti-Nb-based alloys[END_REF][START_REF] Xing | Mechanical twinning and omega transition by ⟨111⟩ 112 shear in a metastable β titanium alloy[END_REF][START_REF] Yang | Stress release-induced interfacial twin boundary ω phase formation in a β type Ti-based single crystal displaying stress-induced α" martensitic transformation[END_REF][START_REF] Wu | 112〈 111〉 Twinning during ω to body-centered cubic transition[END_REF][START_REF] Chen | Omega transitional structure associated with 112<111> deformation twinning in a metastable beta Ti-Nb alloy, revealed by atomic resolution high-angle annular dark-field scanning transmission electron microscopy[END_REF]. A more detailed TEM study would be required to identify clearly the nature of such  phase in the present alloy. Finally, upon unloading at 16% strain, a new reflection corresponding to (200)α'' appears on the XRD pattern. Taking into account the fact that, under load, this reflection was not detected, an hypothesis that could be suggested is that some martensite also forms in the alloy during unloading, in order to accommodate internal strain incompatibilities.

The impact of α precipitates on the mechanical properties is complex. From the alloy design, the  matrix has the same composition in the reference and in the dual-phase alloy. Therefore, it is suggested that the differences regarding the plastic deformation (Figure 3) can be attributed to the  precipitates. They are discussed in the following, starting first with the interaction between the precipitates and the {332}<113> twins, and then, in a second time, with proposed explanations for the mechanical behavior.

Interaction between the precipitates and the {332}<113> deformation twins i. Background on the possible interaction and theoretical study

When a twin intersects a precipitate Pi, 3 theoretical situations may arise.

In the first case, there is a direct gliding system available for Pi (aligned with the incident (332) β twinning plane, or close to it) and the precipitate deforms by dislocation glide.

Dislocation storage in the precipitate may lead to strong strain fields, that can produce orientation gradients in between the sheared area and the rest of the precipitate. In the second case, the precipitate has a twinning system that matches the {332} twinning plane: on a stereographic projection, there should be a {332} plane close to a K1 hcp plane. That would imply that the {332} mirror plane of the matrix and the twin is parallel to a mirror plane for Pi and Pi'. In the third case, the precipitate has no direct available system to transfer the shear carried by the twin into the precipitate, and reacts as an un-deformable object. This would lead to the formation of a strong strain field at the precipitate boundary corresponding to the backstress accommodated by the matrix.

The study of all the possible interactions between the β-twins and the α-precipitates can be reduced to that of one precipitate (fulfilling the BOR with the matrix) with the 12 possible {332}<113> twinning systems. Following the stereographic projections (see in supplementary materials), only one {332} bcc twinning plane matches perfectly with a hcp {11-20} plane, but such plane is not related to a dislocation glide system in hcp structures.

No hcp twinning plane over imposes perfectly with a {332} bcc plane; in the best case, some, such as a {-1012} plane, are not so far away from a {332} plane. Therefore, the bcc twinning shear is supposed to be accommodated in the precipitate by combination of elasticity, to bring a hcp plane in a matching position, and subsequent glide or twinning.

ii. Discussion on the experimental results

Deeper analysis of the experimental results on the interaction between twins and precipitates is performed. The area framed in Figure 5e is taken as an example, as it displays cases representative to what is observed for the other precipitates. More EBSD analyses are performed (11 twin/precipitates interactions studied, not displayed here) to confirm the occurrence of the effects described hereafter.

The orientation map of the considered area is given in Figure 6a. For a better color contrast, the orientation map is given along the tensile direction instead of the normal direction. Orientation changes are visible in the precipitates, confirming the shearing effect of the twin.

As shown on the misorientation profiles plotted in Figures 6c to 6e, the misorientation angle (between Pi and Pi') is rather small (5°) in the case of P5-P5', and larger for the other cases (10° and 15° for P6-P6' and P4-P4' respectively). It is also observed that the orientation change is very sharp for P4 and P5, but that there is an orientation gradient for P6. This was always the case: when the habit plane of the precipitate is perpendicular to the twin plane, there is a sharp orientation change, whereas when the habit plane of the precipitate is rather parallel (or close to) the twin plane, the orientation changes gradually. It is proposed that the abrupt interfaces are found when the incident shear is very localized, confined to a narrow area at the twin/precipitate interface.

To understand further the shearing behavior of the precipitates, stereographic projections for the twin/matrix and the precipitates are given in Figures 6b, 6f, 6g and6h; with the sample virtually rotated to bring the 110 pole common to the twin and the matrix to the center of the stereographic projection, and to bring the (332) twinning plane at the equator of the projection, i.e. with its pole at the north and south poles.

They first allow to confirm that all the precipitates P4, P5 and P6 are in BOR with the matrix, but regarding their sheared variants, only P6' fulfills the BOR with the twin, when P4' is close to the BOR with the twin and P5' has no particular orientation relationship with the twin (for more details see Figure S4 in the supplementary materials). But they also allow to see a new relationship between P4 and P4': the mirror plane that transforms the matrix into the twin also applies to P4, that is transformed into P4' (Figure 6f). However, regarding P5 and P6, no such relationship is found (Figure 6g and6h). It can be noted that P6 and P6' are closer to a mirror relationship, that could be reached by a small rotation around (0001), when it would require a very large rotation of P5' to be put in a mirror relationship with P5.

A mirror relationship between Pi and Pi' is observed in 3 cases out of 11 studied ones (see table 1), for various orientations of the precipitate with respect to the twin -with its (0001) plane parallel to the (110) plane common to the twin and the matrix or not. However, if this mirror plane characterizes a twinning relationship, it does not correspond to the classical hcp twins. The twinning plane could not be identified within the possible low Miller indices planes. Analysis of the orientation of the other planes relevant for twinning or dislocation glide in hcp structures, i.e. {10-11}, {11-22}, {10-10}, {10-12}, {11-21}, {11-22} and{11-24}, does not show any match between these planes in P4, P5 and P6, and the (332) bcc twinning plane.

