Helder Gomes Rodrigues
email: helder.gomes.rodrigues@gmail.com

Fabrice Lihoreau

Maëva Orliac

J G M Thewissen

Jean-Renaud Boisserie

Helder Gomes

Unexpected evolutionary patterns of dental ontogenetic traits in cetartiodactyl mammals

Keywords: Dental eruption, phylogeny, life history, herbivory, Cetartiodactyla

Studying ontogeny in both extant and extinct species can unravel the mechanisms underlying mammal diversification and specialization. Among mammalian clades, Cetartiodactyla encompass species with a wide range of adaptations, and ontogenetic evidence could clarify longstanding debates on the origins of modern specialized families. Here, we study the evolution of dental eruption patterns in early diverging cetartiodactyls to assess the ecological and biological significances of this character and shed new light on phylogenetic issues. After investigation of ontogenetic dental series of 63 extinct genera, our parsimony reconstructions of eruption state evolution suggest that eruption of molars before permanent premolars represent a plesiomorphic condition within Cetartiodactyla. This result substantially differs from a previous study based on modern species only. As a result, the presence of this pattern in most ruminants might represent an ancestral condition contributing to their specialized herbivory, rather than an original adaptation. In contrast, late eruption of molars in hippopotamoids is more likely related to biological aspects, such as increases in body mass and slower pace of life. Our study mainly shows that eruption sequences reliably characterize higher level cetartiodactyl taxa and could represent a new source of phylogenetic characters, especially to disentangle the origin of hippopotamoids and cetaceans.

Introduction

Studying the evolutionary mechanisms that influenced mammalian diversification and specialization is one of the goals of evolutionary biology, and the study of ontogeny in extinct and extant taxa contributed importantly to this goal in the past decades [START_REF] Hautier | Skeletal development in sloths and the evolution of mammalian vertebral patterning[END_REF][START_REF] Weisbecker | Monotreme ossification sequences and the riddle of mammalian skeletal development[END_REF][START_REF] Koyabu | Paleontological and developmental evidence resolve the homology and dual embryonic origin of a mammalian skull bone, the interparietal[END_REF][START_REF] Thewissen | Developmental biology enriches paleontology[END_REF][START_REF] Rodrigues | Ontogenetic variations and structural adjustments in mammals evolving prolonged to continuous dental growth[END_REF][START_REF] Sears | Timing the developmental origins of mammalian limb diversity[END_REF]. Dental ontogenetic parameters, such as the relative timing of dental eruption provide clues to the evolution and biology of mammals in their phylogenetic context [START_REF] Luckett | Ontogenetic Staging of the Mammalian Dentition, and Its Value for Assessment of Homology and Heterochrony[END_REF][START_REF] Van Nievelt | To replace or not to replace: the significance of reduced functional tooth replacement in marsupial and placental mammals[END_REF][START_REF] Asher | Dental eruption in afrotherian mammals[END_REF][START_REF] Hautier | The hidden teeth of sloths: evolutionary vestiges and the development of a simplified dentition[END_REF][START_REF] Rodrigues | Ontogenetic and life history trait changes associated with convergent ecological specializations in extinct ungulate mammals[END_REF]. Dental eruption sequences in mammals can also be used to infer life history and ecological traits, as well as sexual or social characteristics [START_REF] Rodrigues | Ontogenetic and life history trait changes associated with convergent ecological specializations in extinct ungulate mammals[END_REF][START_REF] Smith | Schultz's rule" and the evolution of tooth emergence and replacement patterns in primates and ungulates[END_REF][START_REF] Godfrey | Schultz's Unruly Rule: Dental Developmental Sequences and Schedules in Small-Bodied, Folivorous Lemurs[END_REF][START_REF] Jordana | Ecological and lifehistory correlates of enamel growth in ruminants (Artiodactyla)[END_REF][START_REF] Sallam | Deciduous dentition and dental eruption sequence of Bothriogenys fraasi (Anthracotheriidae, Artiodactyla) from the Fayum Depression, Egypt[END_REF][START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF][START_REF] Asher | Dental eruption and growth in Hyracoidea (Mammalia, Afrotheria)[END_REF]. These data present a strong phylogenetic signal in some mammals [START_REF] Smith | Schultz's rule" and the evolution of tooth emergence and replacement patterns in primates and ungulates[END_REF][START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF][START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF][START_REF] Monson | Breaking the rules: Phylogeny, not life history, explains dental eruption sequence in primates[END_REF][START_REF] López-Torres | Life history of the most complete fossil primate skeleton: exploring growth models for Darwinius[END_REF], and they can clarify phylogenetic issues that are the subject of longstanding discussions for both palaeontologists and molecular biologists.

