Nayana Tusamda Wakhloo

Sebastian Anders

Florent Badique

Melanie Eichhorn

Isabelle Brigaud

Tatiana Petithory

Maxime Vassaux

Jean-Louis Milan

Jean-Noël Freund

Jürgen Rühe

Patricia M Davidson
email: patriciadavidson@curie.fr

Laurent Pieuchot
email: laurent.pieuchot@uha.fr

Karine Anselme

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography

Keywords: Nucleus, Migration, Micropillars, Cytoskeleton, LINC

Cell deformation occurs in many critical biological processes, including cell extravasation during immune response and cancer metastasis. These cells deform the nucleus, its largest and stiffest organelle, while passing through narrow constrictions in vivo and the underlying mechanisms still remain elusive. It is unclear which biochemical actors are responsible and whether the nucleus is pushed or pulled (or both) during deformation. Herein we use an easily-tunable poly-L-lactic acid micropillar topography, mimicking in vivo constrictions to determine the mechanisms responsible for nucleus deformation. Using biochemical tools, we determine that actomyosin contractility, vimentin and nucleo-cytoskeletal connections play essential roles in nuclear deformation, but not A-type lamins. We chemically tune the adhesiveness of the micropillars to show that pulling forces are predominantly responsible for the deformation of the nucleus. We confirm these results using an in silico cell model and propose a comprehensive mechanism for cellular and nuclear deformation during confinement. These results indicate that microstructured biomaterials are extremely versatile tools to understand how forces are exerted in biological systems and can be useful to dissect and mimic complex in vivo behaviour.

INTRODUCTION

Cells reside in intricate 3 Dimensional (3D) microenvironments within which they are subjected to various mechanical constraints that modulate their behavior. The nucleus, which is the largest and stiffest organelle within the cell, represents a physical constraint that limits cell migration in narrow spaces. [START_REF] Davidson | Broken nuclei -lamins, nuclear mechanics, and disease[END_REF][START_REF] Denais | Nuclear envelope rupture and repair during cancer cell migration[END_REF][START_REF] Mcgregor | Squish and squeeze-the nucleus as a physical barrier during migration in confined environments[END_REF][START_REF] Harada | Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival[END_REF] The impact of cell confinement on nuclear deformation and cell mechanics have been extensively studied using physical methods such as micropipette aspiration, micro-indentation or isolating nucleus from cells. [START_REF] Dahl | Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures[END_REF][START_REF] Pajerowski | Physical plasticity of the nucleus in stem cell differentiation[END_REF][START_REF] Lammerding | Nuclear mechanics and methods[END_REF][START_REF] Guilluy | Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus[END_REF] Although these techniques have established some new paradigms on cell-nuclear mechanics, they do not reflect how cells spontaneously deform in vivo. Microfabrication methods have emerged as an extremely versatile tool to engineer and recapitulate various microenvironments, allowing direct visualization of cell spontaneous confinement. [START_REF] Velve-Casquillas | Microfluidic tools for cell biological research[END_REF][START_REF] Berre | Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes[END_REF][START_REF] Malboubi | An open access microfluidic device for the study of the physical limits of cancer cell deformation during migration in confined environments[END_REF][START_REF] Davidson | Design of a microfluidic device to quantify dynamic intra-nuclear deformation during cell migration through confining environments[END_REF] Microfluidic channels have proved very useful to study cell migration. Using these, actin, vimentin and linker of the nucleoskeleton and cytoskeleton (LINC) complex proteins have been implicated in translocating the nucleus through constrictions during migration. [START_REF] Mcgregor | Squish and squeeze-the nucleus as a physical barrier during migration in confined environments[END_REF] Microtubules have also been implicated in nucleus movement in neurons and muscle cells. [START_REF] Wu | Nesprins and opposing microtubule motors generate a point force that drives directional nuclear motion in migrating neurons[END_REF][START_REF] Gimpel | Nesprin-1alpha-Dependent Microtubule Nucleation from the Nuclear Envelope via Akap450 Is Necessary for Nuclear Positioning in Muscle Cells[END_REF].These studies indicate that the nucleus may be pushed from behind or pulled from the front, although it has remained difficult to precisely define the mechanism. [START_REF] Cao | A Chemomechanical Model for Nuclear Morphology and Stresses during Cell Transendothelial Migration[END_REF] We previously demonstrated nuclear deformation ex situ on 3D micron-sized pillars [START_REF] Davidson | Micro-structured surfaces cause severe but non-detrimental deformation of the cell nucleus[END_REF][START_REF] Badique | Directing nuclear deformation on micropillared surfaces by substrate geometry and cytoskeleton organization[END_REF][START_REF] Hanson | Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells[END_REF]: osteosarcoma cells spontaneously remodel their nuclei to fit within the available spaces between 3D micropillars whereas healthy bone cells span the tops of the pillars. [START_REF] Davidson | Topographically induced self-deformation of the nuclei of cells: dependence on cell type and proposed mechanisms[END_REF] These pillar arrays were further used by other researchers to evaluate various cell lines for their ability to deform, or the impact of confinement on differentiation processes. [START_REF] Pan | Control of cell nucleus shapes via micropillar patterns[END_REF][START_REF] Liu | Subcellular cell geometry on micropillars regulates stem cell differentiation[END_REF][START_REF] Ermis | A high throughput approach for analysis of cell nuclear deformability at single cell level[END_REF][START_REF] Hasturk | Square prism micropillars improve osteogenicity of poly(methyl methacrylate) surfaces[END_REF] We show here that our micropillar arrays offer an elegant and simple method to dissect the deformation behaviour of cells in 3-D constrictions. This assay allow us to study nuclear deformation caused by the cell, independently from cell migration, in an assay in which the surfaces can be chemically modified and the cells can easily be treated with drugs and collected for biochemical studies. We combine 3D micropillar topography of controlled surface chemistry with live imaging and in silico cell numerical simulation to understand the mechanism by which cells deform their nuclei. We show that nuclear deformation of osteosarcoma nuclei in confinement is orchestrated by pulling forces which are regulated by actomyosin coupled to LINC complex proteins and is assisted by vimentin intermediate filaments.

MATERIALS AND METHODS:

All products were purchased from Sigma-Aldrich (France) unless specified. CELL CULTURE: SaOs-2 osteosarcoma cells (ATCC, USA) were cultured in a humidified incubator at 37 °C and 5% CO2. McCoy's 5A Modified medium was supplemented with streptomycin (0.1 mg/ml), penicillin (100 U/ml), heat inactivated fetal bovine serum (15% v/v, Biowest, France) and L-Glutamine (2mM). For all experiments 1X10 5 /ml cell density was used in 60mm petri dishes.

MICROPILLAR FABRICATION, CELL SEEDING ON PILLAR TOPOGRAPHY, CHEMISTRY MODIFICATION:

For more details, see previous publications [START_REF] Davidson | Micro-structured surfaces cause severe but non-detrimental deformation of the cell nucleus[END_REF][START_REF] Badique | Directing nuclear deformation on micropillared surfaces by substrate geometry and cytoskeleton organization[END_REF][START_REF] Davidson | Topographically induced self-deformation of the nuclei of cells: dependence on cell type and proposed mechanisms[END_REF].PDMS CAST: silicon templates with square micropillars measuring 7µm (height) x 7µm (length) x 7µm (space) were fabricated in IMTEK (Freiburg, Germany). Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning, USA) negative replica stamps were produced by uniformly mixing the precursor and curing agent in 10:1 ratio and pouring it over the silicon template glued in an aluminum cup. Following degassing in the vacuum pump for 30 minutes to remove air bubbles, PDMS mixture was cured at 60°C in an oven overnight (ON). Hardened PDMS was then separated from template carefully and used to prepare Poly-L-Lactic acid (PLLA) micropillars.

PLLA FILM: PLLA powder (Evonik, Germany) was mixed in chloroform solution and poured in glass petri plate. Uniform PLLA concentration and volume were chosen to keep it constant per unit area of the 10cm glass Petri plate (0.002g/cm 2 ). PLLA film was obtained by allowing the mixture to air dry in a fume hood overnight and then cut into pieces for further use.

PILLAR FABRICATION: Hot embossing was prepared by heating the PDMS negative replica above the glass transition temperature of PLLA (180°C). The piece of PLLA film was placed over the PDMS template and pressed down manually for approximately 5-10 sec. The assembly containing PDMS-PLLA sandwich was quenched in cold water to vitrify PLLA before demolding. SAMPLE PREPARATION: Samples were glued using cell compatible aqua-Silicone glue (Den Bravent Sealants, Netherlands) in 60mm Petri plates and then sterilised using 70% ethanol for 10 minutes and dried under laminar air flow.