Over 11 twin/precipitates interactions studied, all the precipitates are sheared when hit by a twin, and the additional orientation relationships detailed here happen to be representative of what happens which is summarized in table 1 Based on the knowledge on deformation of hcp structures and the present results, the way the precipitates exactly deform is, at the moment, unclear, and more work will be dedicated to unveil the detailed mechanisms. Yet, the fact that they are always sheared indicates a local rotation of the lattice, therefore the activation of several gliding systems, and also indicates that the precipitates are participating to the alloy's plasticity. Special orientation relationships are found after shearing of the precipitate (mirror relationship or BOR between Pi' and the twin) in more than half of the studied cases. Regarding the BOR relationship obtained between the twin and the sheared precipitate, it is obtained in 6 cases out of 11, suggesting that accommodation of the shear in the precipitate may be done with the additional objective of reaching BOR with the twin, which would decrease the interfacial energy between the twin and the sheared precipitate. The fact that the bcc mirror plane corresponds to a hcp mirror plane in 3 cases out of 11 may also suggest the activation of a high index twinning system that could not be identified. This may be favored by the very specific configuration observed here, with high amounts of shear carried by a twin that are localized only on a small part of the precipitate.

The measured misorientations, ranging from 5° to 18° are probably partly correlated to the shearing magnitude. However, P1, P2, P3 and P6 on the one hand and P4 and P5 on the other hand are sheared by the same twin (see Figure 5), but the misorientation measured in these precipitates is not the same. So the shearing provided by the twin does not only depend on the twin and the level of strain, but it is suggested that it may be normalized by the volume of the precipitate to shear, which would explain the different measured values. One can note that this would not apply for the "twinned precipitates" if the hypothesis of a high index twinning system was validated.

Shearing or engulfment of precipitates by/into deformation twins have already been observed in other structures, such as the transmission of twins between the γ matrix (fcc) and γ' precipitates (L12) in superalloys, or between the Mg matrix (hcp) and MnZn' precipitates [START_REF] Stanford | Effect of particles on the formation of deformation twins in a magnesium-based alloy[END_REF][START_REF] Clark | Transmission electron microscopy study of age hardening in a Mg-5 wt.% Zn alloy[END_REF]. However, in these cases, twin growth is carried by the glide of individual partial dislocations at the twin/matrix interface, each partial bringing the shear to twin the next atomic layer. If the transmission mechanism across precipitates is not well understood in Mg alloys, the coherent γ/γ' interface in superalloys allows an easier transmission of the dislocations from one side to the other. {332} twinning in bcc alloys is more complex, as the twinning mechanism itself is still debated [START_REF] Lai | On the mechanism of 332 twinning in metastable β titanium alloys[END_REF][START_REF] Castany | Reversion of a Parent {130}⟨310⟩α" Martensitic Twinning System at the Origin of {332}<113>β Twins Observed in Metastable β Titanium Alloys[END_REF][START_REF] Tobe | Origin of 332 twinning in metastable β-Ti alloys[END_REF]. Therefore, it is too early to better and fully understand the mechanism by which it shears the precipitate.

Understanding the impact of the precipitates on the mechanical properties

The impact of the precipitates on the yield strength, the overall tensile curve, the alloy's strength and work-hardening is now discussed.

A first approach to the yield strength increase could be that the α phase has a higher yield strength compared to metastable β and thus contributes, following a kind of law of mixing, to a total increase of this parameter. However, a general approach for the macro-yield strength is to consider that it corresponds to the moment when the majority of grains are plastically deforming [START_REF] Brandstetter | From Micro-to Macroplasticity[END_REF], and as α are only precipitates and represent as low as 20% of the whole volume, so the behavior of the matrix should still be considered. The general approach presented above is usually applied to materials with dislocations assisted plasticity, where the elastic-to-plastic transition considers the transfer of glide from one grain to its neighbor [START_REF] Saada | Hall-Petch revisited[END_REF]. Transferring this notion to TRIP/TWIP alloys suggests that the macro-yield is characterized as the moment when the majority of grains are subjected to plasticity, possibly including TRIP and TWIP, with a transfer of plasticity from a grain to another. Based on this principle, it is suggested that triggering and propagating the twins and/or the martensite is harder in the case of the dual-phase alloy. The analysis of the plasticity in a 5% strained specimen of the dual phase alloy suggests indeed that twinning is possibly not as massive as in the reference alloy in the first stages of the deformation, by comparing microscopy images of Brozek et al. at 3% strain [START_REF] Brozek | A βtitanium alloy with extra high strain-hardening rate: Design and mechanical properties[END_REF] and the amount of twins found in this alloy by EBSD, at 5% strain.

As the two considered defects are considered to initiate at the grain boundaries (or twin boundaries for the martensite), and then propagate in the grain's interior, it is possible that the nucleation is hardened for the dual-phase alloy. Ribbons of α phase are precipitated at the former / grain boundaries, which may modify the local stress required to trigger the nucleation of a twin or of martensite. Furthermore, the twins are shown to interact with the precipitates, so the latter can be seen as effective obstacles that hinder the twins' propagation.

Finally, transmission of the plasticity from one grain to another may be modified due to the precipitation of αGB. This transmission is easy in the case of single-phase TRIP/TWIP Tialloys, where twins easily trigger another twinning event in the neighboring grain by creating a stress field at the vicinity of the grain boundary [START_REF] Lin | Transmission of {332}〈113〉 twins across grain boundaries in a metastable β-titanium alloy[END_REF], but the presence of the ribbon of α at the grain boundaries with a larger modulus may decrease the stress transmission to the next grain.

Overall, the precipitates are expected to contribute in several ways to the increase of the yield strength, by modifying the glide of dislocations or the TRIP and TWIP mechanisms. At this point, it is unfortunately not possible to know if a single mechanism is occurring during the pre-macro-yield or if all three are active, yet.