Cetartiodactyla constitute a clade of mammals that comprises extant ruminants, camelids, suoids, hippopotamids and cetaceans. The origins and phylogenetic relationships among these have been widely studied, but there is no consensus [START_REF] Naylor | Are the Fossil Data Really at Odds with the Molecular Data? Morphological Evidence for Cetartiodactyla Phylogeny Reexamined[END_REF][START_REF] Theodor | Deciduous Dentitions of Eocene Cebochoerid Artiodactyls and Cetartiodactyl Relationships[END_REF][START_REF] Spaulding | Relationships of Cetacea (Artiodactyla) Among Mammals: Increased Taxon Sampling Alters Interpretations of Key Fossils and Character Evolution[END_REF][START_REF] Boisserie | Evolving between land and water: key questions on the emergence and history of the Hippopotamidae (Hippopotamoidea, Cetancodonta, Cetartiodactyla)[END_REF][START_REF] Gatesy | A phylogenetic blueprint for a modern whale[END_REF][START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF]. Since their origin 55 Ma ago, cetartiodactyls have acquired a wide range of ecological specializations, from cursorial to aquatic adaptations, and from ruminating herbivorous to carnivorous diets. Modern cetartiodactyls show a wide range of body sizes including the largest known mammal, the blue whale, contrasting with their early terrestrial representatives that were mostly small and medium-sized [START_REF] Orliac | Virtual endocranial cast of earliest Eocene Diacodexis (Artiodactyla, Mammalia) and morphological diversity of early artiodactyl brains[END_REF]. In terms of dental ontogeny, a study of dental eruption patterns in extant taxa showed that modern members of early diverging clades of cetartiodactyls (i.e. camelids and suids), and also hippos, have a relatively late eruption of molars compared to ruminants [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF]. These results suggest that the pattern observed in these early diverging taxa is the ancestral state in cetartiodactyls. Besides, it has been proposed that the pattern of eruption of molars before permanent premolars observed in most ruminants is a derived condition, which is associated with a modified and derived masticatory apparatus that allows them to cope with intense chewing strains and forces related to their specialized herbivory [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF]. However, there is little evidence that a correlation exists between life history, body mass and dental eruption pattern in extant ruminants and in other specialized ungulates as well, including perissodactyls (horses, rhinos, and tapirs; [START_REF] Smith | Schultz's rule" and the evolution of tooth emergence and replacement patterns in primates and ungulates[END_REF][START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF][START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF]). This might be explained by phylogenetic inheritance, meaning that features (i.e. inherited traits) observed in crown taxa were selected before the appearance of these groups, and represent plesiomorphic traits.

Therefore, studying dental eruption sequences in relation to skull growth and body mass in extinct cetartiodactyl taxa could contribute to understand the diversity of patterns observed in extant families, and to shed new light on their evolution and their ecological specializations.

Here, we investigate the dental eruption pattern of a wide range of extinct cetartiodactyl families, including the archaeocetes, representing the early diverging cetaceans, which had dental replacement, unlike modern cetaceans. We compare eruption sequences to another ontogenetic parameter, the skull growth, to assess the relative timing of ontogenetic events.

Because most modern superfamilies appeared during the Paleogene, our focus is mostly on taxa from this time period that characterize the appearance and early evolution of dental ontogenetic traits in cetartiodactyls. We also document extant families, such as the understudied hippopotamids and camelids. We put these original data in their phylogenetic context using recent studies on both extant and extinct species. This will allow us to reconstruct the character states of early diverging cetartiodactyl lineages, and to evaluate the influence of body mass on dental eruption in these lineages, such as hippopotamoids (extinct "Anthracotheriidae" + Hippopotamidae) known for their important size increase during their evolutionary history.

This study permits to discuss phylogenetic affinities of specialized herbivores (e.g. ruminants, camelids) and semi-aquatic (hippopotamoids) to aquatic species (cetaceans), and to assess the evolutionary importance of dental ontogeny in extinct mammal species.

