Léonard Dumont
email: leonard.dumont@ugent.be

Stefan Wirth
email: stefan.wirth@u-bourgogne.fr

Professor, Matthieu N Boone
email: matthieu.boone@ugent.be

Iván Josipovic
email: ivan.josipovic@ugent.be

Sylvia Lycke
email: sylvia.lycke@ugent.be

Pieter Tack
email: pieter.tack@ugent.be

Professor, Peter Vandenabeele
email: peter.vandenabeele@ugent.be

Guy De Mulder
email: guy.demulder@ugent.be

Léonard Dumont Corresponding

A Unique Case of 'Counting Marks' Revealed by Tomography on a Middle Bronze Age Sword from Champagneux (France, Savoie)

Keywords: Middle Bronze Age, France, Octagonal-hilted swords, Counting marks, CT-scan, XRF, Metallurgy, Craftsmanship

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Although Bronze Age solid-hilted swords have been studied since the end of the 19th century, very little is known about the workshops they were produced in and the organisation of their production. Except for the very end of the Bronze Age (9th century BC), there are no archaeological remains of the manufacturing sites of these swords [START_REF] Dumont | À la recherche des aires de production des épées de l'âge du Bronze : où sont les moules ? Marcigny & Mordant[END_REF]. Remains of a workshop's location have never been found, nor the moulds used to cast the different parts of these artefacts. Our knowledge concerning the Bronze Age solid-hilted swords' chaîne opératoire, and the organisation of their production, is therefore only indirect and comes from the study of the swords themselves. The case of such a sword found in the Rhône river in Champagneux (France, Savoie) in the 1960s is particularly noteworthy concerning this topic. In the frame of the BOF-funded (Bijzonder Onderzoeksfonds, Ghent University) project 'Production and distribution of Bronze Age solid-hilted swords in Western Europe' (2018)(2019)(2020)(2021)(2022), it was investigated using high-resolution computed-tomography (μCT) in order to study the hilt and the blade and their respective layouts. This technique also revealed the unexpected presence of five so-called 'counting marks' on the part of the blade inserted within the handle. This feature allows us to discuss several hypotheses concerning the production organisation of this kind of weapon. After thoroughly presenting the Champagneux sword and the techniques used to study it, we will try to reconstruct the different steps of its production and determine what was (or were) the role(s) of the marks observed inside the handle.

THE CHAMPAGNEUX SWORD (FRANCE, SAVOIE)

The sword from Champagneux (France, Savoie) was dredged up in a gravel quarry by Mr François Debauge in the Rhône river in the 1960s, upstream of its confluence with the Guiers river. It was given to the Escale Haut-Rhône museum in Brégnier-Cordon (France, Ain) in January 2011 by Mrs Françoise Bousculat. The museum still curates the sword, which has inventory number 2011.1.1. A few studies on the sword were published by A. Bocquet (Bocquet & Haussmann 2001, 305-307;[START_REF] Bocquet | Un grand moment de notre histoire… il y a 3300 ans. À propos de découvertes régionales de la fin de l'âge du Bronze. La Pierre et l'Écrit[END_REF]), but no detailed technical examination has been conducted so far.

Although the blade is broken into two fragments, the 51.2 cm long Champagneux sword is completely preserved (Fig. 1). The visible section of the bl ade is 40.5 cm long from the bottom of the hilt to the tip, with a maximum width of 2.8 cm and an average thickness of 0.7 cm. The blade presents a lenticular-shaped section with a light recess located a few millimetres from the cutting edges. It was found covered with a blackish patina verging on green, which now has been partly removed, exposing the underlying uncorroded golden bronze. The slight deformations of the cutting edges, visible in some places, are associated with alterations of the corrosion layer, indicating that this probably happened after the sword was deposited, for example, when it was taken out of the Rhône river. The hilt is 10.7 cm long and has a 4.5 cm wide oval pommel. Its surface is riddled with small craters, and on its top sits a truncated cone-shaped protuberance. The pommel's edge is flattened on one side. The presence of corrosion and the smooth edge suggests that this happened before the object was deposited in the river, i.e., during its production or period of use. The grip has a convex outline and a circular-shaped cross-section. A series of straight lines carved in the bronze highlights the transition to the pommel. The grip's surface was subject to substantial erosion, especially in its centre, judging from the almost total absence of corrosion on this spot. The erosion may have been caused by the current of the river where the sword remained for more than three millennia or by manual abrasion of the sword's surface after its discovery. The grip ornaments were thereby almost totally erased, except for a circular pattern or a spiral that remains barely visible on the bottom part of the grip, close to the guard (Fig. 1). Finally, the kite-shaped guard is 5.3 cm wide with a semi-circular notch in its centre and two rivets on either side.

TYPOLOGY AND CHRONOLOGY

The Champagneux sword presents singular formal features, making it difficult to fit in the traditional typological framework of Bronze Age solid-hilted swords. It evokes two different types. First of all, the kite-shaped guard, the slightly convex outline of the grip, and the quite massive pommel are typical features of the octagonal-hilted swords (German: Achtkantschwerter). These swords, typical for the second part of the Middle Bronze Age (Bronze C, mid-15th-14th century BC), are equipped with a very distinctive hilt sharing common features with the Champagneux sword, with the addition of an octagonal hilt crosssection that gave them their name (Holste 1953, 16-25;Quillfeldt 1995, 45-94). The patterns of circles or spirals still visible on the hilt of the Champagneux sword is also an element widely found on octagonal hilted swords (Holste 1953, 16-18). However, the circular cross-section of this sword is a feature that only appears at the beginning of the Late Bronze Age (Bronze D, 13th century BC) among Central-European solid-hilted swords. It is then occasionally found on swords belonging to the Riegsee type, such as the one from Král (Slovakia) (Novotná 2014, 33, pl. 5, 24), or on other swords isolated from a typological point of view, as in the case of a sword from Bad Staffelstein (Bayern, Germany) (Quillfeldt 1995, 97, pl. 26, 80).

Despite the lack of features perfectly matching one specific type, the Champagneux sword appears to be related to the octagonal-hilted swords. Although the octagonal cross-section of [START_REF] Holste | Die bronzezeitlichen Vollgriffschwerter Bayerns[END_REF][START_REF] Bianco Peroni | Die Schwerter in Italien. Le spade nell'Italia continentale[END_REF][START_REF] Krämer | Die Vollgriffschwerter in Österreich und der Schweiz[END_REF][START_REF] Kemenczei | Die Schwerter in Ungarn II (Vollgriffschwerter)[END_REF][START_REF] Quillfeldt | Die Vollgriffschwerter in Süddeutschland[END_REF][START_REF] Wüstemann | Die Schwerter in Ostdeutschland[END_REF][START_REF] Laux | Die Schwerter in Niedersachsen[END_REF][START_REF] Winiker | Die bronzeitlichen Vollgriffschwerter in Böhmen[END_REF][START_REF] Bunnefeld | Älterbronzezeitliche Vollgriffschwerter in Dänemark und Schleswig-Holstein: Studien zu Form, Verzierung, Technik und Funktion[END_REF]). © L. Dumont.

the hilt is missing, all the other features, such as the thick, stocky pommel and the kite-shaped guard, belong to the octagonal-hilted swords' characteristics. Furthermore, as mentioned earlier, this sword likely used to have an octagonal cross-section that was altered by its three millennia-long rest in the Rhône river. It is then possible to propose a dating of the Champagneux sword in the second half of the Middle Bronze Age (Bronze C, ca. 1450-1300 BC).

The octagonal-hilted swords are distributed across Europe in two main clusters: one around the Alpine region, especially between the Alps and the Main river; another in Southern Scandinavia, mainly in present-day Denmark and Schleswig-Holstein (Fig. 2). Accepting the affiliation of the sword from Champagneux with this type would make it the most western find from this group. All octagonal-hilted swords are made the same way. Since no hilts are identical to one another and given their rich ornamentation, they are very likely to be produced using the lost-wax casting technique with individually made wax models on clay cores (Hundt 1962, 38).

The hollow-cast hilts were then fixed to the blade using a combination of two techniques. First of all, the hilt is put on the tang located at the top part of the blade. This tang, inserted inside the hilt beyond the guard, is adjusted by the craftsman in charge of the manufacture of both parts so that it can be in contact with the inner walls of the hilt. By doing so, both elements are locked together. The fixation is then strengthened by adding two rivets on the guard piece on either side of the central notch. This technique, which combines the locking of the hilt with the blade and the riveting, is an invention of the Middle Bronze Age that appears with the octagonal-hilted swords and is designated after this type as the 'octagonal-hilted sword principle' (Achtkantschwert-Prinzip) (Hundt 1965, 54). These octagonal-hilted swords form a very consistent group, technically speaking. Examinations performed on this type of sword using X-ray-based imaging techniques such as radiography and computed tomography indicate that almost all are produced the same way, wherever they are found in Europe [START_REF] Hundt | Produktionsgeschichtliche Untersuchungen über den bronzezeitlichen Schwertguss[END_REF]Bunnefeld 2016, 122-125). This technical normalisation and homogeneity suggest that these swords may have been produced in a limited number of workshops whose products were designed for users in the north-Alpine region and in southern Scandinavia, where imitations were also manufactured, probably locally [START_REF] Bunnefeld | Das Eigene und das Fremde -Anmerkungen zur Verbreitung der Achtkantschwerter[END_REF].