Regarding the ductility of the alloy, it is reasonable to consider that it is brought by the combined TRIP and TWIP effects, as in other TRIP/TWIP Ti-alloys. Besides, this study shows that  precipitates display noticeable plasticity, and so are not un-shearable particles.

This can be an additional reason for the good ductility of the alloy (no local high stress concentration leading to failure at the precipitate/matrix interface), and may also explain why the hardening is not higher.

Finally, the work-hardening of the alloy, although very good, differs from that of the reference alloy. The plastic curve of the dual phase alloy shows two very distinct stages, the first one going up to about 10% strain, and the second one from 10% on. In contrast, the reference alloy has a more monotonous tensile evolution. The plateau in the first stage of the dual phase alloy tensile curve is classically attributed to the martensitic transformation [START_REF] Castany | In situ synchrotron Xray diffraction study of the martensitic transformation in superelastic Ti-24Nb-0.5N and Ti-24Nb-0.5O alloys[END_REF].

This translates into a very marked hump in the work-hardening rate curve of the dual-phase alloy. It can be noticed that the martensitic plateau is found at 800 MPa, which is rather surprising, since strain-induced martensitic transformation is usually observed at much lower stress below 600 MPa, a value that has long been considered to be one of the limitations in the yield strength.

This suggests either that, for the dual phase alloy, the twinning is not so significant in the first stage, and does not shield the martensitic plateau on the curve or that the martensitic effect is stronger. The former can be related to the weaker tendency for twinning for the dualphase alloy discussed above. Regarding the latter, recent results showed that martensite may be found as a primary deformation mechanism, taking the shape of needles, or as secondary deformation mechanism where it is rather found at highly strained locations, such as twin/twin or twin/matrix interfaces and intersections, or at grain boundaries [START_REF] Lilensten | On the heterogeneous nature of deformation in a strain-transformable beta metastable Ti-V-Cr-Al alloy[END_REF]. The increased number of interfaces in this alloy compared to the reference one, considering all the / matrix interfaces and the /twin, could explain that the amount of martensite formed as a secondary deformation mechanism is large.

Classical theories for understanding the TRIP/TWIP alloys work hardening are the dynamic Hall-Petch effect and the composite effect [START_REF] Gutierrez-Urrutia | Dislocation and twin substructure evolution during strain hardening of an Fe-22wt.% Mn-0.6wt.% C TWIP steel observed by electron channeling contrast imaging[END_REF][START_REF] Bouaziz | Modelling of TWIP effect on work-hardening[END_REF]. These two effects, the reduction of the dislocation mean free path by formation of interfaces and the necessity of a co-deformation of various domains with different properties, also apply here since both TRIP and TWIP have been confirmed in the alloy. The composite effect should actually be even larger, due to the precipitation of α, possibly inducing an additional mechanical contrast between phases. The slightly weaker work-hardening may be explained by a decrease capability for twinning in the dual-phase alloy, but, as discussed in the section 4.2, a more thorough understanding of the mechanism by which the twins are interacting with the precipitates is needed to proceed further. Further work consisting in decreasing the coherency of the interfaces between the precipitates and the matrix will also be done, to make it more difficult for the twins to cross the precipitates and increase the hardening. A possibility could be to form spheroidal precipitates for instance, as suggested Lee et al., although the interaction twin/martensite/precipitates in this case still has to be investigated [START_REF] Lee | Development of sub-grained α+β Ti alloy with high yield strength showing twinning-and transformation-induced plasticity[END_REF].

Conclusion

An approach based on precipitation strengthening is proposed in TRIP/TWIP Ti-alloys to increase the yield strength. An alloy composition is designed based on Calphad thermodynamical predictions and Bo-Md alloy design, to target a dual phase TRIP/TWIP alloy: the  matrix displays TRIP/TWIP properties, to maintain excellent ductility and workhardening rate, while the precipitates in the matrix bring additional yield strength. The Ti -7 Cr -8.5 Sn (wt.%) composition is proposed. The alloy was prepared and the design strategy is successful: the dual phase alloy containing 20% of α precipitates displays both TRIP and TWIP mechanisms, ensuring a uniform elongation of 0.29 and a good work-hardening rate, while its yield strength is 760 MPa, about 200 MPa above that of the reference alloy that has a full β microstructure and the same matrix composition. It is suggested that α slows down the propagation of the twins, hence the transfer of plasticity from a grain to another, resulting in an increased yield strength. The impact of the intragranular α precipitates on the deformation mechanisms is complex. Based on the tensile behavior, it seems that the TRIP effect is stronger in the first stage for this dual phase alloy than in the reference one, or that the TWIP effect is lower for the dual phase alloy. However, the presence of the precipitates

Figure 1 :

 1 Figure 1: (a) Calphad predictions for the designed nominal composition (1) Ti-7Cr-1.5Sn (wt.%): amount of the predicted phases (bcc-β, hcp-α and C15) as a function of the temperature. The composition of the bcc and hcp phases for a microstructure[START_REF] Sun | A new titanium alloy with a combination of high strength, high strain hardening and improved ductility[END_REF]

Figure 2 :

 2 Figure 2: BSE image of the microstructure of the alloy prior deformation. The α precipitates appear in dark in the β matrix.

Figure 3 :

 3 Figure 3: True stress σ -true strain ε tensile curves (solid lines) and work-hardening rate θ (dashed lines) of the newly designed dual-phase alloy (in red) and the reference 100% β alloy (in grey) [4].

Figure 4 :

 4 Figure 4: synchrotron XRD patterns of the alloy between 0% and 16% strain, and after unloading at 16%. The diffraction reflection positions are labeled in red for the  phase, in green for the  phase, in blue for the '' martensite and in purple for . Note that the vertical bar indicating the position of the (03-30) ω reflection hides that of the (01-13) reflection of α.