MICROPILLAR DIMENSIONS AND CHEMICAL MODIFICATIONS:

Polymeric micropillars were fabricated by double replication. As an original, a structured Si wafer was used. The wafer was microstructured with square micropillars with a width of 10 µm, a height of 10 µm, and spacing of 3 µm. The Silicon wafer was made by the clean room service center of the University of Freiburg (IMTEK). For the first replication step (negative), a commercial PDMS elastomer kit (Sylgard 184, Dow Corning, Midland, USA) was used; base and curing agent were mixed in the ratio of 10:1 (w/w). The mixture was poured over the etched Si wafer and cured at 80 °C for 3 h. The hard PDMS was peeled off the Silicon wafer. The polymer used in the second replication step (positive copy) was obtained by free radical polymerization of n-butyl acrylate (nBA) and 4-(methacryloyloxy) benzophenone (MABP); 5 mol% MABP were used. The resulting P(nBA-co-5%MABP) was dissolved in toluene (100 g/L). A drop of the dissolved polymer was placed on a glass slide (Menzel Gläser cover glasses, round 18 mm, No. 3, VWR, Germany). The PDMS stamp was pressed on top of the dissolved polymer. The combination of all three components (stamp, polymer and glass slide) was crosslinked by UV irradiation (λ = 365 nm, 12.98 J, 90 min). The PDMS stamp was peeled off to liberate the completed microstructured surface. The surface was modified by different methods: To modify the entire surface, spin coating (1000 rpm, 60 sec) with polymer solutions was used either with the cell-repellent (Poly dimethyl acrylamide) P(DMAA-co-5%MABP) (solution in ethanol, 50 g/L), or the cell-attractive (AA-all attractive) P(nBA-co-5%MABP) (solution in toluene, 50 g/L). To modify only the top of the pillars (TA-top attractive, SA-space adhesive), a plane aluminum foil (1 cm× 4 cm) was dip coated (immersion and retraction speed 100 mm/min) with the dissolved polymer. The aluminum foil was then pressed on top of the microstructure and promptly removed. The coated polymer was attached to the surface by UV light crosslinking (λ = 365 nm, 12.98 J, 90 min). For confocal verification of coatings, cell adhesive polymer was tagged with Cyn-5 streptavidin (GE Healthcare, USA) at a 1:500 dilution and cell repellent polymer was tagged with Rhodamine B isothiocyanate (Sigma, France) at 1% concentration with the polymer. DRUG TREATMENT: Concentrations were used as follows: Cytochalasin D (1 µM), Latrunculin B (2 µM), Colchicine (5 µM), Nocodazole (10 µM), 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride [ML 7] (20 µM), Blebbistatin (25 µM), Acrylamide (10 mM) and Iminodipropionitrile [IDPN] (2%). The optimum concentration (more than 50% viability) was determined using an MTT colorimetric assay 6 hours after drug treatment and measuring the resulting absorbance at 570nm (normalised at 620nm) using an EZ READ 400 Plate reader (Bichrom, UK). Percentage viability graph was plotted (S5). Drugs were added in the medium containing cells in suspension and then seeded on pillars for 6 hours. Drug washout experiments were performed at 2, 5 and 20 hours with1X PBS (Gibco, Life technologies, USA).

IMMUNOSTAINING:

Unless specified products were purchased from Sigma Aldrich (France). Cells were fixed with 4% formaldehyde (Electron Microscopy Sciences, USA) and permeabilized using 0.5% Triton X-100 for 15 min and blocked with 3% bovine serum albumin for 1 hour. Incubation with primary antibodies was done for 1 hour with following antibody concentration: anti-vimentin (1:50; V6630; anti-Mouse), anti-βtubulin (1:200; T4026; anti-Mouse), anti-myosin IIA (1:100; M8064; anti-Rabbit), anti-paxillin (1:250; ab32084; anti-Rabbit; Abcam-UK), anti-pericentrin (1:100; ab4448; anti-Rabbit; Abcam-UK), anti-Histone deacetylase 1 (1:200; H3284; anti-Rabbit), anti-Sun 1 (1:100; M8064; anti-Rabbit), anti-Sun 2 (1:100; M8064; anti-Rabbit), anti-SYNE2 (1:50; HPA003435; Atlas, Sweden), anti-Lamin A, specific for lamin A only and not lamin C (1:500; L1293; anti-Rabbit). After short rinses in 1X PBS, secondary antibodies were incubated for 1 hour using Alexa 488 (1:200; ab150073; anti-Rabbit; Abcam-UK), Alexa 488 (1:200; ab150113; anti-Mouse; Abcam-UK), Alexa 647 (1:200; ab150115; anti-Mouse; Abcam-UK). Confocal acquisitions were obtained after PBS rinses. Actin-555 (1:20; A34055; Thermoscientific-France) or phalloidin-FITC (Fluorescein-5-isothiocyanate) (0.4µg/ml) were used to label actin and Hoechst (1:1000; 33258; Thermoscientific-France) was used to label nucleus. TRANSFECTION Dominant Negative-KASH plasmid DNA was kindly gifted by Dr Nicolas Borghi (Jacques Monod Institute, Paris-France). Lamin A-GFP was used to overexpress lamin A (17653, Addgene, USA). Cells cultured up to 60-70% confluency were transfected with plasmids using Lipofectamine 3000 (Thermoscientific, France) upon manufacturer's recommendations. Culture media used for transfection was deprived of antibiotics. After transfection, cells were seeded for 48-72 hours on pillars before imaging.

For the nuclear deformation of the cell over time, cells were transfected with baculovirus kit for actin-RFP and histone-GFP (BacMan 1.0 and 2.0, Thermoscientific-France). The transfection was carried out according to the manufacturer's protocol. The particle per cell (PPC) value was determined to be 40 for SaOS-2. For histone-GFP kit 1.0, cells were incubated for 4 hours with baculovirus, followed by 2 hours incubation with enhancer at 37 °C with 5% CO2 in a humidified incubator. For actin-RFP kit 2.0, the cells were directly transfected with the baculovirus and allowed to grow for 16 hours at 37 °C with 5% CO2 in a humidified incubator.

SiRNA TREATMENTS AND QUANTITATIVE REAL TIME-PCR: SiRNA (Thermo Scientific): non-target negative control (AM4613), SYNE2 (s23330), SUN2 (s24465), SUN1 (s23629) and LMNA (s8221). Transfection was carried out using Lipofectamine® RNAiMAX Transfection Reagent (Thermoscientific, France) following the manufacturer's instructions. 10pmol concentration of siRNA was selected for the knockdown. Vimentin siRNA was used at a 40pmol concentration (sc-29522, Santa Cruz Biotechnology, USA). RNA extraction was carried out after 72 hours of KD treatments using RNAeasy micro-kit (Qiagen, France). The quantity and quality of RNA was determined using a Nanodrop spectrophotometer (Maestro, Taiwan). Retrotranscription was performed using iQ™ SYBRgreen mastermix (Biorad, Switzerland) in iScript cDNA synthesis instrument (Biorad, Switzerland). The data was normalized against the 18S methyltransferase (emg1) housekeeping gene. Primers were designed with melting temperature at 60°C, using the GenScript Real-time PCR Primer Design algorithm (https://www.genscript.com/tools/real-time-pcr-tagman-primer-design-tool). The qRT-PCR efficiency was determined using LinRegPCR software (Heart failure research centre, Netherland). Expression levels were further analyzed based on ΔΔCT method (n=2). The primer sequences are given in S8. The qPCR program was one cycle at {95 C-30 s}, followed by 39 cycles at {95 C for 5 s} and {60 C for 30 s}. PCR products were then sequentially heated to {65 C for 2 s} and to {95 C for 5 s} to measure the dissociation curve. Experiments were independently repeated at least twice. qPCR efficiency was assessed using the LinRegPCR program (version 2015.2). The expression levels of the genes were analyzed based on the ∆∆CT method, as described by Livak et al. [START_REF] Livak | Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF] WESTERN BLOT ANALYSIS: Unless specified, instruments and supplies were all purchased at Bio-Rad (Germany). Cell lysis and protein resuspension were achieved in radio immunoprecipitation assay (RIPA) buffer. Total protein amount was estimated using the colorimetric Micro BCA™ Protein Assay (Thermoscientific-France). 20μg of total protein sample was loaded on a precast Mini-protean TGX stain-free gel and separated according to size by electrophoresis. Protein gel images for normalization were acquired using ChemiDoc™ Imaging Systems before transfer. Proteins were transferred from the gel to 0.2μm polyvinylidene difluoride (PVDF) pre-activated membranes using the Transfer Blot turbo system. Afterwards, unspecific sites were saturated with 3% BSA (Sigma, France) for 1 hour at room temperature (RT). Primary antibody incubation was performed overnight at 4°C, for 1h at RT. Primary antibody incubation was performed overnight at 4°C, using the following antibody concentrations: lamin A (1:500, L1293, Sigma, France), vimentin (1:200; V6630; anti-Mouse; Sigma-France), SUN1 (1:1000; ab124770; anti-rabbit; Abcam-UK), SUN2 (1:100; lot A45573; anti-rabbit; Sigma-France) and nesprin2 (1:1000; ab124770; anti-rabbit; Abcam-UK). After short rinses, blots were incubated with the Horse Radish Peroxidase (HRP) Goat anti-rabbit IgG-HRP conjugate (1:3000, Cat No. 170-6515) and Goat anti-mouse IgG-HRP conjugate (1:3000, Cat No. 170-6515) for 1h at RT. Protein bands were revealed using a chemiluminescent substrate (Clarity™ Western ECL Blotting Substrates) and digitally acquired using the ChemiDoc™ Imaging Systems. Full-sized immunoblots images are given in S9. CONFOCAL IMAGING: Imaging was performed using an upright Carl Zeiss LSM 700 confocal microscope, Germany, using ZEN software. For live imaging z stack were acquired using 63X/1.4 oil; 63X/1.0 VIS-IR water or 20X/1.0 DIC VIS-IR W Plan-Apochromat objective (Zeiss, Germany) equipped with a temperature (Okolab, Italy), CO2/O2 (Okolab, Italy), and humidity controlled chamber.

IMAGE ANALYSIS AND QUANTIFICATION:

NUCLEAR DEFORMATION: 3-D images of nuclei were quantified using ImageJ/Fiji (NIH, version 2.0.0-RC-61/1.51n, USA).( 40) All images to quantify nuclear deformation, were taken via live cell imaging in order to minimize artefacts due to fixation. The area below the pillars was considered as deformed and above pillars was considered undeformed. The deformed area was divided by the total cross-sectional area of the nucleus to obtain the percentage of deformation (see S1).

FOCAL ADHESION LOCALIZATION ON PILLARS: The height, the number of slices and the interval between slices was kept constant for all independent acquisitions. The image was then thresholded and the resulting particles were analyzed. The average number of FA in each section was plotted on the Y-axis showing the height of pillars and X-axis showing the average number of FA/cell.