METHODS AND RESULTS

HIGH-RESOLUTION COMPUTED TOMOGRAPHY (µCT)

A new examination of the Champagneux sword was possible in 2019 and 2020. After a first macroscopic inspection, the sword was brought to the Ghent University Centre for X-ray Tomography (UGCT) in October 2020 for further analysis. Whereas radiography has been used to investigate Bronze Age solid-hilted swords since the 1950s [START_REF] Drescher | Der Überfangguss[END_REF][START_REF] Driehaus | Das Ergebnis der Röntgenuntersuchung der Vollgriff-Bronzeschwerter des Rheinisches Landesmuseums Bonn[END_REF][START_REF] Driehaus | Röntgenuntersuchungen an bronzenen "Vollgriffschwertern[END_REF][START_REF] Hundt | Zu einigen westeuropäischen Vollgriffschwerten[END_REF][START_REF] Hundt | Produktionsgeschichtliche Untersuchungen über den bronzezeitlichen Schwertguss[END_REF], the use of digital radiography and computed tomography is much more recent and enables significant advances in the visualisation of the swords' construction due to increased precision and the opportunity to investigate in 3D the inner structure of the artefacts [START_REF] Mödlinger | Micro-X-ray computer tomography in archaeology: analyses of a Bronze Age sword[END_REF]Bunnefeld & Schwenzer 2011;[START_REF] Bunnefeld | Älterbronzezeitliche Vollgriffschwerter in Dänemark und Schleswig-Holstein: Studien zu Form, Verzierung, Technik und Funktion[END_REF]Dumont et al. 2020a;2020b).

Radiography and high-resolution computed tomography (μCT) were used to investigate the production process of the object. UGCT's HECTOR (High Energy CT Optimised for Research) setup was used [START_REF] Masschaele | HECTOR: A 240kV Micro-CT setup optimized for research[END_REF], suitable for both larger samples and samples with a high X-ray attenuation like the case of this bronze sword. We used an X-RAY WorX XWT 240-SE microfocus source operating at 220 kV and 60 W. The source was equipped with a 1 mm Cu sheet to filter low energy X-rays. Images were acquired with a PerkinElmer 1620 CN3 CS flat panel detector where the 2024 × 2024 0.2 mm pixel range was cropped to fit the sword projection. For the scan of the hilt, the Champagneux sword was placed on a rotary base, and 1501 projections were acquired around the sword's hilt, each with 1 second acquisition time.

As for the digital radiography, projections were acquired over different vertical steps to cover the sword's length. The images were averaged over 100 normalised images, each with 1 s acquisition time.

The digital radiography of the whole sword enables us to assess the global quality of the casting of both parts. Whereas the handle is made of a very homogenous metal, the blade shows inner flaws within the bronze from the upper third (Fig. 1). Thanks to the scan of the hilt area, it is possible to realise a much more precise examination of the hilt structure and the organisation of the different elements. Several sections were made through the reconstructed volume following the axial, coronal and sagittal planes using the 3D Slicer software [START_REF] Fedorov | 3D Slicer as an image computing platform for the Quantitative Imaging Network[END_REF], showing precisely the layout of the blade inserted within the handle (Fig. 3). With the help of the 3D model of the hilt resulting from the μCT, it was also possible to virtually visualise the tang's surface, which is the part of the blade inserted in the hilt, revealing the five marks that were made on it (Fig. 4).

HAND-HELD X-RAY FLUORESCENCE SPECTROSCOPY (HXRF)

When the sword was in Ghent University, non-invasive surface handheld XRF analyses were also conducted. This method allows us to determine the concentration of the element on the sword's surface in a non-invasive way. Although these analyses are limited in precision, not all elements can be detected and the corrosion layer can alter the results [START_REF] Nørgaard | Portable XRF on Prehistoric Bronze Artefacts: Limitations and Use for the Detection of Bronze Age Metal Workshops[END_REF], the impossibility to sample the Champagneux sword and the presence of uncorroded areas on the sword's surface prompted us to use this technique to determine if the blade and the hilt were made from the same type of alloy or not.

The measurements were performed with a commercial instrument, i.e., an Olympus Innov X Delta Premium unit. It is equipped with rhodium (Rh) based X-ray tube and a silicon drift detector (SDD). An aluminium (Al) filter was applied for measuring the higher Z-elements (from Al onwards) with a voltage of 40 kV and a current of 38.7 μA. The measurements were performed in the air for 300 seconds (real-time) with an uncollimated polychromatic X-ray beam for excitation (5 × 5 mm2). The instrument was set on the Geochem mode. As any invasive operations on the Champagneux sword were prohibited by the museum curating it, the measurements were conducted on its surface without any preliminary treatment. Areas where the corrosion layer was missing were targeted (Fig. 5). The collected spectral data were processed using the dedicated XRF spectrum evaluation software AXIL (Analysis of X-ray spectra by iterative least squares) [START_REF] Vekemans | Analysis of X-Ray spectra by iterative least-squares (Axil) -New developments[END_REF]).

The XRF results were quantified using references MBH-32x-lg10g, MBH-32x-pb14e, MBH-32x-sn6b, MBH-32x-sn7b and MBH-33x-gm21b based quantification method. The elemental yield was determined for each certified element in the reference materials by dividing the XRF net peak intensity by the corresponding certified concentration after normalising for measurement time and illuminated mass by division of the Compton scatter intensity. Where an element is present in multiple reference materials, the average elemental yield and its standard deviation were used as a resulting error. For elements not present in any of the used reference materials, the elemental yield was determined by interpolating the closest neighbouring elements by the atomic number -present in the reference materials. Four points were measured on the surface of the Champagneux sword: two on the handle and two on the blade (Fig. 5). The composition of both parts is similar. They are both made out of a binary alloy with copper (Cu) and tin (Sn) with low traces of lead (Pb) (Table 1).

It should be noted that the sum of concentrations listed in Table 1 does not equal 100 -this is because the quantification method used provides concentration values only for the detectable elements, excluding, for instance, the detection of low energetic photons originating from oxygen, carbon, and other elements. Additionally, due to the inability to remove corrosion from the sword in a non-destructive manner, it was opted to measure the sample on the least corroded parts. Corrosion products can partially modify the resulting quantitative information on top of the sword alloy. However, these are expected to be limited due to the limited corrosion layer thickness and relatively lower density than the sample bulk. In any case the corrosion layer thickness is expected to be similar for all measured points, thus providing reliable information on the alloy composition from different measurement locations.

RECONTRUCTING THE PRODUCTION PROCESS OF THE SWORD CASTING

the study of two types of features revealed by the radiography and μCT scanning: blows and shrinkage porosity. Both are two kinds of defects that can occur during the casting and under different conditions. Blows correspond to gas trapped inside the metal during the casting process. They can be caused by insufficient mould ventilation or by too rapid solidification of the bronze, not allowing the gas sufficient time to evacuate. Their almost circular or pearshaped form can easily be identified on the radiographs or the section resulting from the computed tomography. As the gas is lighter than the bronze cast inside the mould, they move upwards while the metal is in a liquid state. The distribution of this kind of fault inside the sword can therefore give us information about the position of the mould and the introduction point of the metal. Shrinkage porosity is another kind of defect inside the casted metallic mass.

The retraction of the bronze causes shrinkage while it is cooling, and the drop in temperature is not homogenous everywhere around the casted objects. The drop is, for example, much faster around the introduction point of the melted metal inside the mould, which is where the bronze comes in contact with the air. This kind of fault is different from blows. It takes the shapes of a series of rims with irregular outlines associated with small uneven cavities. Concentrations of shrinkage porosity and blows are known to be an indication of where the metal introduction point of the mould was located (Wirth 2003, 126-127;[START_REF] Mödlinger | Micro-X-ray computer tomography in archaeology: analyses of a Bronze Age sword[END_REF]2011, 32;Mödlinger & Trnka 2009, 31).

The Champagneux sword's blade has significant inner defects. They are primarily concentrated in the upper quarter of the blade. The flaws are scattered in the central part and almost absent at its tip (Fig. 1). We see a concentration of blows and shrinkage porosity located just below the handle, within the notch of the guard (Figs. 1,3). It suggests that the sword's hilt was cast from its upper extremity, where the tang is located, as most of the swords dating back to the Bronze C period are (Mödlinger & Trnka 2009, 31-32). However, the metal of the tang itself is much more homogeneous than the blade below the hilt. Only very small blows and porosity are visible on this part of the blade inserted in the handle (Fig. 3). The mould used to cast this blade was likely slightly tilted to allow a better pouring and flow of the melted bronze plus a better evacuation of the gas [START_REF] Mödlinger | Micro-X-ray computer tomography in archaeology: analyses of a Bronze Age sword[END_REF]. It is clear from this data that this did not give the expected results. It is difficult to evaluate the impact of these flaws on the solidity of the blade and on its ability to be used in combat. This blade was prepared as a fully functional sword. The cutting edges are not raw and were sharpened. This operation usually involves a series of hammering and heating operations aiming to increase the edges' hardness (Mödlinger 2011, 35-36).