Material and methods

(a) Dentition analyses. Ontogenetic dental series of 63 extinct and 14 extant genera of Cetartiodactyla were investigated (table S1). One Perissodactyla ("Hyracotherium"), an order that appears as the sister taxon of cetartiodactyls in many studies [e.g. [START_REF] Spaulding | Relationships of Cetacea (Artiodactyla) Among Mammals: Increased Taxon Sampling Alters Interpretations of Key Fossils and Character Evolution[END_REF][START_REF] Gatesy | A phylogenetic blueprint for a modern whale[END_REF], and three early eutherian mammals were also added as outgroups ( S1). Our data are based on upper and lower cheek teeth (i.e. premolars and molars), as Monson and Hlusko [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF] did (but only for the lower dentition), because they display high levels of morphological and developmental integration in mammals [START_REF] Kurtén | On the variation and population dynamics of fossil and recent mammal populations[END_REF][START_REF] Hlusko | The integration of quantitative genetics, paleontology, and neontology reveals genetic underpinnings of primate dental evolution[END_REF]. P x and M x refer to the xth upper premolars and molars, Px and Mx to the xth lower premolars and molars, and Px and Mx to both the xth upper and lower premolars and molars. Ontogenetic data on the canine were also considered when available as this tooth is more or less independent from the premolar-molar module, and is important in terms of ecology or social interactions, including mating competition [START_REF] Wv | Diversity of hypsodont teeth in mammalian dentitionsconstruction and classification[END_REF]. The most anterior part of the jaw, which carries the incisors, was not considered here because it is frequently damaged or missing in fossils. In most cases, data were gathered from ontogenetic stages that showed fully erupted teeth. This avoids misinterpretation related to relative eruption of premolars and molars [START_REF] Asher | Dental eruption in afrotherian mammals[END_REF][START_REF] Asher | Dental eruption and growth in Hyracoidea (Mammalia, Afrotheria)[END_REF].

(b) Virtual reconstructions of the dentitions and imaging. Specimens of Diplobune minor, Microbunodon minimum, and Indohyus indirae were scanned using X-ray microtomography (EasyTom 150kV, RX solutions) at 120-130kV, and at voxel sizes of 23.8 and 45.6 μm (mandible and maxilla), 27.8 and 47 μm (mandible and skull), and 45.6 μm (maxilla) respectively. 3D virtual reconstructions of the dentition were made on Avizo version 9.5 (https://www.fei.com/software/amira-avizo/), and then imaged in order to display different states or patterns of dental eruption regarding relative eruption of permanent premolars and molars, and the direction of eruption for permanent premolars (figure 1).

(c) Parsimony reconstruction for character state evolution. The character state reconstruction of early diverging cetartiodactyl lineages was performed using Mesquite version 3.04 [START_REF] Maddison | Mesquite: a modular system for evolutionary analysis[END_REF], with Deltran optimization [START_REF] Agnarsson | Is ACCTRAN better than DELTRAN?[END_REF] for branches with ambiguous state. The first analysis traced the evolution of the dental eruption sequence on a simplified phylogenetic tree derived from concatenation of both molecular and morphological data on some extinct and extant cetartiodactyls, including stratigraphic ranges (modified from [START_REF] Gatesy | A phylogenetic blueprint for a modern whale[END_REF]; figure 2). This analysis, which includes non-cetartiodactyl outgroups (one perissodactyl, two phenacodontids, and one leptictid), and both extinct and extant cetartiodactyls, is relevant for comparison with the analysis based on extant taxa only [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF]. The second analysis explores the relationships between the evolution of dental eruption sequence and estimated body mass ranges. It is realized on a simplified phylogenetic tree of extinct cetartiodactyls based on morphological data only, with a focus on Hippopotamoidea (modified from [START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF]; figure 3). This second analysis is more relevant for assessing the relationships between dental eruption states and body masses, because the phylogeny used includes many more extinct cetartiodactyl families than in the first analysis, and encompasses a higher number of cetartiodactyls in a constrained geological period (mostly Paleogene). The body mass estimations were mostly based on the formula from Martinez and Sudre [START_REF] Martinez | The astragalus of Paleogene artiodactyls: comparative morphology, variability and prediction of body mass[END_REF] using astragalus dimensions (i.e., length and proximal width; or on M1 length when the astragalus is missing; table S2).

(d) Ontogenetic measurements. The stages of dental eruption were reported together with mandibular growth which was used as a proxy for skull growth. In order to maximize the sample size, mandibular depth was measured instead of condylo-incisive length. This was measured at the distal side of M1, for two different eruption stages (see figure 4). The first of these is after M1 eruption, since that stage is significantly correlated to some life history traits in cetartiodactyls, such as age at sexual maturity [START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF]. The second is after eruption of the entire dentition as this generally corresponds to the adulthood (figure 4; table S3).