IN SILICO CELL MODEL:

The cell model was developed and validated previously to analyze the influence of the substrate convexity on cell adhesion and nucleus strain in a previous study.(41) The in silico cell model is extensively described in Vassaux et al. [START_REF] Vassaux | Initial release of the adhesion cell model (Version v1.0.0)[END_REF] In the present study, the only modification to the model was to add the substrate composed of micropillars. The composition of the model and the adhesion process are same as used in Vassaux et al.(41) STATISTICAL ANALYSIS All the results were checked for significance by using one way ANOVA test followed by two-tailed Student's t-test (unequal variance) for pairwise comparison and Dunnet's test for multiple comparisons, using GraphPad Prism software (GraphPad Software, Inc). P-values were calculated by two-tailed unpaired Student's t-test as compared to control untreated cells for comparison between two groups and Dunnett's multiple comparisons test for comparison between more than two groups. The significance value obtained were categorized as P > 0.05 for ns (not significant). The error bars represent the standard deviation of the measurements. All experiments were performed independently three times.

RESULTS

Nuclear deformation is concomitant with chromatin, cytoskeleton and focal adhesion reorganization.

Kinetics of nuclear deformation and chromatin reorganization

We previously showed that SaOs-2 cells deform extensively on micropillars. Here we focus on this cell line and investigate the kinetics of nuclear deformation (S1 and method section) and cell adhesion on micropillars. Deformation of the nucleus is detectable one hour after the cell contacts the surface. The nucleus is progressively inserted in the interpillar space, reaching a maximal and steady deformation at 24 hrs (Fig. 1a,b and movie SM1). Here we define nuclear deformation percentage as the proportion of the nucleus below the tops of the pillars. (See Methods for more details.) Unless otherwise mentioned, all nuclear deformation measurements were performed after 24 hrs of incubation.

We observed that cells increased in number over time within the pillar arrays, which raised the question of cell division in confined cells. We found that the cells avoid confinement during division and ascend perpendicularly to the micropillar substrate to perform mitosis (S2 & SM 2).

The daughter cells subsequently invade the structure quickly after cytokinesis, faster than the initial deformation, likely because the daughter cells retain focal adhesions from mother cells that aid in spreading [START_REF] Thery | Cell shape and cell division[END_REF] .

Focal adhesion localization during nuclear deformation

Cells adhere to surfaces using integrin mediated focal adhesions (FA). We analyzed the FA localization on pillars at different timepoints (Fig. 1c). To facilitate the quantification, we segmented the pillars into three zones (top, middle and bottom) and quantified the average number of FAs for each area (see methods for details). FAs localize initially at the top part of pillars and gradually spread on the lateral sides. FA density increases over the initial 6-8 hours and reaches a plateau, concomitantly with the nuclear deformation kinetics. Nuclear deformation progression is correlated with an increase in FA density on the mid-lateral regions of the interpillar space.

Cytoskeletal organization during nuclear deformation

The cytoskeleton plays a crucial role in adapting the cell to its environment. We thus looked at the organization of cytoskeletal components (microfilaments, intermediate filaments (IFs) and microtubules) in deformed SaOs-2 cells. We observed strong accumulation of actin on the edges of pillars and uniform distribution throughout the cell with an increase in stress fibers across the top of the cell after 24 hours. IFs exhibited higher density around the pillars and accumulated around the nucleus at the center of the cell. Microtubules were uniformly spread throughout the cell (Fig. 1d). The cytoskeleton and FA organization during nuclear deformation is summarized in Fig. 1e.

Nuclear deformation is governed by actomyosin and intermediate filaments dynamics.

We dissected the contribution of microfilaments (actin), myosin, IFs (vimentin) and microtubules (β-tubulin) using cytoskeletal disrupting drugs. Optimal drug concentrations were determined using a cell viability assay (MTT) to determine the concentration at which at least 50% of cells survived drug treatment (S3 and methods). We used the percentage of nuclear deformation as a readout for nuclear confinement. We found that depolymerizing actin (cytochalasin D, latrunculin B), inhibiting the actin motor protein myosin II activity (blebbistatin, ML7) as well as depolymerizing IFs (acrylamide, IDPN) strongly reduced nuclear deformation compared to control cells (Fig. 2a andb). Microtubule depolymerization (colchicine, nocodazole) did not have any effect on nuclear deformation. Because acrylamide can also affect actin and microtubule organization [START_REF] Arocena | Effect of acrylamide on the cytoskeleton and apoptosis of bovine lens epithelial cells[END_REF], we confirmed the results using a gene silencing technique (RNA interference) for vimentin IFs specifically. The silencing efficiency was confirmed by RT-qPCR and western blot techniques (Fig. 2c andd). Nuclear deformation after vimentin knockdown (KD) confirmed the role of IFs in assisting nuclear deformation on micropillars (Fig. 2e). We performed drug washout control experiments for actin, myosin and vimentin. Our results showed that nuclear deformation was regained over time (2, 5 and 20 hours) after the respective drugs were washed out (S4). Our results indicate that acto-myosin and IFs play crucial roles in cell-nuclear confinement, but targeting microtubules does not affect nucleus deformation.

Coupling the cytoskeleton to the nucleus is necessary for nuclear deformation.

The LINC complex is required for nuclear deformation

The nucleus is connected to the cytoskeleton by nuclear envelope proteins which form the LINC complex. (26) Cytoskeletal components (actin, microtubules, IFs) are connected to nesprins, which cross the outer nuclear membrane, and in turn are connected to SUN proteins which cross the inner nuclear membranes. We silenced SUN1, SUN2 and SYNE2 (nesprin2) individually and confirmed the silencing efficiency by immunofluorescence, RT-qPCR and western blot (Fig. 3a-c).

Our results showed a small, but non-significant reduction in nuclear deformation in KD cells compared to control cells (Fig. 3d). Functional redundancy within SUN and Nesprin variants that were not silenced might have played a compensatory role in the process. We attempted to target multiple LINC components at the same time using the same RNAi approach but the silencing efficiency was reduced significantly.

Nesprins have a highly conserved KASH (Klarsicht, ANC-1, Syne homology) domain at their Ctermini that binds SUN proteins while their N-termini associate with different cytoskeletal constituents. Thus, we disconnected the LINC complex by overexpressing a dominant-negative KASH domain (DN-KASH-mCherry) (Fig. 3e and3g), resulting in displacement of endogenous nesprins to the ER.( 27) Nesprin 2 immunostaining confirmed the delocalization of endogenous nesprins after DN-KASH overexpression (Fig. 3f). Cells overexpressing DN-KASH deformed their nucleus less than control cells (Fig. 3g). Strikingly, the effect was dose-dependent: cells accumulating the highest DN-KASH levels presented the lowest nucleus deformation (Fig. 3g).

Altogether, these results highlight the central role of LINC-cytoskeleton coupling in nuclear deformation and cell confinement. Modulation of Lamin A levels does not affect the ability of SaOs-2 cells to deform their nuclei Lamin A proteins play a central role in the regulation of nuclear mechanics. [START_REF] Davidson | Broken nuclei -lamins, nuclear mechanics, and disease[END_REF][START_REF] Swift | Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation[END_REF][START_REF] Denais | Nuclear envelope rupture and repair during cancer cell migration[END_REF] We therefore tested whether modulating their levels has an effect on SaOs-2 nuclear deformation. We downregulated endogenous lamin A/C using siRNAs or overexpressed a lamin A-GFP construct (Fig. 3h-k). Surprisingly, neither the increase nor decrease in A-type lamin levels affected the nuclear deformation ability at 24 hours (Fig. 3k). A-type lamins therefore do not determine the ability of cells to deform their nuclei.

Cell adhesion to the lateral sides of the pillars is necessary for nuclear deformation

We investigated how the cells interact with the substrate to deform themselves to understand whether they attach and pull or push from above to squeeze themselves in narrow spaces. We created three types of surfaces: (1) all adhesive pillars, (2) top adhesive pillars and (3) interspace adhesive pillars, in which the tops of the pillars are cell-repellent but the cells can adhere to the space in between the pillars (Fig. 4 a). We used a more restrictive spacing (3µm) to obtain an intermediate level of deformation and detect increases as well as decreases in the extent of deformation. On "all adhesive" pillars, nuclear deformation at 24 hours reached 30±7% (Fig. 4 b). In contrast, on "top adhesive" pillars, no nuclear deformation was observed (0%). Strikingly, on "interspace adhesive" pillars, the nuclei were completely inserted between the pillars, resulting in a maximum of the nuclear deformation (100%). Altogether, these results show that adhesion to the lateral edges of pillars but not to the top of the pillars is necessary for driving nuclear deformation. Surprisingly, modification of pillar chemistry and topography did not affect hMSC nuclear deformation (S5), indicating that restricting adhesion to the lateral edges of the pillars is not sufficient to induce cell and nucleus deformation.