Reconstructing the casting process of the hilt of this sword is much more complicated. It is indeed made of a much more homogeneous metal in which radiography and μCT scanning only revealed a very limited amount of porosity (Figs. 1,3). Except for a few blows located in the grip, most flaws are concentrated within the pommel. Some are visible on its surface (Fig. 1), which is consistent with a casting made from the upper part of the hilt, especially from the side of the pommel as it was documented on later swords, such as the broken cup-shaped pommel sword from St. Pölten (Austria) [START_REF] Pola | Casting Simulation of an Austrian Bronze Age Sword Hilt[END_REF], as well as on Late Bronze Age moulds for Mörigen type hilts at Erlingshofen (Bayern, Germany) (Wirth 2003, 119-128;Overbeck 2018, pl. 10, 20) and Grésine (Lac du Bourget, Savoie, France) [START_REF] Perrin | Fonderies de bronze des palafittes du lac du Bourget[END_REF]. The flattened edge of the Champagneux sword's pommel might indicate the removal of the metal excess located at the mould pouring gate. The manufacture of a hollow handle means to be cast over a core. This piece was probably made out of clay, which is the most probable option for producing this kind of hilt (Hundt 1965, 43;Quillfeldt 1995, 75-76;Wüstemann 2004, 122-123;Berger 2014, 229;Bunnefeld 2016, 122). The use of a permanent and reusable core, for example, made out of bronze as in the Erlingshofen mould (Overbeck 2018, pl. 10, 20), is to be ruled out. As the hilt's inner room is wider in its centre than at the guard's base (Fig. 3), it would not be possible to take a metallic core out of the hilt after the casting.

On the contrary, a clay core could have been destroyed after the handle was melted down over it. The μCT data reveal the presence of residues on the hilt inner surface (Fig. 3). The residues are likely to come from the core, and since the deposit shows a lower density than the rest of the handle on the tomography, we are confident the core was made out of a different material. Its adhesion to the hilt's inner wall suggests it might be clay. Another exciting feature concerning the production of this hilt is the hollow canal within the pommel connecting the inner room with the outside. There has been much debate concerning these canals, often observed on Bronze Age swords' hilts. First, it has been proposed that this hollow part originated from the gas produced during the casting making its way through the pommel while the bronze was still liquid (Driehaus 1961, 26;Berger 2014, 227). J. Driehaus later suggested that the craftsman may have turned the mould over after casting the bronze in order to remove excess metal, thereby creating at the same time this open room within the pommel (Driehaus 1968, 356-357).

Meanwhile, H.-J. Hundt proposed another explanation, stating that this element could correspond to a vent used for evacuating the gas produced during the casting (Hundt 1965, 42-44). A much more probable hypothesis comes from the study of Early Bronze Age solid-hilted daggers. This feature may originate from using a stand designed to go through the core, the wax model, and the clay mantel to hold the clay core in place during the entire casting process (Schwenzer 2004, 147;Mödlinger 2011, 32). However, this technique would produce only very regular canals, which is not always the case, hence the possibility that some canals and pommel openings could be caused by the combination of the presence of porosity in the bronze and its retraction during the cooling process (Bunnefeld & Schwenzer 2011, 216-217). In the case of the Champagneux sword, the outline of the pommel canal is unmistakable but is not straight: it becomes narrower in the middle of the pommel before it slightly extends just before it reaches the surface, visible on the profile section of the hilt (Fig. 3). It may have been caused by two combining factors: the presence of a stand during the casting and the metal retraction. The regularity of the canal indicates it may not be related to porosity. After the casting, the opening remaining at the centre of the pommel was closed using a small bronze shaft.

ASSEMBLY

After both the hilt and the blade were cast, the two parts needed to be joined together. The fixation was made using two techniques. First, the blade was inserted inside the hilt through the opening located at the guard's base. The contact points between the tang and the inner wall of the handle on the bottom part of the grip show that the blade was forced inside the hilt to block the two elements together. The tang and the guard fit the hilt's inner room, perfectly matching the guard opening and the grip width (Fig. 3). Because we do not know any moulds for casting octagonal-hilted swords' hilts and blades, we are probably dealing with individually made objects and no serial production. Consequently, the blade tang must have been worked after it was cast to fit this specific hilt, such as by hammering to expand it slightly.

Once the blade and the handle were both locked together, the rivets were added to the guard, on either side of the notch in its centre. The role of these two small bronze rods in the fixation of both parts seems somewhat limited. First of all, the μCT data shows that the rivet holes are larger than the shafts inserted in them. Secondly, one of the hole's outlines is broken, making it more like a notch than a real rivet hole. We also see that the two holes in each side of the guard and the perforation made in the tang are not perfectly aligned on one side of the guard, hence a twisted rivet (Fig. 3).

Consequently, it is questionable what the rivets' purposes are. We here propose two hypotheses to this question. The rivets can be seen as a security if the blocking of the hilt and blade fails, for example, following a violent impact. Although there would be a play between the two parts, they would remain joined together. The presence of the rivets might also be considered a cultural remain more than an effective way to bind the handle to the blade. Before the emergence of octagonal-hilted swords in Bronze C, other sword types, such as the Spatzenhausen type, were equipped with a short-tanged blade only fixed to the hilt using rivets (Hundt 1965, 43-44).

The development of the fixation technique mentioned above, blocking and riveting, is indeed a fundamental breakthrough in the swords' conception. As we already saw earlier (see section 'Typology and chronology'), this so-called 'octagonal-hilted swords principle' appeared in the Bronze C period, when craftsmen followed it to produce most of the swords found between the Alpine region and the Carpathian basin from the mid-15th to the 10th century BC. Although this technique has been widely documented through the use of imaging techniques since the 1960s [START_REF] Hundt | Produktionsgeschichtliche Untersuchungen über den bronzezeitlichen Schwertguss[END_REF][START_REF] Ankner | Röntgenuntersuchungen an Riegseeschwertern. Ein Beitrag zur Typologie[END_REF][START_REF] Wüstemann | Die Schwerter in Ostdeutschland[END_REF]Bunnefeld & Schwenzer 2011;[START_REF] Mödlinger | Herstellung und Verwendung bronzezeitlicher Schwerter Mitteleuropas[END_REF][START_REF] Berger | Neue Tauschierungen am atlen Funden. Untersuchungen zur Verzierungs-und Herstellungstechnik zweier Vollgriffschwerter der Hügelgräberbronzezeit[END_REF], the organisation of the crafting process remains undocumented mainly due to the absence of workshops in the archaeological record. The computed tomography performed on the hilt area of the Champagneux sword, besides producing valuable documentation of the sword's casting and assembly, also enabled us to visualise the surface of the tang, which appears to carry a series of five vertically arranged carved marks (Fig. 4). They are believed to come from the craftsman who made the blade. They may be connected to the fixation process of the blade and therefore give us information about how the chaîne opératoire was organised and possibly divided between several individuals.

THE "COUNTING MARKS": MAKING AND FUNCTION(S)

Computed tomography not only allows visualisation of sections of the scanned artefact. It also enables us to virtually reconstruct the object's volume, both from the outer surface and inside.

It is thus possible to 'cut' through this numerical model to get a sight of the inner surfaces, which are impossible to visualise using traditional radiography or CT-scan sections. Thanks to this opportunity, it was possible to glimpse the inside of the Champagneux sword. The most important feature of this examination are five vertically arranged marks on the centre of one side of the blade's tang. They measure 4to5mmlongforawidthbetween2and3mmanda depth of about 1 mm (Figs. 34). They have the shape of an almond, with one sharp edge and a curved one. Each one is slightly tilted and forms an angle between 30 and 45° compared to the blade axis. Each of them shows a thin bulge (Fig. 4), and, with their shallow depth and this thin bulge in mind, it seems clear that these marks were probably made directly on the bronze after it was cast rather than on the wax model (assuming that the lost-wax process was used for the casting).

According to the almond shape and the small size of the marks, they were likely made using the corner of a chisel blade. Their shallow depth and width suggest they were realised with a rather blunt blade. Otherwise, using a sharper edge, the marks would have been deeper and thinner.