Figure 5 :

 5 Figure 5: EBSD analyses of a sample after 5% strain and unloading. (a) phase map over imposed with the quality index. α is in red and β in green. The tensile direction is indicated by the white double arrow. (b) Orientation map in the direction normal to the sample surface. (c) Orientation profile along the black line labelled 1 in (b). The point to point misorientation is plotted in red and the point to origin misorientation is plotted in blue. (d)

Figure 6 :

 6 Figure 6: (a) EBSD orientation map along the tensile direction of the framed zone of Figure 5e. (b) Stereographic projection of the matrix (lilac) and the twin (beige) for the 111, 110 and 332 poles, the sample being virtually rotated to have the mirror plane of the twin along the equator. The (110) plane common to the matrix and the twin is indicated by a black circle. (c), (d) and (e): Misorientation profiles across P4-P4', P5-P5' and P6-P6' respectively. The point to point misorientations are plotted in red and the point to origin misorientations are plotted in blue. (f), (g) and (h): Stereographic projections showing the (0001) pole and the {11-20} poles of P4 and P4', P5 and P5', and P6 and P6', respectively.

Table 1 :

 1 . Summary of the different possible shearing scenarii for a precipitate hit by a twin, depending on its orientation.

	Pi' fulfills BOR	Mirror plane	Misorientation between	Occurrence from the
	with the twin		Pi and Pi'	11 α precipitates
				studied
	No	No	5 to 10°	5
	Yes	No	10 to 13°	3
	Yes	Yes	13 to 18°	3

Acknowledgements

The authors declare no competing interests for this work.

The French national research foundation is acknowledged for the funding through the ANR Titwip. M. Garcia is acknowledged for his help with the Image J software.

The alloy design was developed by L.L and F.P., with the help of J-M. J. for the Calphad part. Sample preparation and mechanical testing was done by L.L., Y. D. and L.P. Synchrotron measurements were performed by R. P. under the supervision of R. S. C. Mocuta is acknowledged for his help about synchrotron data treatments. Analysis of the synchrotron data was done by R. P, F. P, L.L. and R. G. The EBSD analyses was done by L.L, and interpretation of the mechanical and microstructural data was done by L. L., with the help of R.P, Y. D, P.V. and F. P. Manuscript drafting, including the figures, was done by L.L.

does not hinder the twinning and martensitic transformation. Detailed investigation of the orientation relationships between the matrix, the twins and the precipitates shows a wide range of behaviors, yet with the main common point that all the precipitates standing in the path of a deformation twin are sheared. The exact mechanisms by which the deformation twins and martensite interacts with the precipitates remain unclear, and more work will be dedicated in understanding that aspect, as well as evaluating the exact contribution of the α precipitates to the mechanical behavior. Overall, these new results open the way to a new branch of phase-to-phase alloy development, where each phase can be tuned individually to bring specific properties to the alloy as a whole.


Sebastien Houy

Gaël Nicolas

Fanny Momboisse

Magali Malacombe

Marie-France Bader

Nicolas ; Vitale

Marie-Christine Lecomte

Stéphane Ory

Stephane Gasman
email: sgasman@unistra.fr

; Inci

Sébastien Houy

Stéphane Gasman

Stéphane Gasman

αII-spectrin controls calcium-regulated exocytosis in neuroendocrine chromaffin cells through N-WASP interaction

Keywords: vesicular exocytosis, PC12 cells, chromaffin cells, N-WASP, neuroendocrine secretion Spectrin, exocytosis, PC12 and chromaffin cells, N-WASP

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

Neuroendocrine cells release hormones and neuropeptides through calcium-regulated exocytosis, a process that is coordinated by a fine remodelling of the actin cytoskeleton. We describe here that calcium-regulated exocytosis is impaired in chromaffin and PC12 cells with reduced II-spectrin expression levels. Using yeast two-hydrid screening, we show that N-WASP is a partner of the II-spectrin SH3 domain and demonstrate that secretagogue-evoked N-WASP recruitment at cell periphery is blocked in absence of II-spectrin. Additionally, experiments performed with ectopically expressed II-spectrin mutant unable to bind N-WASP indicated that the interaction between SH3 domain and N-WASP is pivotal for neuroendocrine secretion. Our results extend the list of spectrin interactors and strengthen the idea that IIspectrin is an important scaffold protein that gathers crucial actin related players of the exocytic machinery.

Abbreviations

Introduction

Most secretory cells display a dense network of filamentous actin beneath their plasma membrane. In neuroendocrine cells, actin dynamics play an important and composite function in calcium-regulated exocytosis in order to achieve secretory vesicle recruitment, docking and fusion with the plasma membrane. At first, cell stimulation with a secretagogue leads to a loss of cortical actin filaments. Thus, for a long time actin filament network was uniquely considered to be a physical barrier preventing the reserve pool of secretory vesicles to gain access to the plasma membrane. However today this view has evolved towards a dynamic role of actin in which a complex process of actin filament remodelling is critically required for efficient exocytosis [START_REF] Gasman | Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASPinduced actin filaments[END_REF][START_REF] Malacombe | Exocytosis in neuroendocrine cells: new tasks for actin[END_REF][START_REF] Gabel | Annexin A2dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis[END_REF][START_REF] Li | Actin Remodeling in Regulated Exocytosis: Toward a Mesoscopic View[END_REF]. Particularly, we have shown that N-WASP is recruited at exocytic sites where its ability to promote actin filament assembly is necessary for exocytosis [START_REF] Gasman | Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASPinduced actin filaments[END_REF].

Actin filaments are thought to be physically linked to spectrin family proteins, which represent another important element of the membrane skeleton network in neuronal cells [START_REF] Unsain | The Actin/Spectrin Membrane-Associated Periodic Skeleton in Neurons[END_REF].