In silico analysis of nuclear stress during cell confinement

To attain further insight on the effect of micropillar topography on the mechanical stress on the nucleus during self-confinement, we used an in silico cell model published by Vassaux et al. [START_REF] Vassaux | Initial release of the adhesion cell model (Version v1.0.0)[END_REF] It assumes that the cell's mechanical homeostasis results from the equilibrium between contraction forces generated by the actomyosin network and compression forces originating from microtubules, intermediate filaments and the cytosol. The LINC complex is modeled as connections between the nucleoskeleton and the three cytoskeletal networks (microfilament, microtubules and IF). Briefly, the nucleus was represented as a dense packing of particles in contact, enclosed by a stretchable envelope. The model represents the mechanical stress during nuclear deformation depending on variables such as presence and absence of: i) LINC ii) apical actin cap and iii) pillars dimensions and chemistry modulations. The mechanical stress is assumed to be caused by nuclear components themselves and the cytoskeletal elements (Fig. 5,S6). In the absence of the LINC complex, nuclear deformation and mechanical stress on the nucleus are reduced compared to the reference model comprising the LINC complex (~4-fold and 2-fold less respectively) (Fig. 5a-c) which recapitulates the experimental data. This shows that in the absence of LINC complexes, the cytoskeleton is unable to transmit forces (observed by least mechanical stress) required for nuclear deformation. We modeled the pillar dimensions and chemistry constraints to quantify the forces on the nucleus. We used the "all adhesive" pillars as a reference for calculation. The mechanical stress experienced by the nucleus during deformation was at a minimum on "all adhesive" and "top adhesive" pillars, whereas it was at a maximum on "interspace adhesive" pillars, reaching twice the value observed with the reference surface (Fig. 5d-f). Taken together with the experiments, the highest mechanical stress observed on the interspace adhesive pillars configuration suggest that cells probably self-confine by pulling themselves on the substrate. Besides pulling forces, cells in confined spaces also experience pushing forces that can be generated by the apical actin cap.( 31) Lastly, we examined the influence of actin cap on both nuclear deformation and mechanical stress. We observed that loss of apical actin has no impact on mechanical stress or deformation of nucleus which also coincides with the experimental results (Fig. 5g-i, S7), suggesting that the pushing down forces might not play a significant role in deforming the nucleus.

DISCUSSION

The mechanisms by which cells exert forces to confine their cell body and particularly their rigid nucleus into narrow constrictions is not yet fully understood. We dissect here the role of different cytoskeletal elements during spontaneous nuclear confinement using a multidisciplinary approach that combines cellular and molecular biology techniques, 3D surface chemistry modifications and in silico cell modeling. We show that the nucleus is pulled down by the cell through its LINC complex using actomyosin contractility assisted by vimentin (Fig. 6). Micropillar arrays are a versatile tool to study nuclear deformation:

Here we study nucleus deformation due to cell-generated forces using our micropillar-based selfconfinement assay. This strong deformation of the nucleus is maintained for hours, yet it is reversible: as cells divide, they lose then regain their deformation. This assay allows a high degree of versatility and is easily tunable as we show in this manuscript. 1) We can easily change the size of the constrictions to modulate the extent of deformation. 2) We can change the surface chemistry to determine the directionality of forces. 3) We can rapidly add drugs and other chemicals to determine the actors responsible for the phenomenon. It is important to note that the phenomenon we study is not due to migration into the surface (migration can only occur perpendicularly to the surface). We are thus studying nuclear deformation by the cell specifically and decoupled from migratory cues. Nonetheless, these mechanisms may be used by the cell during cell migration to deform the nucleus.

The forces on the nucleus involve the LINC complex, vimentin, actin and myosin contractility:

We found that actin, myosin activity and vimentin are necessary for nucleus deformation and confinement by the cell. This is in agreement with studies that have linked migration through narrow constrictions with vimentin and actin activity (reviewed by McGregor et al.(3)). We demonstrate that disruption of the LINC complex using a dominant negative KASH construct results in severe loss of deformation of the nucleus. This strongly indicates that the LINC complex is involved in exerting force on the nucleus during the deformation. These results reflect studies in which the LINC complex is involved in moving the nucleus in 2-D [START_REF] Lee | LINC complexes and nuclear positioning[END_REF], nesprin-2 knock-down increases transit time through narrow constrictions [START_REF] Thomas | Non-muscle myosin IIB is critical for nuclear translocation during 3D invasion[END_REF] and a linker of actin and the LINC complex is necessary for nucleus translocation during migration through narrow constrictions. [START_REF] Jayo | Fascin Regulates Nuclear Movement and Deformation in Migrating Cells[END_REF] The nesprins that are likely to exert forces on the nucleus are the two giant isoforms that bear actin-binding domains, however we found that knocking down these isoforms individually did not affect nucleus deformation. This may be due to redundancy between the two giant nesprins, or may indicate that other nesprins could contribute. Indeed, Nesprin-3α can aid force transmission to the nucleus through vimentin. [START_REF] Petrie | Generation of compartmentalized pressure by a nuclear piston governs cell motility in a 3D matrix[END_REF] Further study may thus be necessary on the individual nesprins and intermediary proteins involved in this deformation.

Cells pull on their nucleus:

Cells that only adhere to the tops of pillars are unable to deform their nuclei and the extent of deformation is strongly increased when adhesion is constrained to the sides of the pillars (fig 4). Thus, contractile forces from above the pillars are not required to deform the nucleus. This striking result does not agree with proposed mechanisms by which nucleus deformation during migration through narrow constrictions is caused by contraction at the rear of the cell.(3) Other studies have suggested that the nucleus is being pulled during migration (3), also based on the shape of the nucleus as it is deformed. [START_REF] Cao | A Chemomechanical Model for Nuclear Morphology and Stresses during Cell Transendothelial Migration[END_REF] It is important to note that here we study the deformation of the nucleus by the cell decoupled from migratory cues, and thus contraction at the rear of the nucleus may be necessary for forward migration of the cell but not for nuclear deformation. is therefore likely that the mechanisms are dependent on the particular circumstances of the nuclear deformation, but multiple mechanisms may also be involved, which may synergistically contribute to forward migration through constrictions. Lamin A/C does not affect the extent of deformation: Previous studies have found that cells with reduced lamin A/C levels translocate their nucleus through narrow constrictions more quickly than their wild-type counterparts. [START_REF] Davidson | Nuclear Deformability Constitutes a Rate-Limiting Step During Cell Migration in 3-D Environments[END_REF][START_REF] Harada | Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival[END_REF] We found that increasing or decreasing nuclear deformability by reducing or increasing lamin A/C expression does not affect the extent of deformation by the cell at 24 hours. However, future studies should focus on whether lamin A/C levels may affect the speed of deformation on micropillars, which would be in agreement with results obtained in the migration studies.

The mechanisms by which healthy cells prevent deformation: had previously shown that MSCs react to the pillars by spanning the top of the pillars rather than adhering between the pillars and deforming the cell body and nucleus. We show here that even in circumstances in which the adhesion to the top of the pillars is prevented, the MSCs adhere in between the pillars without deformation. Thus MSCs likely have mechanisms to prevent the deformation of the nucleus. We observed that the A-type lamin levels were comparable in between the SaOs-2 cells and MSCs, indicating that lamins are not the sole determinants of nuclear deformation. The difference may thus lie in the coupling between the nucleus and the cytoskeleton, or the manner in which the cells engage contractility at the LINC complex. Direct mechanical coupling between adhesions and the nucleus may be lacking in these cells.

CONCLUSIONS AND OUTLOOK

Our 3D micropillar array platform provides a simple highly tunable approach to access nuclear deformation using cell-generated forces decoupled from migration. We have already shown nuclear deformation in a variety of cell types. [START_REF] Pan | Control of cell nucleus shapes via micropillar patterns[END_REF][START_REF] Liu | Subcellular cell geometry on micropillars regulates stem cell differentiation[END_REF][START_REF] Ermis | A high throughput approach for analysis of cell nuclear deformability at single cell level[END_REF][START_REF] Hasturk | Square prism micropillars improve osteogenicity of poly(methyl methacrylate) surfaces[END_REF] Recently the nuclear deformability of human pluripotent stem cells during early germ layer specification was investigated using 3D micropillar array platforms and found to be lost during ectoderm differentiation. [START_REF] Grespan | Effect of geometrical constraints on human pluripotent stem cell nuclei in pluripotency and differentiation[END_REF] Nuclear deformability on micropillars could thus be used to determine differentiation. This technique could be further exploited to explore the 3D organization of chromosome territories and gene expression pattern modifications and unravel other molecular players/cell signaling pathways related with confinement-induced nuclear deformation.

DATA AVAILABILITY

The processed data required to reproduce these findings are available to download from http://dx.doi.org/10.17632/psfmnh8n2y.1. The raw data are available upon request. --------------------------------------------------------------------- - --------------------------------------------------------------------

S8: Western blot

Total protein gel images for KD of lamin A, SUN1, SUN2, nesprin 2 and vimentin used for protein normalization and corresponding immunoblots.

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Figure 1 .

 1 Figure 1. Nuclear deformation involves focal adhesions, chromatin and cytoskeletal dynamics. a) SaOs-2 deformation kinetics on pillars. Orthogonal images show cells expressing actin-RFP (red) and histone H2B-GFP (green) seeded on micropillars at different time points during cell adhesion (for video see supplementary movie 1). b) Quantification of nuclear deformation. (For a description of the analysis see supplementary figure S1). The mean value is indicated by a red line. Pvalues compared to initial cell adhesion on pillars at 2 hours. (n=150/time point, 3 independent experiments.) c) FA localization and nuclear deformation on pillars, z-stack projection of SaOs-2 cells (actin-green; paxillin-red; nucleus DNA-blue) at different time points. Subsets represent the average number of FAs at different heights (see figure on left). (n=45/time point, 3 independent experiments). d) Immunostaining (vimentin & β-tubulin) and phalloidin (actin) labelling showing cytoskeleton

Figure 2 .

 2 Figure 2. Actomyosin and IF drive nuclear deformation. a) Immunostaining of SaOs-2 cells on micropillars 24 hrs after drug treatments. Z stack projection of cells immunostained with actin (green) and nucleus (magenta); myosin IIA (green); vimentin (green), β-tubulin (green). b) Quantification of nuclear deformation of SaOs-2 6 hours after drug treatment (n=160/drug treatment) with mean (red line). c) RT-qPCR was used to access vimentin expression in knockdown and non-target control (nontarget RNA) cells (grey) normalised to emg1 gene expression, after 48 hours. d) Western blot was used to access vimentin protein levels after 48 hrs in knockdown and control cells. e) Quantification of nuclear deformation in vimentin knockdown and non-target control cells, after 48 hours, (n=150 nuclei/siRNA treatment). All experiments were performed independently three times.