Although this kind of feature has, to our knowledge, never been observed on a Bronze Age solid-hilted sword, elements of comparison are known on other kinds of bronze artefacts. Armrings are the best-known case, with several examples dating back to the Late Bronze Age, mainly from the north and east Alpine regions [START_REF] Thrane | Counting marks on Urnfield Culture arm rings[END_REF][START_REF] Müller-Karpe | Neue Bronzefunde der späten Urnenfelderzeit vom Bleibeskopf im Taunus[END_REF][START_REF] Weidmann | Ein reicher Ringfund der Spätbronzezeit aus Sursee[END_REF][START_REF] Janssen | Ein urnenfelderzeitliches Brandgrab von der befestigten Höhensiedlung "Bullenheimer Berg[END_REF][START_REF] Janssen | Ein urnenfelderzeitliches Brandgrab von der befestigten Höhensiedlung "Bullenheimer Berg[END_REF]Primas 2008, 192-193;[START_REF] Windholz-Konrad | Ein neues Bronzeschmuckdepot von Bad Aussee im steierischen Salzkammergut. Zum ausgeprägten Hortphänomen im Alpendurchgang sudöstlich von Hallstatt[END_REF]. Two kinds of marks are known on bracelets: thin, long and engraved marks (German: Strichmarken) and more extensive, deeper and shorter chiselled marks (German: Schlagmarken) (Windholz-Konrad 2008, 385-386). Armrings with such signs are more commonly found in hoards than graves (Janssens 1989(Janssens -1990, 88), 88). Usually, the bracelets with marks form sets where each bracelet wears a unique number mark. It was first proposed that they were made in the framework of intense exchanges to enable the 'tradesman' to 'keep account of his rings' or for the user to check if a set was complete (Thrane 1962, 99). In the case of the rings found in the Late Bronze Age Bleibeskopf settlement (Bad Homburg, Hesse, Germany), the number of marks can be related to the size of the bracelets: the more marks there are, the smaller the circumference of the armring is. As the use-wear marks show, the bracelets were probably used together as a unique set, and the marks were probably made by the craftsman for the user, indicating the place of each ring within this set and the order they should be worn (Müller-Karpe 1974, 204-207). The same interpretation is proposed concerning the marks on the nine bracelets from Bad Aussee (Austria), where they are believed to correspond to the positioning order of the rings when worn (Windholz-Konrad 2008, 386-387).

The rings coming from the Landzunge Zellmoos lake-shore settlement (Sursee, Switzerland) is different. Here, bracelets are matched by a certain number of marks forming pairs. The rings from the same pair are decorated with precisely the same patterns arranged identically. Therefore, the marks are interpreted as signs put on the raw bracelet by the founder before the decoration was chiselled on the ring's surface (Weidmann 1983, 182-185). Marks found on bracelets can be explained in different ways and be linked with the production process, the exchange/trade, and the use of the rings.

More complex marking systems are also documented on Late Bronze Age tools between the Alps and the Carpathian Basin, especially at salt or ore mining sites [START_REF] Mayer | Zur Herkunft der Marken auf urnenfelder-und hallstattzeitlichen Bronzegeräten des Ostalpenraumes[END_REF]Primas 1997, 124). They are interpreted as property marks related to mining activities or quality control by workshops (Mayer 1976, 378-379). Finally, another kind of marking system exists on Late Bronze Age sickles, even though the marks are here very different from what we discussed above since they consist of ribs that were not made directly on the objects but the moulds before they were cast (Brunn 1958, 43-50;Thrane 1962, 99;Sommerfeld 1994;Jahn 2013, 197-202).

In the light of the different examples presented so far, the marks on the Champagneux sword's tang can be interpreted in various ways. First, as these signs are not visible on the sword's surface, we can rule out the hypothesis of marks targeting the weapon user. It is, therefore, more likely to be related to the production process. The simplest explanation could be to consider these signs as workshop or craftsman's marks, simply indicating this blade is the fifth produced in a series. As we already saw, the sword's tang was probably slightly modified after it was cast to fit in the inner space of the handle. The marks seem unrelated to the tang adaptation process (often involving hammering to widen this part slightly). In this case, the signs chiselled on the tang's surface did not affect the tang outline as the induced deformation is local and very limited. One possibility is that these signs could be part of a marking system used to match a blade with a hilt.

Very little, or next to nothing, is known about the workshops where the octagonal-hilted swords were produced. About 250 swords belonging to this type are known -covering approximately 150 years [START_REF] Bunnefeld | Das Eigene und das Fremde -Anmerkungen zur Verbreitung der Achtkantschwerter[END_REF], indicating an average production of fewer than two swords per year. Although it is difficult to give a precise estimation of the number of octagonal-hilted swords that were produced in total, the production of this kind of weapon was probably somewhat limited. We should not picture large workshops with many craftsmen working on a large number of swords simultaneously, but instead small workshops or singular craftsmen mastering the whole or at least most of the chaîne opératoire. The similar composition of the Champagneux sword's hilt and blade (tab. 1) is consistent with the hypothesis whereby a single craftsman or workshop is in charge of production. Within this framework, the marks may have proven helpful for the craftsman to identify which blade has to be adapted to which hilt when working on a few swords at once, meaning that a similar marking system should also exist on the handle, which was not observed on the sword from Champagneux. For example, these marks could have been included in the ornamentation of the hilt, which is not preserved here. Comparing these marks with the ones known on Bronze Age bracelets and tools may provide other explanations, such as a marking related to the length of the blade or a way to test the casting quality. As the Champagneux sword is so far the only sword where such marks were observed on the tang, it is difficult to go further in their interpretation.

CONCLUSION

Although the sword from Champagneux does not easily fit in the typological classification of this kind of object, the technical study shows that it perfectly matches the technical standards of the solid-hilted swords coming from the north-Alpine region between Bronze C and Hallstatt B1 (mid-15th-10th century BC). The blade was likely cast from the top of its elongated tang, as indicated by the distribution of the inner blows and shrinkage porosity concentrated in its upper third. It is associated with a hilt that probably was cast from the side of its pommel using the 'cire perdue' (lost wax) process. The wax model was likely built around a clay core and may have included a vertical stand going through the pommel -at the origin of the canal between the handle's inner space and the pommel's surface. Both parts were then fixed together by blocking one another and then adding two rivets on the guard. The 'counting marks' observed on the sword's tang may relate to the fixation process where the tang needs to fit the shape of the hilt so that both elements can be blocked together. The marks may have helped the craftsman identify the blade to be adapted to one specific hilt. There could also be other explanations for the marks; related to the length of the blade, for example, or the quality of the metal, but it is, of course, difficult to discuss these hypotheses based on only one sword. It would be interesting to conduct an extensive and systematic CT-scan campaign on octagonalhilted swords handles and re-examinations of previous scans to look for similar marks on other swords, hopefully producing substantial advances in identifying the production of specific workshops and the way their production was organised.

Figure 1 .

 1 Figure 1. General view, details, and radiography of the Champagneux sword (Musée Escale Haut-Rhône, Brégnier-Cordon, Ain, France, no. 2011.1.1). © L. Dumont, UGCT.

Figure 2 .

 2 Figure 2. Distribution of Middle Bronze Age octagonal-hilted swords in Europe (after Holste[START_REF] Holste | Die bronzezeitlichen Vollgriffschwerter Bayerns[END_REF][START_REF] Bianco Peroni | Die Schwerter in Italien. Le spade nell'Italia continentale[END_REF][START_REF] Krämer | Die Vollgriffschwerter in Österreich und der Schweiz[END_REF][START_REF] Kemenczei | Die Schwerter in Ungarn II (Vollgriffschwerter)[END_REF][START_REF] Quillfeldt | Die Vollgriffschwerter in Süddeutschland[END_REF][START_REF] Wüstemann | Die Schwerter in Ostdeutschland[END_REF][START_REF] Laux | Die Schwerter in Niedersachsen[END_REF][START_REF] Winiker | Die bronzeitlichen Vollgriffschwerter in Böhmen[END_REF][START_REF] Bunnefeld | Älterbronzezeitliche Vollgriffschwerter in Dänemark und Schleswig-Holstein: Studien zu Form, Verzierung, Technik und Funktion[END_REF]). © L. Dumont.

Figure 3 .

 3 Figure 3. Different sections through the Champagneux sword's hilt produced by µCT scanning. The red arrows show the limits of the core residue deposited on the hilt's inner wall. © UGCT.

Figure 4 .

 4 Figure 4. Different views of the cropped reconstructed volume of the Champagneux sword's hilt. The tang carries a series of five carved marks which are vertically arranged. © UGCT.

Figure 5 .

 5 Figure 5. Locations of the measured points using hand-held XRF. © S. Lycke & L. Dumont.

Table 1 .

 1 Concentrations of the elements measured by hand-held XRF. © S. Lycke & P. Tack.

		Champagneux_bl1	Champagneux_bl2	Champagneux_hd3 Champagneux_hd4
		C[w%] C_err[w%] C[w%] C_err[w%] C[w%] C_err[w%] C[w%] C_err[w%]
	Cu	74,6	7,42	79,47	7,9	80,19	7,98	77,83	7,74
	Sn	13,48	1,62	14,44	1,73	16,22	1,95	14,82	1,78
	Pb	0,21	0,05	0,22	0,06	0,22	0,06	0,25	0,07
	Mn	0,01	0,01	0,01	0,01	0,01	0,01	0,01	0,01
	Fe	0,01	0,02	0,01	0,02	0,01	0,03	0,02	0,03
	Co	0,02	0,02	0,02	0,02	0,02	0,03	0,02	0,02
	Ni	0,06	0,02	0,06	0,02	0,06	0,02	0,06	0,02
	Zn	0,12	0,08	0,13	0,09	0,13	0,09	0,12	0,09
	As	0,05	0,01	0,04	0,01	0,05	0,01	0,04	0,01
	Ag	0,03	0,06	0,05	0,09	0,01	0,01	0,08	0,16
	Bi	0,03	0,01	0,04	0,01	0,03	0,01	0,04	0,01

ACKNOWLEDGEMENTS

This work was made possible thanks to a subsidy coming from the Direction Régionale des Affaires Culturelles (DRAC) of the Auvergne-Rhône-Alpes region (France). The study is taking place within the frame of the BOF-funded project 'Production and diffusion of Bronze-Age solid-hilted swords in Western Europe' (2018)(2019)(2020)(2021)(2022), supervised by Guy De Mulder. Pieter Tack is funded by a BOF grant (BOF20/PDO/037). The authors wish to thank Mr Jean-Paul Thevenot, former senior researcher at the CNRS and experienced metalworker, for his precious help and guidance concerning the reconstruction of the Champagneux sword's chaîne opératoire. They are also grateful to Sylvie Boulud-Gazo, and Thibaud Poigt for their help in finding comparisons to the counting marks observed on the Champagneux sword.