Results

The most striking result is that the wide majority of Paleogene families of cetartiodactyls (Amphimerycidae, Anoplotheriidae, Cainotheriidae, Cebochoeridae, Choeropotamidae, Diacodexeidae, Dichobunidae, Lophiomerycidae, Merycoidodontidae, Mixtotheriidae, Raoellidae, Xiphodontidae) has a pattern with the M3 erupting before most permanent premolars, like most extant ruminants (figures 2 and 3). This also includes early cetaceans (Dorudon, Zygorhiza) and early camelids (Poebrotherium), one of the closest sister taxa of cetartiodactyls, the perissodactyl "Hyracotherium", and early diverging eutherian mammals, the Phenacodontidae and Leptictidae. Conversely, the anthracotheres, most suoids, Choeropotamus and Gobiohyus show an M3 erupting last, like Ovis (an extant caprine ruminant). Extant Camelidae, as well as Hippopotamidae, generally display an intermediate, but variable eruption pattern in which M3 erupts at the same time or after P4, while only strict intermediate eruption (i.e. M3 erupting at the same time as P4) is observed for Archaeotherium, even if data are scarce.

The parsimonious reconstruction of character state evolution performed on the first phylogeny shows that the plesiomorphic cetartiodactyl state corresponds to M3 erupting before the permanent premolars (figure 2). Additionally, despite the absence of a non-cetartiodactyl outgroup, the ancestral state reconstruction analysis using the second phylogeny also recovers the same result for the basal most cetartiodactyl node (figure 3). In contrast, M3 erupts later than all or most of the premolars in medium to large-sized Paleogene certartiodactyls such as Hippopotamoidea, and many Suoidea (figure 3). Nonetheless, many medium-sized Paleogene taxa do not present this pattern (e.g. Anoplotheriidae, Lophiomerycidae, Merycoidodontidae, Mixtotheriidae), as well as large-sized archeocetes, and medium to large-sized Neogene to extant cetartiodactyls (e.g. many ruminants).

The precise relative eruption sequences of the molars and of the premolars also show variation. In general, M1 erupts immediately after the deciduous premolars, but the eruption of M2 is more variable, either erupting before or after some of the permanent premolars. This pattern is obvious in Hippopotamidae (table S1). If we except P1, the replacement of which is difficult to identify for most taxa, M2 erupts before P2 in most studied cetartiodactyls, while in extant hippopotamids, it erupts at the same time (Choeropsis) or after P2 (Hippopotamus).

Permanent premolars erupt (or mineralize) sequentially in the mesio-distal direction (from P1 to P4) in most extinct and extant cetartiodactyls. However, the opposite pattern (from P4 to P1) is observed in a few Paleogene taxa, such as Indohyus (Raoellidae), Archaeotherium, Entelodon (Entelodontidae), Dorudon and Zygorhiza (Cetacea; figures 2 and 3, table S1). It is also observed in a few extant cetartiodactyls (e.g. suoids).

The eruption of the permanent canine is also highly variable. This tooth erupts at the same time or after M2 or P2 in early mammals (Phenacodontidae) and early cetartiodactyls (e.g.

Dichobune, Merycoidodon

). Alternatively, it erupts simultaneously or after M1 in extinct and extant hippopotamids (e.g. Archaeopotamus, Hexaprotodon) and suoids (e.g. Palaeochoerus).

In derived anthracotheres (e.g. Libycosaurus), the canine erupts after M3, as it does in extinct cetaceans, and Diplobune. It is also the case in extant camelids and some high-crowned ruminants (e.g. Tragulidae, Moschidae) and early cetartiodactyls (e.g. Mixtotherium).

Measurements on ontogenetic series show that anthracotheres, suids, early diverging hippopotamids and the extant ruminant Capra show the lowest value for the ratio of the depth of the mandible at M1 between juveniles and adults (figure 4). This also includes some Eocene cetartiodactyls, Cebochoerus and Diplobune, for which samples are small (less than five specimens measured). Values for hippopotamids, including extant species, as well as anthracotheres, show high variation. Camelids display the highest values, slightly higher than extant hippos, extinct and extant ruminants, and other Eocene cetartiodactyls (e.g.

Dacrytherium, Dorudon).

Discussion (a) Early eruption of molars is the plesiomorphic state in cetartiodactyls and in eutherians

Eruption of M3 before permanent premolars occurs in most ruminants, representing the vast majority of extant cetartiodactyls [START_REF] Smith | Schultz's rule" and the evolution of tooth emergence and replacement patterns in primates and ungulates[END_REF][START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF][START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF]. Monson and Hlusko [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF] (figure 2b) proposed that a late eruption of the molars is the ancestral state in cetartiodactyls based on its presence in early diverging crown families (camelids, suids, tayassuids) and hippos. However, our analyses of dental eruption sequences of extant camelids and hippos rather show that intermediate to late eruption of molars occurs in these taxa, as it does in most suoids (table S1).