Figure 3 .

 3 Figure 3. Nuclear deformation requires LINC-cytoskeletal coupling. a) SaOs-2 nuclei on micropillars 24 hours after siRNA treatments. Immunostaining for SUN1, SUN2 and Nesprin2 (SYNE2) at right top corner and Hoechst staining for nucleus (blue). All images are Z stack projection and grey squares represents the position of top part of pillars. b) RT-qPCR was used to access SUN1, SUN2 and SYNE2 expression in knockdown and control cells (grey) normalized to emg1 gene expression. c) Western blot was used to access SUN1, SUN2 & syne2 protein levels in knockdown and control cells. d) Quantification of nuclear deformation after 48 hrs of siRNA treatment (n=150/SiRNA treatment) showing mean (red line). e) Confocal images of SaOs-2 transfected with DN-KASH mCherry showing low and high DN-KASH expression. f) Displacement of endogenous nesprin (grey) after DN-KASH transfection compared to untreated cells. g) Quantification of nuclear deformation 48 hours after transfection of DN KASH in cells presenting high, low and no (CTRL) mCherry signal (n=150/SiRNA treatment). The mean value is shown with a red line. h) Confocal images of SaOs-2 after 48 hrs of lamin A knockdown (green) and lamin A-GFP overexpression (green). i) Lamin A/C expression in knockdown and control cells (grey) evaluated by RT-qPCR. j) Lamin A protein levels in knockdown and control cells. k) Graph showing nuclear deformation percentage after 48 hrs of lamin A knockdown, high lamin A-

Figure 4 .

 4 Figure 4. Effect of micropillars chemo-topographic modelling on nuclear deformation. a) Left, Sketch representing chemo-topographic alteration of 3/10 µm (space/height) micropillar surface chemistry (Blue: cell-adhesive PnBA coating, red: cell-repellent PDMA coating). Middle, Fixed cells stained for actin (green) and DNA (blue). Right, Orthogonal images of nuclei (blue) on 3/10 µm (space/height) micropillars. b) Quantification of nuclear deformation of SaOs-2 nuclei on different chemistry coated surfaces compared to 'All adhesive' surface; (n=30 nuclei/surface over three independent experiments).

Figure 5 .

 5 Figure 5. In silico cell model to analyze mechanical stress on nucleus during deformation. Computational model images showing the nucleus deformation and mechanical stress in mPa on pillars with and without LINC (a-c), after chemistry and topography surface modifications (d-e) and with and without LINC (g-i). Computational model images of adhesion on the pillars showing the nucleus deformation and overall stress. The values of the deformation of the nucleus obtained from the in-silico model simulations were calculated the same way as in vitro experiments using a cross-sectional view and dividing the area above pillars by the total area of the nucleus (see Fig. S1).

Figure 6 .

 6 Figure 6. Proposed mechanism underlying nuclear deformation. Actomyosin pulling force, coupled to FAs and LINC complexes, drives cell-nuclear deformation on pillar topography. In this system, vimentin IF also assist in nuclear deformation.

  The forces exerted on the nucleus are on the lateral sides of the pillars: Intriguingly, forces exerted from the sides of the pillars are sufficient to pull on the nucleus: there are no focal adhesions at the bottom of the pillars and the cells deform extensively in tall pillars (10 µm, fig. 4). observations of the cytoskeletal architecture and the progression of focal adhesion formation indicate that, as focal adhesions form on the edges of the pillars, the nucleus is pulled down by the actin cytoskeleton, aided by intermediate filaments. The progression of the nucleus between the pillars thus closely follows the appearance of focal adhesions, suggesting closely-related mechanisms for cell adhesion and nucleus pull-down, and may indicate direct coupling between focal adhesions and the LINC complex.

  CREDIT AUTHOR STATEMENT N.T.W. Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writingoriginal draft; S.A. Investigation, Methodology; F.B. Investigation, Methodology; M.E. Investigation, Methodology; I.B. Investigation, Methodology, Validation; T.P. Investigation, Methodology, Validation; M.V. Investigation, Methodology, Software; J-L.M. Methodology, Supervision, Validation; J-N.F. Writing -review & editing; J.R. Funding acquisition, Methodology, Supervision; P.M.D. Conceptualization, Methodology, Writing -review & editing; L.P. Conceptualization, Methodology, Supervision, Writing -review & editing; Conceptualization, Funding acquisition, Supervision, Writing -review & editing. 41. Vassaux M & Milan JL (2017) Stem cell mechanical behaviour modelling: substrate's curvature influence during adhesion. Biomech. Model. Mechanobiol 16(4):1295-1308.

-S2:

  Mitosis on pillars a) Orthogonal view of the SaOS-2 cell dividing on the micro-pillars with time, transfected with histone actin-RFP and histone-GFP. b) The drawing illustrates the mitotic phases performed by SaOS-2 on pillars. c) Confocal z stack projection images showing mitotic phases and centromere position during division (pericentrin-red, β-tubulin-green and DNA-blue).

  Graphs showing the MTT viability test and the respective concentrations chosen (red box) from different concentrations of drugs after 6 hours. Error bars represent ±SD resulting from three replicates over three independent experiments.

ACKNOWLEDGEMENTS

We are grateful to Dr. Nicholas Borghi (Jacques Monod Institute, Paris-France) for providing DN-KASH mcherry plasmid and Dr Tanmay Lele (University of Florida, USA) for Lamin-A GFP plasmid. This work was supported by Region Alsace [NTW and FB PhD grants], Deutsche Forschungsgemeinschaft [SA and ME PhD grants], IRTG Soft Matter [NTW, SA, ME, PMD], La Ligue contre le Cancer [REMX17751 to PMD and CCIR-GE120193 to KA] and the Fondation ARC [PDF20161205227 to PMD].

SUPPLEMENTARY FIGURES

S1: Calculation of nuclear deformation

Image shows the cross sectional area of nucleus (blue) on micropillar surface. The area above (A1) and below (A2) pillar is calculated using ImageJ/Fiji. The deformation of nucleus is calculated in percentage by the formula A2/A1+A2*100.

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M B Kozlikin

W Rendu
email: wrendu@u-bordeaux.fr

H Plisson
email: hugues.plisson@u-bordeaux.fr

M Baumann

M V Shunkov
email: shunkov77@gmail.

Unshaped Bone Tools from Denisova Cave, Altai

Keywords: Altai Mountains, Denisova Cave, Initial Upper Paleolithic, Denisovans, bone industry

HAL is

Introduction

The issue of the Upper Paleolithic culture origins still remains debatable, since the picture of evolutionary development created on the basis of European materials is not considered universal any more. As shown by the results of studies conducted in Africa, Eurasia, and Oceania in recent decades, the model of succession of cultures in the European Paleolithic just summarizes the local scenario of peopling processes. The context of Denisova Cave and other multilayered Paleolithic sites in the northwestern Altai does not agree with the European concept of Paleolithic development. The results of investigation of these sites indicate the concurrent existence of Denisovans, an earlier unknown population of the genus Homo, with Neanderthals in southern Siberia. Besides, they point to a relatively early (about 50-45 ka BP) appearance of stone and bone working technologies, as well as non utilitarian items, which correspond to the behavior of anatomically modern humans, though no evidence of the presence of such humans in Altai in the Initial Upper Paleolithic has so far been found. The concept of "modern behavior" itself is based upon analyses of the activities of Homo sapiens sapiens, whose representatives were the founders of the Upper Paleolithic traditions in the territory of Europe. Certain modern behavioral traits were refl ected in earlier materials in the African continent [START_REF] Henshilwood | The origin of modern human behavior: Critique of the models and their test implications[END_REF]. Before the appearance of anatomically modern humans, some groups of European Neanderthals seem to have experienced innovations, in particular in symbolic fi eld [START_REF] Hoffmann | Symbolic use of marine shells and mineral pigments by Iberian Neanderthals 115,000 years ago[END_REF]. The semantic shift between biological and cultural modernity, resulting from the historical background of prehistoric researches, could lead to a circular reasoning when enlarging the focus geographically and chronologically. To avoid such a situation, the context of any site must be analyzed taking into account its specifics, whatever took place in the western part of Eurasia at that time. Archaeological and anthropological materials from Denisova Cave are indicative of the gradual evolutionary development of behavioral traits typical of anatomically modern humans on the basis of local culture [START_REF] Derevianko | Three scenarios of the Middle to Upper Paleolithic transition. Scenario 1: The Middle to Upper Paleolithic transition in Northern Asia[END_REF]. This process is reflected not only by the stone industry but also by the bone. Denisova Cave is situated in the upstream flow valley of the Anuy River, in the low-mountain and mid-mountain zone of the northwestern Altai, at 690 m a.s.l. (Fig. 1). The cave consists of several narrow dark chambers, interconnected through the Main Chamber. The cultural and chronological range of Pleistocene deposits at the site is the greatest such range for North and Central Asian sites; it covers a period from the Early Middle Paleolithic (about 300 ka BP) to the Final Late Paleolithic [START_REF] Jacobs | Timing of archaic hominin occupation of Denisova Cave in southern Siberia[END_REF]. Ornaments and tools made from organic materials have been discovered in deposits aged 50-35 ka BP (layers 11.5-11.1 in the Main Chamber, layers 11.2 and 11.1 in the East Chamber, layer 11 in the South Chamber). Their creation at the early stage of the Upper Paleolithic is evidenced not only by the dates of enclosing sediments, but also by those of pendants made of elk incisors and bone points [START_REF] Douka | Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave[END_REF]. Stones, bones, and animal teeth, mammoth tusks, ostrich eggshells and mollusk shells were used as raw materials for manufacturing ornaments. These were treated using various techniques, such as scraping, grinding, polishing, sawing, and drilling [START_REF] Derevianko | A Paleolithic bracelet from Denisova Cave[END_REF][START_REF] Shunkov | Personalniye ukrasheniya ranney stadii verkhnego paleolita iz yuzhnoy galerei Denisovoy peshchery[END_REF]. Along with ornaments and stone tools belonging to the initial Upper Paleolithic, formal bone tools, mainly eyed needles and awls, were discovered in Denisova Cave. Mitochondrial and nuclear DNA recovered from anthropological remains and cave deposits belongs to Denisovans and Neanderthals [START_REF] Slon | Neanderthal and Denisovan DNA from Pleistocene sediments[END_REF]. This fact suggests that the transition to the Upper Paleolithic proceeded here on the basis of local Middle Paleolithic culture, as for example at the Arcy-sur-Cure site in France, judging by the results of analysis of the bone industry and ornaments from the Chatelperronian layer of this cave [START_REF] D'errico | Many awls in our argument. Bone tool manufacture and use in the Châtelperronian and Aurignacian levels of the Grotte du Renne at Arcy-sur-Cure[END_REF].