Aouatef Abaza

Jean-Marc Soleilhac

Joanne Westendorf

Matthieu Piel

Isabelle Crevel ||

Aurélien Roux

Dr Fabienne Pirollet
email: fpirollet@cea.fr

M-Phase Phosphoprotein 1 is a human plus-end directed kinesin-related protein required for cytokinesis

The human M-Phase Phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (1), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubulesstimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves towards the microtubule plus-end at a 0.07 µm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis.

Introduction

Eukaryotic cells exhibit dramatic changes of microtubule organization and dynamics as they enter mitosis [START_REF] Dustin | Microtubules, Second Ed[END_REF][START_REF] Kirschner | [END_REF]. These changes are timely and spatially coordinated with nucleus and membranes alterations by the tight control of M-phase promoting factor (MPF) 1 , whose catalytic component, the p34 cdc2 or cdk1 kinase becomes activated at the G2-M transition (4,5). Extensive progress has been made to describe the complex circuitry of phosphatases and kinases, which regulates cdk1 activation (6,7) and the downstream molecular pathways (8,9).

Microtubule dynamics are an intrinsic property of the polymer of tubulin and are highly regulated by the balance of the activity of different factors throughout the cell cycle (10)(11)(12). Several Microtubule-Associated Proteins (MAPs) have been described to promote tubulin assembly and polymer stabilization or destabilization (13,14). Besides their roles in intracellular trafficking of organelles and vesicles during interphase, dyneins and Kinesin-Related Proteins (KRPs), microtubule-based molecular motors, play important roles in cell division. At each stage of mitosis or meiosis, dyneins and various KRPs interact with microtubules in order to insure centrosome separation, spindle formation and maintenance, chromosome congression and cytokinesis completion (15)(16)(17)(18)(19).

However, whereas it is well established that the p34 cdc2 kinase is centrally involved in the regulation of microtubule dynamics during mitosis (20), only a few cdc2 substrates with plausible involvement in the control of microtubule dynamics have been identified so far. The p34 cdc2 kinase phosphorylates the ubiquitous MAP4 during M-phase (21) and this phosphorylation abolishes MAP4 microtubule stabilizing activity (22). There is evidence that the phosphorylation of the microtubule destabilizing protein Stathmin/Op18 by p34 cdc2 is important for mitotic progression (23). Similar phosphorylation of the mitotic KRP Eg5 is required for Eg5-dependent centrosome migration and bipolar spindle formation in vivo (17).

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These data suggest that mitotic kinases regulate microtubule dynamics and organization by phosphorylating various microtubule-interacting proteins and this has been an incentive for the systematic search of mitotic phosphoproteins.

We have recently identified a subset of M-phase phosphoproteins by expression library screening using the MPM2 monoclonal antibody, which recognizes a phosphoepitope present on a set of 40 to 50 proteins that become phosphorylated at the G2-M transition (1,(24)(25)[START_REF] Davis | Molecular regulation of nuclear events in mitosis and meiosis[END_REF]. Among the 11 proteins identified, we show here that M-Phase Phosphoprotein 1 (MPP1) has extensive homology with proteins of the kinesin superfamily. We demonstrate that MPP1 is a plus-end directed molecular motor with an important role in cytokinesis.

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Experimental procedures

Cloning and sequencing of full-length human MPP1 cDNA Human MPP1 cDNAs were obtained by screening two λzapII cDNA libraries made from HeLa, one a generous gift from P. Chambon, the other a Uni-ZAP XR library from Stratagene, following standard procedures [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. Starting with the 645 bp N-terminal fragment of the previously described partial-length MPP1 cDNA 6-1 (24) as a probe, the human MPP1 cDNA, 1L9 (4265 bp) was isolated. The overlapping MPP1 cDNA, 1C12 (2822 bp) was obtained by a second round of screening using a N-terminal fragment of pBS-1L9. Fulllength MPP1 plasmid, pBS-MPP1, was obtained by subcloning the 2408 bp N-terminal fragment of the pBS-1C12 in pBS-1L9. The HsMPP1 sequences are available under accession numbers AY282406, AY282407. Sequencing was performed by the Genome Express Company, Grenoble. The sequences were analyzed and database searches were performed with GCG, FASTA, BLAST programs available at NCBI or INFOBIOGEN ressources. Specific motifs were searched using PESTFIND, COIL, PredictNLS, pSORT programs.

FLAG epitope tagging of MPP1 by mutagenesis

The MPP1 coding sequence starting at base 70 was tagged using mutagenesis technique based on M13-phage ssDNA protocol (Amersham Sculptor Kit). 1C12 ssDNA was obtained using standard procedures [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. A 66-mer oligonucleotide was designed that introduced between bases 69-70, 36 bp containing a NotI restriction site and encoding MDYKDDDDK amino acids which correspond to the FLAG epitope upstream of the cleavage sequence of enterokinase. After sequencing of the 5' end of a selected clone, the 3' end fragment NsiI-BamHI (2484 bp) was replaced by the similar fragment obtained from the original 1C12 to ensure that no other mutations were introduced in plasmid pBS-m1C12.

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Orientation of the mutated Nterminal EcoRI fragment (484 bp) was inverted in the pBluescript vector and the 5145 bp NsiI fragment of pBS-MPP1 was subcloned into this plasmid to construct pBS-mMPP1 which encoded full-length MPP1 tagged with the FLAG epitope.

Expression and purification of recombinant MPP1 mutants in insects cells

A recombinant full-length MPP1 (rMPP1) and a truncated form (rMC1), both tagged with a N-terminal FLAG epitope, were produced by baculovirus expression following the manufacturer's instructions of the Bac-to-Bac system (Life Technologies). The mutated NotI-KpnI fragment of pBS-mMPP1 and the NotI-XhoI of pBS-m1C12 were respectively subcloned into the pFastBac HTb vector in phase with its 6His coding sequence to generate doubly tagged recombinant viruses in Sf9 cells. Recombinant proteins were then expressed in High-Five cells, a generous gift of Dr B. Goud. Cells were harvested at 48 h after viral infection at MOI=2, frozen in liquid nitrogen and stored at -80°C. Frozen cell pellets were resuspended in ice-cold lysis buffer (50 mM Tris, pH=8, 0.5 M NaCl, 2 mM MgCl 2 , 5 mM CaCl 2, 1 mM DTT, 0.02%(v/v) Triton X-100, in presence of Complete TM inhibitors (Boehringer). After sonication, the lysate was cleared by centrifugation at 90,000 g for 45 min at 4°C and loaded onto an Anti-FLAG M2-agarose column (Sigma). After washing, the adsorbed proteins were eluted with 3.5 M MgCl2 and buffer exchanged on a PD-10 column (Pharmacia) equilibrated in 50 mM Tris, pH=7.4, 0.2M NaCl. For biochemical studies, the 6His tag, which induces protein precipitation, was removed by cleavage with the TEV protease as described in the technical information (Life Technologies). The rMPP1 and rMC1 proteins were then concentrated on Ultrafree-4 centrifugal filters (Millipore). The final fractions were aliquoted, frozen in liquid nitrogen and stored at -80°C. For gliding assays, the proteins were complemented with 1 mM ATP, 2 mM MgCl 2 , 0.1mg/ml casein and frozen inserm-00353257, version 1 -21 Jan 2009 without concentration. Protein concentration was determined colorimetrically using BSA as a standard and Bio-Rad Protein Assay (Bio-Rad).

Anti-MPP1 antibody production and purification

A polyclonal anti-MPP1 antibody was raised by Eurogentec using four injections of 100 µg of 6His-rMPP1 proteins in rabbit. The antibody was affinity-purified by three-step positive-negative affinity purification. The antiserum was filtered through 6His-FLAGunrelated protein and 6His-rMC1 affinity-columns in order to remove any antibodies reacting with tags and conserved motifs present in the MPP1 motor domain. Specific anti-MPP1 antibody, which recognizes epitopes present in the C2 to tail domains, was then purified by passage of the filtrate onto a 6His-rMPP1-affinity column. Purified anti-MPP1 antibody was eluted with Tris 50 mM, MgCl 2 3.5 M, pH=7.5, dialyzed overnight against PBS and stored at 4°C.

Fluorescent microtubule spindown assay

Purification of bovine brain tubulin [START_REF] Mitchison | [END_REF], polymerisation and purification on glycerol cushion of taxol-stabilized microtubules (MTs) were performed using standard procedures.