Spectrins are long stretchable proteins composed of intertwined  and  subunits to form  heterodimers. Spectrin exists as elongated tetramers resulting from self-association of heterodimers. These tetramers form filaments that have been involved in various processes including membrane stabilisation, membrane protein-sorting, vesicular trafficking and neurite outgrowth. Two and five genes encode  and  chains, respectively. The involvement of IIspectrin (previously named fodrin) in calcium-regulated exocytosis has been previously suggested both in neurons and neuroendocrine cells [START_REF] Perrin | Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells[END_REF][START_REF] Featherstone | Drosophila alphaand beta-spectrin mutations disrupt presynaptic neurotransmitter release[END_REF]. However, besides these studies, no further evidence has been provided and the underlying molecular mechanism by which IIspectrin may control exocytotis remains unknown.

The present study was undertaken to investigate the role of II-spectrin in calciumregulated exocytosis. Using neuroendocrine PC12 and chromaffin cells as a secretory cell models, we describe here the interaction between II-spectrin and N-WASP and propose that this interaction is pivotal for the exocytotic process.

Material and methods

DNA constructs, cell culture and transfection

Plasmids encoding Flag-8-11, Flag-8-11 P1017L , N-WASP-EGFP, and the vectors pGHsuper and pEGFP-RNAi were described previously [START_REF] Gasman | Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASPinduced actin filaments[END_REF][START_REF] Rotter | AlphaIIspectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts[END_REF][START_REF] Malacombe | Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42[END_REF][START_REF] Zeniou-Meyer | Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage[END_REF]. DNA fragment encoding the unrelated siRNA sequence (ATTCTATCACTAGCGTGAC) or the siRNA sequence of rat/bovine IIspectrin (CCAAGCTTATTCAGAACAA /CCAAGCTGATTCAGAACAA) separated from it reverse complement by a short spacer were annealed and cloned in the BglII and HindIII sites in front of the H1 promoter of either the pGHsuper vector or the pEGFP-N2-RNAi [START_REF] Malacombe | Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42[END_REF][START_REF] Momboisse | betaPIXactivated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells[END_REF] Chromaffin cells were isolated from fresh bovine adrenal glands by retrograde perfusion with collagenase, purified on self-generating Percoll gradients and maintained in culture as previously described [START_REF] Bader | Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture[END_REF]. PC12 cells culture and transfection conditions were as described [START_REF] Gasman | Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASPinduced actin filaments[END_REF][START_REF] Momboisse | betaPIXactivated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells[END_REF].

Antibodies and immunofluorescence

The following antibodies were used: rabbit polyclonal anti-II-spectrin antibodies (6); mouse monoclonal anti-flag antibodies (Sigma); rabbit polyclonal anti-N-WASP antibodies (Tebu Santa Cruz); mouse monoclonal anti-actin antibodies (Sigma) and rabbit polyclonal anti-GH antibodies (kindly provided by Dr. A. F. Parlow, NIDDK, NIH, Bethesda, MD). Alexa-labelled secondary antibodies were obtained from Molecular Probes. Blots were processed using the Super Signal detection system (Pierce). For immunocytochemistry, chromaffin cells grown on fibronectine-or PC12 cells grown on poly D-lysine-coated glass coverslips were fixed and immunostained as described previously [START_REF] Gasman | Identification of a potential effector pathway for the trimeric Go protein associated with secretory granules. Go stimulates a granule-bound phosphatidylinositol 4-kinase by activating RhoA in chromaffin cells[END_REF]. Stained cells were visualized using a confocal microscope LSM 510 (Carl Zeiss, Jena, Germany).

Growth hormone (GH) release assay

PC12 cells were washed twice with Locke's solution (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 11 mM glucose and 15 mM Hepes, pH 7.2) and then incubated for 10 min with Locke's solution (basal release at resting) or stimulated with an elevated K + solution (Locke's containing 59 mM KCl and 85 mM NaCl). The supernatant was collected, and the cells were harvested by scraping in 10 mM phosphate-buffered saline. The amounts of GH secreted into the medium and retained in the cells were measured using an ELISA assay (Roche). GH secretion is expressed as a percentage of total GH present in the cells before stimulation.

Carbon fiber amperometry

Carbon-fiber electrode of 5 µm diameter (ALA Scientific) was held at a potential of +650 mV compared with the reference electrode (Ag/AgCl) and approached closely to chromaffin cells. Catecholamine's secretion was induced by 10 seconds pressure ejection of 100 mM K + solution from a micropipette positioned at 10 µm from the cell and recorded during 100 seconds. Amperometric recordings were performed with an AMU130 (Radiometer Analytical) amplifier, sampled at 5 kHz, and digitally low-passed filtered at 1 kHz. Analysis of amperometric recordings was done as previously described ( 14) with a custom-written macro (obtained from Dr. R. Borges laboratory) running under Igor software (Wavemetrics), allowing automatic spike detection and extraction of spike parameters. The number of amperometric spikes was counted as the total number of spikes with an amplitude >5 pA within 100 seconds.

The spike parameters analysis was restricted to spikes with amplitudes of 5 pA. Data of amperometric spikes were averaged by individual cell.

Yeast two-hybrid screening

Yeast two-hybrid screening using the II-spectrin SH3 domain as a bait was performed as described in [START_REF] Nicolas | Tyrosine phosphorylation regulates alpha II spectrin cleavage by calpain[END_REF][START_REF] Rotter | AlphaIIspectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts[END_REF]. The sequence encoding from residues E970 to P1026 (accession number U83867) was cloned into the pLEX10 vector in frame with the C-terminus of the LEXA DNAbinding domain. The yeast strain L40 established with the II-Sp-bait was transformed with 100 µg of plasmid cDNA originating from a rat kidney library made in the laboratory. As the baits did not induce background, His + clones were selected on medium lacking tryptophan, leucine and histidine (DO-WLH) after 3 to 4 days of growth at 30°C. Beta-galactosidase activity was analysed on filter and liquid assays using X-Gal (5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside) and ONPG (o-nitrophenyl-β-D-galactopyranoside) as substrates, respectively. Recombinant pGAD plasmids were recovered from His + lacZ + phenotype yeasts and selected after transformation of an HB101 E. coli strain grown on a selective medium lacking leucine. Analyses of interaction specificity were performed by yeast mating of the L40 and AMR70 strains.