Lithic industries from Denisova Cave and Arcy-sur-Cure reflect the continuity of development: Middle Paleolithic types of tools were used along with new shapes. Meanwhile, traces left by H. s. sapiens at the early stage of the Upper Paleolithic are absent at these sites. The bone artifacts found here are not only a new group of artifacts, but also the evidence of using a new type of raw material that was traditionally considered to be culturally specific to anatomically modern humans [START_REF] Henshilwood | The origin of modern human behavior: Critique of the models and their test implications[END_REF]. Is there a contradiction?

Informal bone tools in the Paleolithic

Tools made of organic materials have been recorded in the Early and Middle Paleolithic assemblages unrelated to anatomically modern humans [START_REF] D'errico | Additional evidence for bone technology in the Southern African Middle Stone Age[END_REF]Backwell et al.;2008;[START_REF] Li | Use-wear analysis confirms the use of Palaeolithic bone tools by the Lingjing Xuchang early human[END_REF][START_REF] Mozota | El hueso como materia prima: El utillaje óseo del fi nal del Musteriense en el sector central del norte de la Península Ibérica[END_REF][START_REF] Soressi | Neanderthals made the first specialized bone tools in Europe[END_REF][START_REF] Stout | Late Acheulean technology and cognition at Boxgrove, UK[END_REF][START_REF] Julien | Characterizing the Lower Paleolithic bone industry from Schöningen 12 II: A multi-proxy study[END_REF][START_REF] Zutovski | The use of elephant bones for making Acheulian handaxes: A fresh look at old bones[END_REF][START_REF] Doyon | Discovery of circa 115,000-year-old bone retouchers at Lingjing, Henan, China[END_REF]. The Middle Paleolithic bone industries are characterized by small typological variability, the Upper Paleolithic ones by a great variety of shapes and manufacturing techniques (cutting, scraping, grinding, and polishing). If formal bone tools typical of the European Upper Paleolithic are discovered in earlier assemblages, they are considered as a sign of "modernity" [START_REF] Backwell | Palaeolithic bone tools[END_REF].Comparative analysis of bone industries is based on series of items, wherein unshaped tools are usually not taken into account. However, such items are recorded not only in the Middle Paleolithic assemblages, but also in the Upper Paleolithic ones. Solutrean assemblages of the last glacial maximum, state of the art of Paleolithic flint knapping, contain eyed needles that are the most ancient in Europe. At the same time, a considerable part of the bone industry in this culture consists of unshaped tools; among these, pressure tools for manufacturing thin leaf-shaped bifaces. These tools revealed by means of technological and use-wear analysis do not have evident signs of social or symbolic specialization. However, since they performed an important technical function, they should be considered one of the type specimens in the Solutrean industry [START_REF] Baumann | A l'ombre des feuilles de laurier, l'équipement osseux solutréen du Sud-ouest de la France: Apports et limites des collections anciennes[END_REF]. Manufacture of blanks for Aurignacian tools was based mainly on percussion [START_REF] Tartar | La fracturation du bois de renne à l'Aurignacien, mise en évidence d'une nouvelle modalité d'exploitation[END_REF]. Objects of the Aurignacian portable art and antler spearheads with split bases are known better than informal bone tools of this culture. Informal bone tools have become the subjects of special studies rather recently, for several reasons. First, owing to the absence of standardization in their shaping, it is difficult to identify them by typological criteria; second, bone items are often subject to mechanical and chemical destruction, and so are usually fragmented; third, they are hardly distinguishable from bones that were purposefully splintered by humans to extract bone marrow or were gnawed by carnivores. Thus, even if bone fragments are well preserved, it is often very difficult to distinguish shaping from other causes of anthropic or natural fracturing, in the absence of an appropriate method. Within the framework of this study, noteworthy are needles and awls of the Initial Upper Paleolithic from Denisova Cave. The uniqueness of these tools is determined by their function rather than by shaping technology. Eyed needles and awls are generally associated with sewing clothes, weaving and plaiting, i.e. with the ability of treating hides and plant fibers, which suggested the use of various types of tools. Discovery of a series of items of the same type in a single stratigraphic context can be considered the evidence of production areas at the site and its prolonged occupation. This is the reason why we were expecting more than eyed needles and awls in the bone assemblage of the Initial Upper Paleolithic of Denisova Cave.

Material and methods

Faunal materials from layers 11.4-11.2 in the East Chamber of the cave were analyzed in the course of this study. In layer 11.2, which was accumulated during the period of the fi rst half of MIS 3, artifacts of the Initial Upper Paleolithic have been found. Layers 11.3 and 11.4, formed during the time corresponding to MIS 4 and 5, yielded Middle Paleolithic finds. Bone fragments (over 10,000 spec.) up to 1 cm long were studied visually, without technical aids, and also using a Nikon SMZ-1 stereoscopic microscope. Photo-recording of materials was performed using a Canon 1000D and 100D SLR cameras, with a Canon EF-S 60 mm macro lens. The bone fragments were divided into three groups by the types of traces on their surface: taphonomic, technical, and from use. Earlier described materials [START_REF] Semenov | K izucheniyu tekhniki, tekhnologii izgotovleniya i funktsiy orudiy mustyerskoy epokhi[END_REF][START_REF] Fisher | Bone surface modifi cations in zooarchaeology[END_REF][START_REF] Villa | Bone and ivory points in the Lower and Middle Paleolithic of Europe[END_REF][START_REF] Maigrot | Etude technologique et fonctionnelle de l'outillage en matières dures animales, La station 4 de Chalain (Néolithique final[END_REF][START_REF] Pickering | Experimental patterns of hammerstone percussion damage on bones: Implications for inferences of carcass processing by humans[END_REF][START_REF] Baumann | Ideas no longer written in antler[END_REF][START_REF] Baumann | A l'ombre des feuilles de laurier, l'équipement osseux solutréen du Sud-ouest de la France: Apports et limites des collections anciennes[END_REF] and experimental data on manufacturing unshaped and formal bone tools were used as a comparative base. Post-sedimentary organic (microorganisms, animals, plants) and inorganic (weathering, soil subsidence, water courses) modifications, resulting in dissolution, cracking, striations, erosion, disintegration, vermiculation, concretions, were looked for. One of the most destructive factors in Denisova Cave was the activity of carnivores, especially hyenas. There are certain criteria that allow damage on bones left by animals to be distinguished from the traces of human activities [START_REF] Blumenshine | On the marks of marrow bone processing by hammerstones and hyenas; their anatomical patterning and archaeological implications[END_REF][START_REF] Villa | Flaked bone from a hyena den[END_REF][START_REF] Villa | Human and carnivore sites in the European Middle and Upper Paleolithic: Similarities and differences in bone modification and fragmentation[END_REF]. The traces associated with activities of hyenas are bites, gnawed out areas, deep scratches, and pitted or glazed surfaces formed during digestion-the most widespread mark of the vital activities of hyenas. Gnawed bone fragments are quite numerous; however, bone surfaces and edges have been little altered, probably because of a fast embedding and low temperature of the sediment, which was also propitious for DNA preservation. Consequently, the distinction between natural and anthropic traces is relatively easy there. The overall condition is good, and some samples with a well-preserved spongiosa would even look fresh, without spattering of oxide deposits on most pieces, particularly manganese. Crushed long tubular bones were most frequently the result of brain marrow extraction. The produced splinters could have been used as blanks for bone tools. However, without systematic refitting it is not possible to establish whether they were resulting from operating sequences included in or distinct from the butchering process. Primary spalls on bones and notches from percussions, are considered as traces of man-induced reduction. Smooth surface, curved or V-shaped outlines, oblique angle of the fracture facets [START_REF] Villa | Breakage patterns of human long bones[END_REF] are regarded as evidence of fresh-bone knapping.

Results

During sorting bone fragments from layers 11.4-11.2 in the East Chamber, 51 unshaped tools were found (see Table ; Fig. 2) on the basis of 30 % of faunal materials from this portion of section. The general state of preservation of bone materials from Denisova Cave is good, and spongiosa is in perfect condition. Unshaped tools were manufactured from the long tubular bones of ungulates of large (57 %) and middle (43 %) size. One item made of a vertebra, and two items made of rib fragments, have been found. The bones identifiable to a species belong to bison Bison priscus and red deer Cervus elaphus. Tibias were used as a basis. Humeri, femura, and metatarsi were used more rarely. Unshaped bone tools were divided into three categories in terms of localization of wear traces and manifestations of characteristics that allow the functional purpose of items to be determined. The bones showing wear traces on their cortical surfaces are assigned to the group of tools that, since the end of the 19th century, are classified as retouchers [START_REF] Daleau | Sur des lésions que présentent certains os de la période paléolithique[END_REF]. The items with wear traces at one or both ends are subdivided into two groups. The first group consists of tools with a smoothed end, like awls, designed to handle soft materials. The second one includes intermediate tools with axial damage to both ends; these tools had the same operating process, but, judging by the variability of wear traces and the morphology of their ends, were used for different purposes. Obviously, tools with retouch and/or wear traces on their longitudinal edges differed functionally.