MTs concentration (i. e., concentration of tubulin dimer in MT polymer) was determined according to Desai et al (29). Recombinant MPP1 proteins, routinely 0.1 µM, were cleared by ultracentrifugation and mixed with taxol-MTs (1 µM) in 100 µl BRB80 buffer supplemented with 10 µM taxol. After incubation for 15 min at 25°C, reaction was fixed with 1ml of MEM-50% sucrose containing 1% glutaraldehyde. MTs were diluted and sedimented through a glycerol cushion onto coverslips as described in (30). Coverslips were post-fixed in -20°C methanol for 6 min and washed three times for 10 min in PBS containing 0.1% NaBH4 to prevent glutaraldehyde autofluorescence. MTs and recombinant proteins were stained with inserm-00353257, version 1 -21 Jan 2009 rabbit anti-Glu and ∆2 tubulin antibodies (1/1,000; a generous gift of Dr D. Job (31) and a mouse anti-Flag M2 antibody (1/500, Sigma). Fluorescent labeling was performed with Alexa 488-labeled anti-rabbit (1/4,000) and TRITC-labeled anti-mouse (1/1,000) IgGs antibodies from Molecular Probes and Jackson.

Measurement of steady state ATPase rates

Steady-state MT-activated ATPase rates were measured using a pyruvate kinase/lactate dehydrogenase-linked assay, mainly as described in (32). Briefly, ATPase activities were assayed at 30°C in 1 ml reaction volume of 100 mM K-Pipes, pH=6.8, 4 mM MgCl 2 , 1 mM EGTA, 1 mM PEP, 0.3 mM NADH, 40 U of pyruvate kinase and 55 U of lactate dehydrogenase. NADH oxidation was followed at 340 nm in a temperature-controlled UVIKON 923 spectrophotometer. Rates were determined during the linear phase after 5 min for attainment of steady state, using ε-NADH = 6220 M -1 .cm -1 . The kinetic parameters k cat , K 1/2 MT (the concentration of MTs required for half-maximal activation) and Km for ATP were obtained by least squares fitting the MT activation or ATP dependent data to rectangular hyperbolae using Sigmaplot.

Motility assay with polarity-marked microtubules

A standard motility assay (33) was performed with recombinant MPP1 proteins using the fluorescence based kinesin motility kit from Cytoskeleton. Polarity marked MTs with a bright seed and a dim elongated segment at their plus-end were prepared by inclusion of NEM-treated tubulin, according to previously described protocols (see Methods page at the kinesin website http://www.proweb.org/kinesin). Briefly, acid-washed flow cells were coated with motor protein (typically 0.15 and 0.3 µM for rMPP1 and rMC1 respectively) in motility assay buffer (MAB = BRB80 buffer supplemented with 0.1 mg/ml casein, 1 mM ATP, 20 µM inserm-00353257, version 1 -21 Jan 2009 taxol and an oxygen scavenging mix). After 5 minutes at room temperature, non-adsorbed motor was washed out with two flow cell volumes of MAB. Asymmetrically labeled MTs (0.2 µM) were then flowed through the cell and allowed to interact with the motor for 5 minutes at room temperature. Finally, unbound MTs were washed out with 2 flow cell volumes of MAB. Video images of MTs were acquired in a thermostated room with a Princeton CCD Micromax RTE 1317K1 camera on a Zeiss Axioscop with a 100 x 1.3 NA Plan-Neofluar lens using IPLab software (Ropper Scientific). Measurement of MT velocities was performed using the RETRAC program available at http://mc11.mcri.ac.uk.

Preparation of a GFP fused mutant of MPP1

The 70-981 bp portion of MPP1 cDNA was amplified by recombinant PCR using TAcloning kit (Invitrogen). This fragment was cloned into the XhoI site of pEGFP-C2 eukaryotic expression vector (Clontech), using an XhoI restriction site introduced upstream of the initiation codon. The pEGFP-sM plasmid obtained encodes the GFP protein fused to the N-terminus region of MPP1 extending from aa 1 to aa 304. The C-terminal HindIII-KpnI fragment of pBS-MPP1 was subcloned into this plasmid to construct pEGFP-MPP1-FL, which encodes the full-length fusion protein GFP-MPP1.

Cell culture and cloning

HeLa and HCT116 cells were grown in RPMI-1640 and McCoy medium (Gibco) supplemented with 10% FCS, respectively. HeLa cells were transfected with the various pEGFP-MPP1 plasmids using the FuGENE TM 6 transfection reagent as described by the manufacturer (Boehringer Mannheim). Stable clones expressing the GFP-MPP1-FL fusion protein were then isolated, by using the limited dilution method in the presence of 500 µg/ml inserm-00353257, version 1 -21 Jan 2009 G418. In some instances, cells were analyzed with treatment with 250 nM of trichostatin A overnight to increase expression (34).

SiRNA preparation and transfection

MPP1-specific small interfering RNA (siRNA) duplexes were designed according to Harborth et al (35). Sequences of the type AA(N 19 )UU (N, any nucleotide) were searched in the open reading frame of MPP1-mRNA and submitted to a BLAST search to ensure their specificity. Selected 21-nt sense and 21-nt antisense oligonucleotides targeting MPP1 from position 424-446 (siRNA1) or 4782-4804 (siRNA2) relative to the start codon were purchased from Dharmacon (Lafayette, CO) in deprotected and desalted form. As unspecific siRNA controls, we used an unrelated sequence that failed to target p160ROCK mRNA (siRNAU) (36) or a siRNA1 sequence mutated on two nucleotides (siRNA1m). Annealing and transfection was performed as previously described (37). HCT116 cells were transfected with siRNAs using Oligofectamine (InVitrogen). Mock-transfections were also performed using control buffer instead of oligonucleotides. At different time points after transfection, cells were harvested and either fixed and processed for FACS analyzis or analyzed by Western blot after addition of SDS-PAGE sample buffer. Time-lapse imaging was also performed.

Immunofluorescence microscopy

Exponentially growing cells were plated on glass coverslips and incubated for 24-36 hours. Cells were fixed in methanol at -20°C for 8 min and processed with primary and secondary antibodies diluted in PBS with 1mg/ml bovine serum albumin. The primary antibodies used were purified anti-MPP1 IgGs (5 µg/ml, this study), a mouse monoclonal anti ß-tubulin 2-3 B11 (1/5,000, a generous gift of Drs A. Giraudel and L. Lafanechère, unpublished results) or mouse monoclonal anti-mitosin 14C10 (1 µg/ml, from GeneTex).

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Suitable Cy3-(Jackson, 1/1,000) or Alexa 488-(Molecular Probes, 1/500) conjugated antibodies were applied as secondary antibodies. DNA was stained with Hoechst 33258 (1 µg/ml) or Topro 3 (1/1,500, Molecular Probes). The coverslips were examined on a Zeiss microscope by using a 100 x 1.4 oil immersion objective. Confocal images were obtained on a TCS-SP2 Leica laser scanning microscope. Z series were collected and displayed images correspond to projections of optical sections (0.2 µm thick), which number varied in relation to the cell depth.

Flow cytometric analysis

For standard analysis of DNA content, cells were washed once with PBS, trypsinized, fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.2% triton X-100 in PBS. DNA was stained overnight at 4°C with 2 µg/ml of Hoechst. Cells were sorted on a FACS Star Plus cytometer (Beckton and Dickinson Co). After collection of 20, 000 events, results were analyzed with CellQuest software and aggregated cells were gated out.

For double staining of DNA and specific antigen, cells were fixed with ice-cold 70% ethanol.

Labelling of MPP1 or mitosin was performed before the DNA counterstaining step by incubation with anti-MPP1 or anti-mitosin antibodies followed by Alexa 488-conjugated antirabbit or anti-mouse IgGs (1/500, Molecular Probes).

Time-lapse imaging

For phase-contrast imaging, control or MPP1 siRNA transfected cells were trypsinized 6 h after transfection and transferred into a multiple well chamber of a poly-dimethyl siloxane gel fixed on a glass chamber coated with collagen and fibronectin. Sequential phase-contrast images of the various samples were recorded on a Leica DMIRBE microscope controlled by Metamorph software (Universal Imaging) for 66 h. The microscope was equipped with an inserm-00353257, version 1 -21 Jan 2009 open chamber equilibrated in 5% CO 2 and maintained at 37°C, and images were taken with a 20x objective using a cooled MicroMax 1MHz CCD camera (Roper Scientific).

For GFP-MPP1 imaging during mitosis, stably transfected cells were plated on coated coverslips and maintained at 37°C in sealed chambers containing complete phenol red-free RPMI medium supplemented with 20 mM Hepes. Rounded cells were searched and time lapse Z-sequences were collected as described by Savino et al (38)

Results

Identification of MPP1 as a kinesin related protein present in several human tissues

The entire human MPP1 cDNA (6325 bp) was cloned using two rounds of conventional cDNA library screening, starting with the previously obtained partial clone 6-1 (24). Sequence comparison in databases showed that MPP1 belongs to the kinesin superfamily of motor proteins with the characteristic organization into three domains (15) To test the force-producing capability of MPP1, we used a multiple-motor assay using polarity-marked MTs. Protein rMC1 induced MT motility with the minus end leading and most of the MTs (>90%) were seen gliding (Fig 3C). MT gliding was also observed with rMPP1 but, curiously, only a subset of relatively short microtubules (1-5 µm in length) was seen moving (data not shown). In both cases, gliding was abolished in the presence of 1 mM AMP-PNP (data not shown). The average velocity of microtubule gliding was 0.07 ± 0.01 inserm-00353257, version 1 -21 Jan 2009 µm/s and 0.071 ± 0.007 µm/s for rMPP1 and rMC1, respectively. These data demonstrate that MPP1 is a slow plus-end directed KRP, when compared to already described motors (43).