Expression and purification of recombinant peptides in E Coli

The sequences of human II-spectrin corresponding to the SH3 domain (E970 to P1026) and to the 9 repeat containing the SH3 domain (residues E885 to K1089) were cloned into pGEX- [START_REF] Guan | Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione Stransferase[END_REF]. GST-tagged peptides were expressed in BL21 Gold E. coli strain after induction with 0.5 mM IPTG and purified according to the manufacturer's instructions.

In Vitro protein-protein interactions and immunoprecipitation

In vitro interactions were performed overnight at 4°C with 20 µg of recombinant GST-tagged peptides immobilised on Sepharose 4B glutathione (Amersham Biosciences) and S 35 labelled-N-WASP obtained by transcription and translation in vitro (TNT® T7 Quick kit for PCR DNA, Promega) in 100 mM KCl, 0.2 mM EDTA, 20 mM Hepes buffer, pH 7.9, containing proteases inhibitors. After five washs in 0.01% NP 40, Hepes buffer, bound proteins were eluted by GSH (10 mM). Amounts of eluted, radiolabelled proteins were evaluated after SDS-PAGE using "Instant Imager" apparatus (Packard).

Anti-2 spectrin or anti-flag antibodies were covalently immobilized on AminoLink coupling resin and co-immunoprecipitation experiments were performed using Pierce™ Direct IP Kit (ThermoFisher) according to the manufacture instructions.

Data analysis

In all the Figures, data are

Results

Calcium-regulated exocytosis requires II-spectrin

To directly assess the functional importance of endogenous spectrin in the exocytotic machinery, we employed a short interference RNA (siRNA) strategy in PC12 cells, using a plasmid (pGHsuper) that expresses full-length human growth hormone (GH) and a siRNA targeting the sequence of II-spectrin. GH specifically stored into secretory granules is used as a reporter for exocytosis in the subpopulation of cells that transiently express the siRNA [START_REF] Malacombe | Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42[END_REF]. As revealed by immunocytochemistry analysis, cells expressing II-spectrin siRNA, identified by the expression of GH, presented significant loss of spectrin staining but no alteration of the expression level of GH or its distribution in secretory granules (Fig. 1A). Fluorescence quantification revealed that the level of II-spectrin in cells expressing siRNAs was reduced by ~ 90 % (Fig. 1B). The effect of spectrin silencing on PC12 cell secretory activity was then analyzed. Reduction of endogenous spectrin significantly inhibited the secretion of GH stimulated by a depolarizing concentration of K + (59mM; Figure 1C), indicating that IIspectrin is a key component of the exocytic machinery allowing large dense core vesicle to release hormones and proteins from neuroendocrine cells.

To further analyze the role of II-spectrin in hormone secretion, we monitored real-time single granule catecholamine exocytosis by carbon fiber amperometry. Figure 2A illustrates representative amperometric traces obtained from chromaffin cells expressing either an unrelated siRNA or a II-spectrin siRNA. The number of amperometric events was found to be significantly reduced in II-spectrin knocked down cells (Figure 2B), indicating that IIspectrin plays a role in the number of granules undergoing exocytosis. Figure 2C summarizes the detailed analysis of each individual amperometric spikes. None of the spike parameters were significantly altered by the reduction of endogenous II-spectrin levels (Table 1), suggesting that II-spectrin is not directly implicated in the fusion pore expansion or in the size and catecholamine content of the secretory granules.

Interaction between II-spectrin and N-WASP

To further investigate the putative mechanisms by which II-spectrin could regulate exocytosis, we looked for potential partners using a yeast two-hybrid. The backbone of II-spectrin is composed of 20 repeat units (1-20) and the 9 unit contains a SH3 domain (Figure 3A). This SH3 domain (residues E970 to P1026) fused to the DNA binding domain of LexA was used as a bait to screen a rat kidney cDNA library. Six clones containing incomplete sequences of Wiskott-Aldrich Syndrome protein N-WASP (accession number D88461/BAA21534) were retrieved. The largest sequence encodes a peptide starting from residue D67. Proline1017 of the II-spectrin SH3 corresponds to a highly conserved proline residue involved in SH3 domain interactions with its binding partners [START_REF] Nicolas | Tyrosine phosphorylation regulates alpha II spectrin cleavage by calpain[END_REF]. We probed interaction of II-spectrin SH3 peptides with N-WASP D67-A190 using the yeast two-hybrid system and found that P1017L mutation totally abolished the interaction as revealed by the absence of growth on selective medium (Figure 3B).

The interaction between spectrin and N-WASP was then validated through an independent biochemical approach using 35 S-labelled N-WASP and the II-spectrin SH3 domain fused to GST. As shown in figure 3C, the GST-SH3 domain bound N-WASP peptide whereas GST alone did not, thus confirming that the SH3 domain of II-spectrin is able to interact with N-WASP. Interestingly, a spectrin peptide containing the SH3 domain and the adjoining 9 repeat retained higher amount of N-WASP suggesting that the sequence surrounding the SH3 domain improved the spectrin-N-WASP interaction (Figure 3C). This interaction was further confirmed ex-vivo as endogenous N-WASP was co-immunoprecipitated with II-spectrin from PC12 cells (Figure 3D).