Retouchers. Items of this type are the most widespread bone tools of the Stone Age. These are known from the Early Paleolithic [START_REF] Smith | Taphonomic resolution and hominin subsistence behavior in the Lower Palaeolithic: Differing data scales and interpretive frameworks at Boxgrove and Swanscombe (UK)[END_REF][START_REF] Kolfschoten | Lower Paleolithic bone tools from the 'Spear Horizon' at Schöningen (Germany)[END_REF], occur in the greatest number in the Middle Paleolithic industries [START_REF] Costamagno | Bone retouchers and site function in the Quina Mousterian: The case of Les Pradelles (Marillac-Le-France, France)[END_REF], and were still used in the Upper Paleolithic (Tartar, 2012b;[START_REF] Guadelli | The retouchers from the Gravettian Levels in Kozarnika Cave[END_REF] and Neolithic [START_REF] Taute | Retoucheure aus Knochen, Zahnbein, und Stein vom Mittelpaläolithikum bis zum Neolithikum[END_REF]. All retouchers have marks of percussions against sharp edges of stone tools. The function of retouchers was studied for the first time by H. Martin, using materials from La Quina, France (1906). According to him, these items could have been used as hammerstones to apply retouch and as anvils to perform cutting work. Later on, experiments were conducted to study the functions of these tools [START_REF] Semenov | K izucheniyu tekhniki, tekhnologii izgotovleniya i funktsiy orudiy mustyerskoy epokhi[END_REF]Shchelinsky, 1983;[START_REF] Bourguignon | Apports de l'expérimentation et de l'analyse techno-morpho fonctionnelle à la reconnaissance de processus d'aménagement de la retouche Quina[END_REF][START_REF] Rigaud | Retouchoirs sur éclats diaphysaires ou "affûtoirs" de la Labastide (Hautes-Pyrénées)[END_REF]. Most scholars agree that they were used for shaping lithic edges by percussion. Bone retouchers in layers 11.4-11.2 of Denisova Cave are rare; however, these are the first tools of this type found at the site. Blanks vary from long and narrow to short and large. Their length is 4.7-9.9 cm, width 1.1-3.6, and thickness 0.4-1.0 cm. Such heterogeneity is probably partly due to the small size of the sample and the diversity of the blanks, but also to the fragmentation of the artifacts: on at least 5 of the 8 samples, the impacted area was cut by a fracture, which occurred when the bone was fresh. In terms of density of percussion marks, the tools are distributed non-uniformly. The impact traces include both separate percussion marks (Fig. 3,b), and dense areas of percussion marks with microflaking (1-4 cm long) on the cortical surfaces of bones (Fig. 3,a). Retouchers are typical not only for layers 11.4-11.2: six such tools have been discovered in underlying Middle Paleolithic layer 12.

Tools with rounded end. The collection contains three small points with marks of smoothing. The lengths of the items are 5.6, 3.8, and 6.3 cm, widths 1.2, 2.1, and 1.0 cm, and thicknesses 0.2, 0.3, and 0.2 cm. Two tools are manufactured from ribs of ungulate animals of middle or large size, and the third from a small splinter of diaphysis. On one rib, an oblique break forming a natural trihedral point without traces of additional treatment is a working edge (Fig. 4, a-c). Working edges of other items are shaped by fi ne abrupt single-or double-sided retouch, as on lithic borers (Fig. 4,d).These tools correspond to the general definition of awls: "elongated objects made from bone material, partially or completely shaped, dimensions and section of which are variable, with a more or less acute point, sometimes smoothed or rejuvenated" [START_REF] Camps-Fabrer | Fiches typologiques de l'industrie de l'os préhistorique. Cah. III: Poinçons, pointes, poignards, aiguilles[END_REF]. Awls are commonly considered as instruments for piercing hides or other soft materials [START_REF] Maigrot | Etude technologique et fonctionnelle de l'outillage en matières dures animales, La station 4 de Chalain (Néolithique final[END_REF][START_REF] Christidou | Hide working and bone tools: Experimentation design and applications[END_REF]. Except sewing needles and projectile points, determined by their smooth surface and specific basal end (an eye or slots for fastening), any other pointed items are usually interpreted as awls, irrespective of their actual functions. Sharp ends of these three items became blunted, most probably as a result of working soft organic materials (see Fig. 4,a,b). Wear traces on two tools similar to lithic micro-borers are very small and correspond to the function of perforating medium-soft organic materials (see Fig. 4,d). Wear traces on the third item are more pronounced; undoubtedly, the tool was used for tailoring clothes, but not as an awl. The smooth morphology of the rounding, its distribution and extension, giving a spatula aspect to the active end (Fig. 4, a, b), strongly suggest a tiny hide burnisher, like the ones used for compacting the seam or for leather folding. Unlike the Initial Upper Paleolithic awls found in the cave, these tools were manufactured without scraping and grinding. Similar unshaped tools possibly lie in the Middle to Upper Paleolithic transition layers at the sites of Arcy-sur-Cure (Chatelperronian horizon), Cavallo, Cala, and Castelcivita (Uluzzian layers) in Italy, where numerous awls manufactured by scraping and grinding have been found [START_REF] D'errico | Les poinçons en os des couches châtelperroniennes et aurignaciennes de la grotte du Renne (Arcy-sur-Cure, Yonne): Comparaison technologiques, fonctionnelles et décor[END_REF][START_REF] D'errico | Uluzzian bone technology and its implications for the origin of behavioral modernity[END_REF]. Unshaped points have also been recorded in the earlier assemblages, such as Mousterian horizons of the Combe-Grenal site in Dordogne [START_REF] Tartar | L'utilisation des matières osseuses au Moustérien[END_REF], in deposits aged about 80-100 ka BP at the Lingjing site in the Chinese province of Henan [START_REF] Li | Use-wear analysis confirms the use of Palaeolithic bone tools by the Lingjing Xuchang early human[END_REF], and in the Early Paleolithic horizons of the Schöningen site in Lower Saxony [START_REF] Julien | Characterizing the Lower Paleolithic bone industry from Schöningen 12 II: A multi-proxy study[END_REF].

Tools with axial damage. These items form the most numerous group of unshaped tools (32 spec.). The majority of them have utilization traces, typical for intermediate tools. One (basal) end of tool was for striking, while the opposite one (apical) was for working. Striking against the basal end resulted in compaction of bone tissue that was sometimes with a crushed flange and, more frequently, by appearance of edge spalls. The bevelled apical end can be compacted and have spalls, while scratches caused by contact with the work material cover its cutting edge and polished areas. The degree of concentration and the types of marks at both ends of the tool depends on the worked material, the hammerstone, and the striking angle [START_REF] Rigaud | Utilisation du ciseau dans le débitage du bois de Renne à La Garenne-Saint-Marcel (Indre)[END_REF][START_REF] Provenzano | Fiche générale des objets à biseau distal[END_REF]Tartar, 2012a). Intermediate tools are well known from numerous Upper Paleolithic finds, whose identification caused no difficulty because of similarity with ethnographic analogs [START_REF] Lartet | Reliquiae Aquitanicae: Being contributions to the archaeology and palaeontology of Perigord and the adjoining provinces of Southern France[END_REF][START_REF] Chauvet | Os, ivoire et bois de renne ouvrés de la Charente, Hypothèses palethnographiques[END_REF].Upper Paleolithic intermediate tools were predominantly made of reindeer antlers. All such fi nds from Denisova Cave are made of bone. The length of intact or nearly intact items is 4.5-16.1 cm (9.2 on the average), width 0.9-4.5 cm (2.0 on the average), and thickness 0.5-1.6 cm (1.0 on the average). The tools are mostly long and massive (relationship between width and thickness is usually 2:1). In terms of morphology, these items can be divided into two groups. The first group involves tools on relatively large blanks, with a convex or straight cutting edge. Wear traces are small overlapping subparallel detachments (Fig. 5). The second group consists of longer and narrower blanks as compared to the above. Their working edge is thicker and blunted as a result of impact loads (Fig. 6,a,b). The basal part is covered by tangential detachments related probably to the shaping of tools (Fig. 6,c,d).More than a hundred such items were discovered in the Early Aurignacian assemblages (Tartar, 2012a). However, they are not specific to the beginning of the Upper Paleolithic, and they also were mentioned in Late Mousterian sites, such as Gazaria in Basque Country, France (Ibid.), Axlor in Biscay, Spain [START_REF] Mozota | El hueso como materia prima: El utillaje óseo del fi nal del Musteriense en el sector central del norte de la Península Ibérica[END_REF], Karabi Tamchin in Crimea, Russia [START_REF] Burke | A Middle Palaeolithic bone tool from Crimea (Ukraine)[END_REF]. As in Denisova Cave, they were manufactured mainly using tibias of large ungulates or straight and thick splinters of diaphyses. Tools made from tibias, which are distinguished by their length, thickness, and tissue density, were the most resistant to impact loads. Several finds from the Early Paleolithic assemblage of Schöningen can also be assigned to the items of this type [START_REF] Julien | Characterizing the Lower Paleolithic bone industry from Schöningen 12 II: A multi-proxy study[END_REF]. The results of experimental use-wear analysis and comparison with ethnographic materials have suggested several assumptions about the function of the intermediate tools, including their use as chisel-like tools and barking instruments. Some use-wear signs and morphology of the working edges of items from Denisova Cave (see Fig. 2,10,13,14) confirm the possibility of such use [START_REF] Semenov | K izucheniyu tekhniki, tekhnologii izgotovleniya i funktsiy orudiy mustyerskoy epokhi[END_REF][START_REF] Rigaud | Utilisation du ciseau dans le débitage du bois de Renne à La Garenne-Saint-Marcel (Indre)[END_REF][START_REF] Maigrot | Tracéologie des outils tranchants en os des Ve et IVe millénaires av. J.-C. en Bassin parisien, Essai méthodologique et application[END_REF]Camps-Fabrer et al., 1998;[START_REF] Provenzano | Fiche générale des objets à biseau distal[END_REF]. The efficiency of these tools has been proved experimentally. However, the functional range was probably larger. Not all the traces fit with the work of woody material, such as on the tip of the sample shown on Fig. 2, 10, which has been crushed by a repeated contact against a harder material.