MPP1 distribution during the cell cycle

Knockdown of MPP1 induces apoptosis and cytokinesis defects

We used small interfering RNA (siRNA) duplexes (37) to assess the consequences of

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These data indicate that MPP1 is important for cell growth and completion of cytokinesis. Whereas it does not appear to be necessary for initiation of furrowing and cleavage furrow ingression it seems to play an important function for further cell separation.

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Discussion

In this study, we find that MPP1, a member of a set of mitotic phosphoproteins specifically recognized by the MPM2 antibody at the G2/M transition (1), is a slow plus-end directed KRP that plays critical roles in cytokinesis.

As other KRPs, MPP1 exhibits MT activated-ATPase activity and binds to MTs (43).

Additionally, MPP1 apparently bundles microtubules in vitro and behaves as a slow molecular motor in gliding assays. Phylogenic analysis of KRP motor domains (FP, unpublished results, (45,46)) shows that MPP1 belongs to the N6-class of KRPs, which includes RB6K (18,47,48) and orthologs of MKLP1 (49)(50)(51)(52). As these KRPs, MPP1 displays an insertion in the N1-N2 sequence landmark of the motor domain when compared with conventional KHC (53). MPP1 insertion (77 aa) is the longest and this determines a N1-N2 spacing of 190 aa compared to less than 132 in other KRPs classes (53). MPP1 also displays an insertion (28 aa) at the end of the core motor domain, suggesting an extended neck linker region in the protein. Both MKLP1 and RB6K display similar insertions. Interestingly, motor domain and neck linker insertions concern loops (L6 and neck linker loop, respectively) whose interaction is important for motility behaviour (54) and this suggests concerted evolution of the two loop domains. As MPP1, MKLP1 cross-bridges microtubules in vitro and induces a slow (0.066 µm/s) plus-end movement of the tubulin polymers in gliding assays (50). These common functional features may be related to the structural characteristics shared by the two proteins.

MPP1 has a specific pattern of localization and expression during the cell cycle, being mostly nuclear in interphase cells with a sharp increase in expression in G2, diffuse in metaphase cells, with subsequent association to the central spindle and the midbody, at the end of mitosis. Motors with mitotic functions show similar cell-cycle regulated localization on mitotic structures and subsequent concentration in the midbody during cytokinesis inserm-00353257, version 1 -21 Jan 2009 (18,48,50,(55)(56)(57). The midbody localization of MPP1 was not evident in previous works (46,58) but seems to be a consistent property of MPP1 in different localization assays (IF, GFP-MPP1 distribution in vivo) and different cell lines, HeLa, HCT116 (this work) and HUVEC (data not shown). This new localization of MPP1 seems important in light of our siRNA studies indicating a vital role of the protein in the correct completion of cytokinesis.

Cytokinesis depends on the presence of a number of proteins (59-61) including the other N6-class KRPs, RB6K and MKLP1 orthologs (18,19,48,49,51). Given their properties in vitro, these KRPs may be involved in both midzone microtubule bundling and subsequent microtubule sliding that are required for successful completion of cell cleavage (62). It is remarkable that MPP1, RB6K and MKLP1 are all independently required for cytokinesis, despite apparent functional and structural redundancy. Maybe, the rational for this multiplicity of motors will be revealed by detailed studies of molecular interactions between these motors themselves and between these motors and other major components of the midbody such as INCENP (63), survivin (64,65), TD60 (66), PRC1 (67). The three motors may also be included in different signalling pathways, involving different GTPases (68)(69)(70)(71) and kinases (72)(73)(74).

In conclusion, our data show that MPP1 is a novel KRP whose activity is required for proper progression of cytokinesis in human cells. Further work is needed to see if this newly discovered function of MPP1 is related to its mitotic hyperphosphorylation. inserm-00353257, version 1 -21 Jan 2009

Figure legends

Fig. 1. Structural organization of human MPP1

A. Human MPP1 cDNA representation and genomic organization. The three cDNAs 6-1, 1L9 and 1C12 that were cloned as detailed under Experimental procedures are drawn as thin lines above the schematic representation of the full-length MPP1 cDNA. Horizontal lines indicate 5' and 3' -untranslated regions. The enlarged 70-5412 sequence encodes a kinesin family member, 1,780 amino acid residues long whose predictive mass is 206,187. It exhibits a conserved organization into three regions (15). The dark grey box (M), which corresponds to the hyper-conserved motor domain, indicates that MPP1 is an NH2-terminal motor. In the central region, four α-helical domains (C1 to C4) shown in light grey are predicted to form coiled-coil conformations as determined by the program of Lupas et al (75). The tail region, which is supposed to interact with cargos, exhibits five putative MPM2-recognition sites and one presumptive NLS as indicated with grey and black triangles (24,76). No putative PEST and cyclin or DNA-binding domains sequences were found. HsMPP1 is identical to the recently described KRMP1 found independently by another laboratory and the hypothetical protein CAB55962, with the exception of 9 and 2 aa, respectively (46,77). The very high sequence identity suggests that the same gene encodes them all. The human genomic MPP1 locus (gb AL 157389) was identified using the MPP1 cDNA as a query in the Blast algorithm against the human genome database. The intron-exon boundaries were defined by direct residues are in bold and the positions of secondary structure elements are indicated according to KHC crystallographic data (39,40). The four conserved motifs, which interact with ATP, are drawn as boxes N1-4 as defined by Sablin et al (78). The open triangle indicates the limit between the KHC motor core domain and its neck region, which consists of β9 and β10 strands and a weak coiled-coil region extending at aa 352 (79). The heptad repeat positions predicted by Coils are underlined.

Fig. 2. Purification of recombinant MPP1 mutants and immunoblot analysis of MPP1 distribution in various human tissues

A. Preparation of recombinant full-length protein and MC1 mutant. Recombinant full-length MPP1 protein, rMPP1, or the deletion mutant rMC1 (aa 1-650), which contains the putative motor domain and the first α-helical domain C1, were synthesized as FLAG-tagged fusion proteins and purified to near homogeneity from insect cells as described under Experimental procedures. They were run on an 8% PAGE-SDS gel and stained by Coomassie. rMPP1 migration corresponds to the predicted size of MPP1 and to its previously identified apparent molecular mass (1). On the other hand, rMC1 migrates anomalously large with an apparent MW ~15 kDa larger than MW calculated, suggesting an elongated form of the mutant. A schematic representation of the two constructs is drawn. B. Immunoblot of recombinant MPP1 mutants and of various human cell and tissues extracts. Tissue homogenates were aliquots of Clontech's human protein medleys.

Recombinant proteins (10 ng) or extracts (20 µg) were run on 8% PAGE-SDS gel, transferred to Immobilon P (Amersham) in the presence of 0.01% SDS and immunoreacted with an affinity-purified anti-MPP1 antibody (0.5 µg/ml) prepared as described under Experimental procedures. ECL substrat (Amersham) was used for detection. Lack of reactivity with rMC1 inserm-00353257, version 1 -21 Jan 2009 mutant indicates that we selected antibodies, which recognize epitopes present in the C2 to tail domains, the most specific portion of the protein when compared to other KLPs.

Fig. 7. MPP1-depleted cells exhibit mitotic defects

A. Quantitation of cell progression phenotypes of control and MPP1-siRNA transfected cells. HCT116 cells were transfected as in Figure 6, transferred to live-cells chambers and three different fields were imaged every 5 min as described under Experimental procedures. Fifty to 60 cells were picked up randomly on the three images taken 18h after transfection and were followed until 54 h after transfection. HCT116 cells were scored for the various phenotypes observed in their cell cycle progression. When cells were dense, inserm-00353257, version 1 -21 Jan 2009 blebbing, with non-uniform membranes they were scored as apoptotic. Most of the cells become rounded and a well formed midbody between the two daughter cells appeared indicating that they were undergoing mitosis. When two viable daughter cells were formed, the cells were scored as going through successful mitosis. However, various defects in mitotic exit, daughter cells separation and further appearance of apoptosis were sometimes observed and the cells were then scored as undergoing failure of mitosis. In other cases, either mitosis or apoptosis were observed and we hypothesize that cells may be blocked. C. MPP1 expression in siRNA-transfected cells. Indirect immunofluorescence of HCT116 cells stained with the anti-MPP1 antibody, 24 h after transfection with the indicated duplexes. The pattern obtained after siRNAU transfection is similar to the one after mockand siRNAi1m transfection (data not shown). As the cells were stained and the pictures acquired and processed under exactly the same conditions, this indicates that the level of transfection with siRNAi1 was very high and we can assume that the cells that we looked at in Central Domain Tail N-ter Motor

  on a Leica DMIRBE microscope controlled by Metamorph software (Universal Imaging). This microscope was equipped with a piezoelectric device for rapid and reproducible focal changes, a 100 x 1.4 NA Plan Apo lens, a cooled CCD camera (Micromax 5MHz; Roper Scientific) and a DG4 illumination device. Z-stacks were deconvoluted and maximal-intensity projections were constructed. inserm-00353257, version 1 -21 Jan 2009