II-spectrin/N-WASP interaction is required for secretagogue-induced hormone release

In PC12 cells, upon K + -stimulation ectopically expressed GFP-N-WASP is efficiently recruited from the cytoplasm to the cell periphery to stimulate exocytotic activity by promoting actin filaments formation (1). To explore the functional importance of the N-WASP/spectrin interaction during exocytosis, we used two different approaches. First, the distribution of GFP-N-WASP was investigated in cells expressing II-spectrin siRNA. We found that the translocation of N-WASP to the plasma membrane in response to 59 mM K + -stimulation was strongly inhibited in cells depleted for II-spectrin compared to cells expressing an unrelated siRNAs (Figure 4A,B). These data strongly suggest that II-spectrin actively participates to the recruitment and/or stabilization of N-WASP at the plasma membrane upon cell stimulation for secretion. Second, we expressed in PC12 cells a flag tag-fused peptide of truncated IIspectrin corresponding to the region between 8 and 11 repeat units (Figure 3A) containing the N-WASP interacting domain. This peptide should sequester N-WASP and prevent its interaction with endogenous II-spectrin. Overexpressed Flag-Spectrin 8-11 displayed a predominant cytosolic staining in resting PC12 cells and was efficiently translocated to the cell 4C). On contrary, the mutant defective in N-WASP binding, Flag-Spectrin 8-11 P1017L , remained cytosolic upon cell stimulation (Figure 4C). Moreover, endogenous N-WASP was found to co-immunoprecipitate with flag-spectrin 8-11 peptide, but not with the corresponding peptide carrying the P1017L mutation (Figure 4D). These results confirm that the peptide 8-11 could act as a dominant negative truncated mutant by sequestering N-WASP and thus by preventing the endogenous interaction II-spectrin-N-WASP.

Expression of flag-spectrin 8-11 in PC12 cells significantly inhibited GH release, by nearly 60% as efficiently as the knockdown of II-spectrin (Figure 4E). This inhibitory effect was dependent on the ability of flag-Spectrin 8-11 to bind N-WASP since it was not observed with the peptide carrying the P1017L mutation (Figure 4E). Altogether, these results demonstrate for the first time that in neuroendocrine cells II-spectrin is able to recruit N-WASP to the plasma membrane and that this interaction is required for large dense core granule exocytosis.

Discussion

The potential involvement of II-spectrin in adrenal chromaffin cell exocytosis was first suggested in the late 80's in studies showing that injection of antibodies raised against IIspectrin inhibits calcium-mediated secretion [START_REF] Perrin | Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells[END_REF]. Using a loss of function approach, we have observed here that knocking down expression of II-spectrin inhibits calcium-regulated exocytosis in chromaffin and PC 12 cells, directly demonstrating that endogenous -spectrin is pivotal for neuroendocrine hormone release. A role for spectrin in neurotransmitter release from neurons has also been proposed in neuromuscular junctions of fruit flies Drosophila as presynaptic neurotransmitter release is impaired in -spectrin null Drosophila mutants or in mutants displaying a large decrease of pre-synaptic level of -spectrin [START_REF] Featherstone | Drosophila alphaand beta-spectrin mutations disrupt presynaptic neurotransmitter release[END_REF][START_REF] Wang | Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction[END_REF]. Yet, until now the mechanism by which -spectrin regulates calcium-regulated exocytosis of synaptic and large dense core vesicles remains unexplored.

We show here by amperometric analysis of catecholamine secretion from chromaffin cells that silencing of -spectrin expression decreases in the number of individual exocytic events/spikes without changing the shape of the remaining spikes. This observation rules out a direct role for II-spectrin in the membrane fusion process itself, but is consistent with a role in earlier stages of exocytosis such as the recruitment and/or docking of large dense core secretory granules to the exocytotic sites. Sikorski and collaborators have previously proposed in cultured hippocampal neurons that brain spectrin is involved in synaptic vesicle recruitment to the presynaptic active zone through its interaction with the vesicle-bound synapsin [START_REF] Sikorski | Spectrin (betaSpIIsigma1) is an essential component of synaptic transmission[END_REF].

Whether synapsin interacts with II-spectrin in chromaffin cells is not known and remains to be tested, although synapsin has been proposed to negatively regulate catecholamine release from chromaffin cells [START_REF] Villanueva | Synapsin II negatively regulates catecholamine release[END_REF]. II-spectrin has been also shown to interact with syntaxin1, a member of the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery involved in granule docking and fusion [START_REF] Nakano | Interaction of syntaxin with alpha-fodrin, a major component of the submembranous cytoskeleton[END_REF], but the importance of this interaction during exocytosis has never been explored. On the other hand, spectrin was first described as a regulator of actin polymerization [START_REF] Brenner | Spectrin/actin complex isolated from sheep erythrocytes accelerates actin polymerization by simple nucleation. Evidence for oligomeric actin in the erythrocyte cytoskeleton[END_REF][START_REF] Elbaum | Modulation of actin polymerization by the spectrin-band 4.1 complex[END_REF][START_REF] Stromqvist | Effect of spectrin dimer on actin polymerization[END_REF]. II-spectrin is also known to interact with several actin-binding proteins including Tes and the Ena/VASP protein family members, EVL and VASP [START_REF] Rotter | AlphaIIspectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts[END_REF][START_REF] Bournier | Spectrin interacts with EVL (Enabled/vasodilator-stimulated phosphoprotein-like protein), a protein involved in actin polymerization[END_REF][START_REF] Benz | Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes[END_REF]. The present findings describing the actin binding protein N-WASP as a functional partner of II-spectrin in the secretory pathway, replaces spectrin in the frame of the cortical actin organization that supervises the exocytotic machinery. We have previously described that N-WASP is recruited to the plasma membrane upon PC12 cell stimulation thanks to its interaction with the Rho-GTPase Cdc42 which is activated at the plasma membrane during hormone release [START_REF] Gasman | Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASPinduced actin filaments[END_REF]. Once located at the plasma membrane, N-WASP may interact with the secretory granule-bound actin nucleating complex ARP2/3 to promote actin polymerization at the exocytotic site and then enhance hormone release [START_REF] Gasman | Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASPinduced actin filaments[END_REF]. Here, we observed that knocking down II-spectrin prevents N-WASP recruitment to the cell periphery of stimulated PC12 cells suggesting that II-spectrin is also implicated in the recruitment of N-WASP to the exocytotic sites. Whether N-WASP interacts simultaneously with Cdc42 and II-spectrin is not known. However, N-WASP is known to be maintained in an autoinhibited conformation through intramolecular bounds that are relieved upon Cdc42 binding [START_REF] Rohatgi | Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate[END_REF]. As activation of N-WASP by Cdc42 unmasks its proline-rich domain, it is tempting to imagine that Cdc42 binding occurs first hence allowing the subsequent binding with II-spectrin SH3 binding domain. II-spectrin is a long protein (200 nm for tetramers) with a SH3 domain that might constitute an active spot to recruit the exocytic machinery including N-WASP. In line, several presynaptic proteins from the active zone seem to be mislocalized in the absence of spectrin [START_REF] Featherstone | Drosophila alphaand beta-spectrin mutations disrupt presynaptic neurotransmitter release[END_REF]. It is worth to mention that annexin-A2 has also been identified among the spectrin interacting partners [START_REF] Gerke | Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to nonerythroid spectrin and F-actin[END_REF]. Annexin-A2 is a cytosolic protein that triggers the formation of actin bundles essential for the formation of active exocytotic sites in stimulated chromaffin cells [START_REF] Gabel | Annexin A2dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis[END_REF]. Whether spectrin is a hub involved in the recruitment to the exocytotic sites of the actin-binding proteins (N-WASP, annexin-A2) required for building the actin architecture necessary for exocytosis remains to be determined.