Tools with damaged edges. Two intact tools are made of long tubular bones (tibia and humerus), most probably belonging to a bison; six items, different in size and morphology, were fragmented before drying of the bones. They are made from long bones (tibia and humerus) of a large ungulate (bison). The dimensions of two biggest items are 16.1 × 21.5 × 1.0 and 11.1 × 3.5 ×× 1.0 cm, respectively. Wear traces (Fig. 7,a) typical of intermediate tools have been revealed at their transverse edges adjoining the side working edges, which suggests multi-functionality of the items. All items show retouch at one longitudinal edge. It occupies 1/3 of the length at intact artifacts. Retouch is double-side, marginal on the dorsal side and covering the marrowy canal or spongiosa on the internal face (Fig. 7,c). Retouch on two items is bifacial. The shape of one item resembles a burin spall (Fig. 7,d) produced possibly by accident during utilization or rejuvenation of the tool. The functional purpose of the retouch is confirmed by some longitudinal removals produced during its use as a knife, and, in one case, by a light rounding of the cutting edge and a luster, probably from plant cutting (Fig. 7,b).Such tools are mentioned rarely. The most known retouched bone items are Acheulean bifaces found in Africa, Europe, and in the Near East [START_REF] Zutovski | The use of elephant bones for making Acheulian handaxes: A fresh look at old bones[END_REF]. These are fully shaped pieces rather than tools with rejuvenation of one of the edges. Middle Paleolithic tools made of bones with retouched edges are known from materials of such sites as Vaufrey [START_REF] Vincent | L'outillage osseux au Paléolithique moyen: une nouvelle approche[END_REF], Combe-Grenal, and La Ferrassie [START_REF] Tartar | L'utilisation des matières osseuses au Moustérien[END_REF] in France, Axlor [START_REF] Mozota | El hueso como materia prima: El utillaje óseo del fi nal del Musteriense en el sector central del norte de la Península Ibérica[END_REF] and Bolomor [START_REF] Blasco | Using bones to shape stones: MIS 9 bone retouchers at both edges of the Mediterranean Sea[END_REF] in Spain, and also from the Late Mousterian horizon finds in Fumane Cave in Italy [START_REF] Romandini | A retouched bone shaft from the Late Mousterian at Fumane cave (Italy). Technological, experimental and micro-wear analysis[END_REF]. As in Denisova Cave, retouched bone artifacts at the majority of sites were discovered mostly along with smoothed points and intermediate tools.

Discussion

Unshaped bone tools discovered in layers 11.4-11.2 in the East Chamber of Denisova Cave are small in number; however, these are evidence of a developed industry rather than randomly picked bone splinters remaining after butchering animal carcasses and used to retouch stone tools. According to the morphology of tools, they were used to work different materials, including sewing of clothes from leather; severing or cutting of plants; splitting, barking, or carving of wood. In order to determine functions of each tool, it is necessary to expand the database of experimental studies. The variety of blanks, morphology of working edges, and microwear traces suggests that this toolkit was a part of established manufacturing system. The fact that it was produced only with percussive techniques is not a satisfactory argument for refusing the term "industry", or this term should also be declined for the lithic assemblages until the introduction of stone polishing. Being part of the process of meat production, in contrast to lithic procurement, the obtaining of bone material, nevertheless, is not more opportunist than the use of beef tallow by modern industry. Concerning the absence of standardization emphasized by many researchers who reject unshaped bone tools as an industry, it should be noted that the use of elongated bone blanks to manufacture such items is no different from the use of stone blades. The lithic industries of Denisova Cave reflect not only the continuity of development as a whole, but also the establishing of the Upper Paleolithic on the basis of the local Middle Paleolithic. In the materials from lithological layers 11.4-11.2 in the East Chamber of the cave, along with Middle Paleolithic tools, Upper Paleolithic shapes are present. While bone items among the Late Middle Paleolithic tools in layer 11.4 were noteworthy as evidence of the use of a new type of raw material, the eyed needles, pipe-shaped beads, pendants, and other ornaments among unshaped tools of the Initial Upper Paleolithic in layer 11.2 are a part of a developed industry. Bone items are very well preserved, which rules out mechanical impact and the probability of considerable displacement in the thickness of cave deposits. Anthropological materials and paleogenetic data pertaining to these deposits suggest the association of the innovations with the activities of (most probably) Denisovans, though the presence of Neanderthals is also observed in layer 11.4. There are no traces of anatomically modern humans at the initial Upper Paleolithic in Denisova Cave.

Conclusions

Until recently, it was impossible to record unshaped tools in bone industries owing to the complexity of differentiating items of this type from mass faunal materials. Such items can only be classified as tools by their use-wear traces: spalls, compacted areas, microflaking, smoothness, impressions, etc. A unique preservation of organic materials in Denisova Cave simplifies identification of such traces. The results of analysis of the Middle Paleolithic unshaped tools from the East Chamber allow a conclusion to be drawn that items of the said type were rather archaic. Starting from the Middle Paleolithic, they were made on chipped out bone fragments, but the scraping technique was not used; it gained widespread use during the subsequent Upper Paleolithic epoch. The bone industry under consideration, like the stone one, reflects the continuous process of cultural development and formation of the Upper Paleolithic traditions on the basis of the local Middle Paleolithic. Since there is no evidence for the habitation of anatomically modern humans either in Denisova Cave or in the whole of Altai in the Paleolithic, the obtained results suggest that the manufacture of bone tools and the development of Upper Paleolithic traditions in this territory were connected with the culture of Denisovans. , 11, 12, 15, 16, 20), 11.3 (4, 13, 18, 22, 24), and 11.2 (2, 3, 5-10, 14, 17, 19, 22, 23) in the East Chamber of Denisova Cave (photos by M. Baumann). 1-14 -tools with axial damage; 15-17 -tools with damaged edge; 18-22 -retouchers; 23, 24 -tools with rounded end. 24 -tools with rounded end.

NB :

The initial manuscript, validated by all the authors, was largely modified without informing the first two signatories before being formatted for publication. To not block the release of the issue in press, the order of the authors has been changed, the first two authors (becoming respectively the 4th and 3rd) acknowledging the raw data but not their interpretation nor their overall perspective.

Le manuscrit initial, validé par tous les auteurs, fut largement modifié à l'insu des 2 premiers signataires avant mise en page pour publication. Afin de ne pas bloquer la sortie du numéro sous presse, l'ordre des auteurs fut modifié, les deux premiers (devenus respectivement les 4ème et 3ème) ne reconnaissant que les données brutes mais pas leur interprétation ni leur mise en perspective.

The version here transcribed is the published version. La version retranscrite ici est la version publiée. M. Kozlikin, W. Rendu, H. Plisson, M. Baumann, M. Shunkov. Unshaped Bone Tools from Denisova Cave, Altai. Archaeology, Ethnology and Anthropology of Eurasia / Arkheologija, Ehtnografija i Antropologija Evrazii, MAIK Nauka/Interperiodica, 2020, 48 (1), pp.16-28. ⟨10.17746/1563-0110.2020.48.1.016-028⟩

Fig. 1 .

 1 Fig. 1. Denisova Cave on the map of Altai.

Fig. 2 .

 2 Fig.2. Unshaped bone tools from layers11.4 (1, 11, 12, 15, 16, 20), 11.3 (4, 13, 18, 22, 24), and 11.2 (2, 3, 5-10, 14, 17, 19, 22, 23) in the East Chamber of Denisova Cave (photos by M. Baumann). 1-14 -tools with axial damage; 15-17 -tools with damaged edge; 18-22 -retouchers; 23, 24 -tools with rounded end. 24 -tools with rounded end.

Fig. 3 .

 3 Fig. 3. Bone retouchers with traces of long-term (a) and short-term (b) use from layer 11.3 in the East Chamber of Denisova Cave (photos and drawing by M. Baumann).

Fig. 4 .

 4 Fig. 4. Tools with rounded end from layers 11.2 (a-c) and 11.3 (d) in the East Chamber of Denisova Cave (photos and drawing by M. Baumann). a -smoothed cortical surface and traces of distal end modifi cation; b -smoothed inner surface of the distal end; cworking edge; d -retouched edge.

Fig. 5 .

 5 Fig. 5. Intermediate tool from layer 11.3 in the East Chamber of Denisova Cave (photos and drawing by M. Baumann).

Fig. 6 .

 6 Fig. 6. Intermediate tool from layer 11.2 in the East Chamber of Denisova Cave (photos and drawing by M. Baumann). a -compaction of the apical end; b -spalls at the apical end; c -compaction of the basal end; d -longitudinal and transverse spalls at the basal end.

Fig. 7 .

 7 Fig. 7. Tools with damaged edges from layers 11.2 (a, b) and 11.4 (c, d) in the East Chamber of Denisova Cave (photos and drawing by M. Baumann). a -wear traces at the distal end; b -polish of the cutting edge; c -retouched edge; d -burin spall from the retouched edge.

Acknowledgement

This study was supported by the Center of French-Russian Studies in Moscow (2015), a postdoctoral fellowship provided by the FYSSEN Foundation (2016), International Associated laboratory ARTEMIR (2015)(2016)(2017)(2018), and the Russian Foundation for Basic Research (Project No. 18-09-40100).