  , as detailed in Fig 1A. A search of genome resources indicated that a unique human MPP1 gene located on chromosome 10 in the 10q23.31 region spreads at least 73 kb and consists of 33 exons. A mouse ortholog (82% similarity) encoded by a conserved syntheny was found on murine chromosome 19. Alignment of the conserved motor domains of MPP1 and conventional kinesin heavy chain, KHC (Fig 1B) showed that MPP1 motor exhibits two large insertions (186-263, 480-507), which span respectively between alpha helix 2 and β sheet 4 and alpha helix 6 and β sheet 9 when compared to KHC structural data (39,40). For immunoblot analysis of MPP1 distribution, we used an affinity-purified MPP1 antibody directed against the C2 to tail domains (aa 651 to 1780) of MPP1 (Fig 2). This antibody reacted with a single 200 kDa band in HeLa cell extracts (Fig 2B, lane 3), which comigrates with purified recombinant full-length MPP1 (Fig 2A and 2B, lane 1). MPP1 was detected in several human tissues, including brain, ovary and kidney (Fig 2B). In the testis extract, a strong signal corresponding to a slightly lower MW band was detected and may correspond to a testis specific splicing variant of MPP1. Recombinant MPP1 behaves as a genuine molecular motor To assay MPP1 motor activity, we used recombinant proteins corresponding either to the complete MPP1 (rMPP1) or to a deletion mutant of the protein containing the putative inserm-00353257, version 1 -21 Jan 2009 motor domain and the first α-helical domain (rMC1, Fig 2A). The proteins were assayed for the characteristic activities of genuine KRP i.e. regulated ATPase activity, binding to MTs and ability to induce microtubule gliding on motor coated coverslips (15,41) (Fig 3). Both rMPP1 and rMC1 exhibited a basal ATPase activity, which was activated by addition of MTs (~ 280 or 630-fold for rMPP1 and rMC1, respectively; Fig 3A). The k cat and KmATP values of rMPP1 and rMC1 were close to each other and the MT concentration required for halfmaximal activation was ~6-fold higher for rMC1 mutant than for rMPP1. The microtubule binding activity of rMPP1 and rMC1 could not be assayed by conventional MT pelleting assays (42) due to partial insolubility of the unbound proteins. For visualisation of microtubule binding, control microtubules or microtubules incubated with rMPP1 or with rMC1 were centrifuged on coverslips and subsequently double stained with tubulin and FLAG antibodies (Fig 3B). In control samples short individual MTs were observed. Incubation with rMPP1 or rMC1 prior to centrifugation induced extensive MTs cross-linking. Recombinant proteins were associated with microtubule bundles, while being undetectable on single polymers. When ATP was added in the incubation medium, microtubule bundling was inhibited in a dose dependent way and many individual MTs could be observed on the coverslip (Figure 3B and data not shown). These results indicate that both rMPP1 and rMC1 bind to MTs in an ATP-dependent way and induce microtubule bundling, in vitro.

  MPP1 expression and localization during the cell cycle was investigated by immunofluorescence analysis of fixed HeLa cells (Fig 4). In interphase cells double stained with MPP1/tubulin antibodies, MPP1 was mainly localized in the nucleus. MPP1 was also detected in the cytoplasm as a punctuated pattern without clear association with microtubules (Fig 4A). The nuclear staining varied from cell to cell, suggesting a cell cycle dependent expression of the protein MPP1. This possibility was tested using indirect immunofluorescence and FACS analysis of HeLa cells double stained for MPP1 and mitosin, a centromere-associated-protein whose expression is strongly enhanced in G2 cells (44), (Fig 4B). The strongest MPP1 staining was observed in cells with bright mitosin-labelling, indicating enhanced MPP1 expression in G2. Accordingly, FACS analysis indicated a two to three fold increase of MPP1 expression as cells progress from G1 to G2/M (Fig 4B). During mitosis, in both prophase and metaphase cells, MPP1 staining showed fine punctuations diffuse throughout the cytoplasm (Fig 4C). At anaphase, MPP1 staining accumulated at the mid-plan of the cell and formed a distinct band extending across the spindle midzone (Fig 4C). In telophase cells, MPP1 was sharply concentrated in the midbody (Fig 4C). We further used GFP-tagged MPP1 to visualize the dynamics of MPP1re-distribution during the cell cycle (Fig 5). We observed extensive cell death during establishment of the cell line, suggesting a toxic effect of MPP1 overexpression. In cells with a GFP-MPP1 level apparently similar to that of the endogenous protein (Figure 5A), the pattern of expression was similar to that of the endogenous protein (data not shown). During cytokinesis, time lapse imaging of GFP-tagged MPP1 showed MPP1 in the midbody first as a single spot and inserm-00353257, version 1 -21 Jan 2009 then as two dots, four dots and again as two spots. MPP1 concentration at the midbody then decreases asymetricaly, with a spot staying visible in only one of the daughter cell until abscission occurs (Fig 5B). This behavior suggests that the plus-end directed motor activity of MPP1 may play a role in MTs organization during cytokinesis exit.

MPP1 suppression (Fig 6 and 7 )

 7 . Human HCT116 epithelial cells were transfected with two independent MPP1-specific siRNAs. Results were similar with both siRNAs and are shown in the case of siRNA1 in Figures 6 and 7. Immunoblot analysis of cell extracts showed extensive depletion of MPP1 24 hours after transfection (Fig 6A). FACS analysis showed no clear modification at this time point, but after 48h, a hypodiploid peak appeared, indicating accumulation of apoptotic cells. Cell apoptosis was further enhanced at the point 72 hours following transfection with specific MPP1 oligoduplexes (Fig 6B). We then used phasecontrast videomicroscopy to examine the behavior of the cells over 36h duration, starting 18 hours after transfection (Fig 7). Over this period of time, most cells, which have lost MPP1 (Fig 7C), underwent at least one M-phase (Fig 7A). In MPP1-siRNA1 treated cells, a high proportion of cytokinesis failure was observed (Fig 7A and B). Although a midbody formed, abscission did not occur. Either the midbody regressed with appearance of a binucleated cell, which further underwent apoptosis during a second round of mitosis or the midbody persisted and the two ill separated daughter cells finally underwent apoptosis. Such behaviour was observed in 42 % of siRNA treated cells whereas less than 10% control cells showed similar cytokinesis defects.

  comparison of the cDNA sequence against the genomic sequence. The corresponding exonintron structure of the human MPP1 gene is shown below with exons as numbered boxes and introns as connecting lines. B. Comparison of MPP1 and conventional KHC motor domains. Sequences of the first 550 and 350 amino acids of MPP1 and HsuKHC (sp P33176) were aligned using the Blast 2 sequences program. Sequences are 40% similar with 25% identity. Conserved inserm-00353257, version 1 -21 Jan 2009

Fig. 3 .

 3 Fig. 3. Activity of recombinant MPP1 and MC1

Fig. 6 .

 6 Fig. 6. MPP1-depleted cells undergo cell death

B

  . Cell cycle progression of control and MPP1-siRNA transfected cells. Selected frames of the time-lapse phase-contrast microscopy videos were shown: top row after transfection with siRNAU duplex, one typical control siRNA-transfected cell; middle and bottom rows after transfection with specific siRNA1 duplex, two typical siRNA1-transfected cells undergoing mitosis failure. The black arrow indicates the cell observed. m1 and m2 indicate entry into first or second mitosis, daughter cells are labelled with white arrows. Symbol marks ( , ) show daughter cells from the same mitotic cell. md indicates a mitotic defect, bin, a binucleated cell and a, an apoptotic cell. Time shown is in hours and min.

Fig

  Fig 7B are actually transfected. inserm-00353257, version 1 -21 Jan 2009

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The abbreviations used are: MPF, M-phase promoting factor; MAPs: Microtubule-Associated Proteins; KRPs, Kinesin-related proteins; MTs, Microtubules; MPP1, M-phase phosphoprotein 1; GFP, Green fluorescent protein; siRNA, small interfering RNA. inserm-00353257, version 1 -21

Jan 2009

Acknowledgments

The authors thank D. Job for helpful discussion and comments during the writing of the manuscript. We are grateful to R. Cross, B. Goud and M. Bornens for their support. We are indebted to D. Grunwald and V. Collin for assistance with confocal microscopy and FACS. This work was supported by grants from LNCC (Ligue nationale contre le cancer, N° 10V04) and from ARC (Association pour la recherche contre le cancer, N° 9857) to F. Pirollet. J.M. Westendorf was supported by fellowships from ARC and FRM (Fondation pour la recherche médicale). A. Abaza was a recipient of French Research Ministry and a FRM fellowship. inserm-00353257, version 1 -21 Jan 2009