2 Abstract

 2 Besides a fundamental structural role at the plasma membrane, spectrin-and actin-based skeletons have been proposed to participate to various processes including vesicular trafficking.

  Abbreviations:N-WASP: Neuronal Wiskott-Aldrich Syndrome protein Ena/VASP: Ena/Vasodilator-stimulated phosphoprotein EVL: Ena/Vasodilator-stimulated phosphoprotein-like protein

  given as the mean values ± S.D. obtained at least in three independent experiments performed on different cell cultures. n represents the number of experiments. Statistical significance has been established using ANOVA test and data were considered significantly different when p value was lower than 0.05. Gaussian distribution and variance equality of the data were verified.

  mM K + -stimulation of the cells (Figure

Figure 1 :Figure 2 :

 12 Figures legends:

Figure 3 :

 3 Figure 3: In vitro interaction between N-WASP and SH3 domain of αII-spectrin. (A) Schematic representation of II-spectrin. II-spectrin is composed of 20 repeat units (1-20) flanked by a small N-terminal region and C-terminus EF-hand motifs. Repeat 9 contains the SH3 domain. SH3-9 and 8-11constructs are indicated. Numbers represent amino acid position. (B) L40 yeast cells transformed with pLEX vector empty or containing the indicated spectrin constructs were mated with AMR70 yeast cells transformed with pGAD vector empty or containing N-WASP (D67-E501). As revealed by yeast growth on triple-selective medium (DO-WLH), αII-spectrin SH3 domain (SH3 WT ) interacts with N-WASP. Note that the mutation (P1017L) abolished the interaction with N-WASP. (C) GST, GST fused to αII-SH3 or fused to αII-SH3-α9 were immobilized on sepharose 4B beads and incubated with rat N-WASP (D67-E501) expressed as 35 S-methionine labelled peptide using in vitro transcription and translation. After extensive washings, retained N-WASP peptide were eluted with 10 mM glutathione then analyzed by SDS-PAGE. 35 S-methionine-labelled N-WASP peptide was analyzed by autoradiography. (D) Immunoprecipitation experiments from lysates of PC12 using IIspectrin antibodies. Endogenous N-WASP, revealed by anti-N-WASP antibodies, coprecipitated with II-spectrin. Control with non-immune (NI) serum is shown.

Figure 4 :

 4 Figure 4: (A-B) PC12 cells transfected with pGHsuper encoding unrelated shRNA (control) or with pGHsuper encoding II-spectrin shRNA were plated on four-well plates and transfected again, 48 hours later, with the N-WASP-EGFP plasmid. 24 hours after the second transfection, cells were stimulated for 10 minutes with 59 mM K + (stimulated) or incubated for 10 minutes in Locke's solution (resting). Cells expressing siRNA were identified by GH staining (not shown) whereas II-spectrin was observed with anti-II-spectrin antibodies visualized with Alexa-647 conjugated anti-rabbit antibodies. Bar, 5 µm. The effect of II-spectrin knocking down on intracellular distribution of N-WASP was then analyzed by confocal microscopy and quantified in B. (C) PC12 cells expressing the Flag-tagged spectrin 8-11 peptide or the P1017 mutant unable to interact with N-WASP (Flag-tagged 8-11 P1017L ) were labelled with the monoclonal anti-flag antibodies revealed by Alexa-488 conjugated anti-mouse antibodies. Bar, 5µm. (D) Immunoprecipitation (IP) experiments from lysates of PC12 cells expressing flagtagged spectrin 8-11 peptides using anti-flag antibodies. Precipitated spectrin 8-11 peptides were revealed by western blot with anti-flag antibodies and N-WASP with anti-N-WASP antibodies. Control with non-immune (NI) serum is shown. (E) GH secretory activity was measured in PC12 cells co-expressing GH and either Flag-8-11 or Flag-8-11 P1017L  GH

Table 1 : Table summarizing the amperometric parameters of the exocytic events recorded from chromaffin cells knocked-down for II-spectrin.

 1 11 or Flag-8-11 P1017L  GH Value are given as the mean ± SEM. Q, quantal size or charge; t half, half-width, I max, spike amplitude.

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Acknowledgement:

We thank the municipal slaughterhouse of Haguenau (France) to provide bovine adrenal glands. We acknowledge T. Tahouly for her helpful technical assistance for chromaffin cells culture and Valérie Calco for the design of the constructs coding for the II-spectrin siRNA.

release is expressed as the percentage of total GH (n = 3). **P<0.01, compared to control cells (ANOVA).