More surprisingly, the parsimonious reconstruction shows that M3 erupting before the permanent premolars is the ancestral state reconstructed for cetartiodactyls (figures 2). The fact that most Paleogene genera present this pattern contradicts the conclusions of Monson and Hlusko [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF], who also considered the origin of this state going back to 55-40 Ma, around their hippopotamid-ruminant divergence.

Veitschegger and Sánchez-Villagra [START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF] proposed that Cainotheriidae, an extinct group of small European cetartiodactyls, retain ancestral mammalian eruption sequences (i.e. early eruption of M3), consistent with our finding on a large sample of Paleogene cetartiodactyls (figure 2a). The early molar eruption also occurs in some early diverging eutherian mammals (phenacodontids, leptictids, and also arctocyonids; figure 2a, table S1) and concurs with the hypothesis that this pattern is a plesiomorphic mammalian character [START_REF] Schultz | Eruption and decay of the permanent teeth in primates[END_REF]. That hypothesis is further supported by data on other mammals such as early diverging primates (i.e. non anthropoids; [START_REF] Godfrey | Schultz's Unruly Rule: Dental Developmental Sequences and Schedules in Small-Bodied, Folivorous Lemurs[END_REF][START_REF] Monson | Breaking the rules: Phylogeny, not life history, explains dental eruption sequence in primates[END_REF][START_REF] López-Torres | Life history of the most complete fossil primate skeleton: exploring growth models for Darwinius[END_REF]) and rodents [START_REF] Bryant | Cranial anatomy and phylogenetic position of Tsaganomys altaicus (Mammalia: Rodentia) from the Hsanda Gol Formation (Oligocene), Mongolia[END_REF]. The early eruption of molars is likely plesiomorphic in ruminants, and previous functional interpretations [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF] should be revised. As a result, the early eruption of molars in ruminants might not have represented an adaptation related to modification of the masticatory apparatus.

(b) Dental eruption sequences in cetartiodactyls as a potential source of phylogenetic characters

Dental eruption sequences can offer insights into mammalian higher phylogeny, especially considering the position of Hippopotamoidea (both "Anthracotheriidae" and Hippopotamidae), Suoidea and Cetacea. Several authors have proposed that "Anthracotheriidae" are closely related to taxa originally assigned to Asian "Heloyidae", such as Gobiohyus [START_REF] Coombs | Dentition of Gobiohyus and a reevaluation of the Helohyidae (Artiodactyla)[END_REF][START_REF] Ducrocq | First discovery of Helohyidae (Artiodactyla, Mammalia) in the Late Eocene of Thailand: a possible transitional form for Anthracotheriidae[END_REF][START_REF] Foss | Family Heloyidae[END_REF] or to European Choeropotamidae [START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF][START_REF] Hooker | A new species of Amphirhagatherium (Choeropotamidae, Artiodactyla, Mammalia) from the Late Eocene Headon Hill formation of Southern England and phylogeny of endemic European anthracotherioids[END_REF][START_REF] Orliac | Early Miocene hippopotamids (Cetartiodactyla) constrain the phylogenetic and spatiotemporal settings of hippopotamid origin[END_REF] (figure 3). Late eruption of M3 (after the permanent premolars) occurs in anthracotheres, Gobiohyus and Choeropotamus.

Although some morphological features do not suggest that anthracotheres are directly derived from these families [START_REF] Hooker | A new species of Amphirhagatherium (Choeropotamidae, Artiodactyla, Mammalia) from the Late Eocene Headon Hill formation of Southern England and phylogeny of endemic European anthracotherioids[END_REF][START_REF] Ducrocq | The late Eocene Anthracotheriidae (Mammalia, Artiodactyla) from Thailand[END_REF], our evidence is consistent with close phylogenetic ties between these taxa, as it was suggested for anthracotheres and Choeropotamus by recent studies [START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF][START_REF] Orliac | Early Miocene hippopotamids (Cetartiodactyla) constrain the phylogenetic and spatiotemporal settings of hippopotamid origin[END_REF][START_REF] Boisserie | Basal hippopotamines from the upper Miocene of Chorora, Ethiopia[END_REF].

Interestingly, the suoids studied here (including Palaeochoerus), show that the canine erupts early, which is also observed in hippopotamids. This character could support close affinities of Suoidea with Hippopotamidae as suggested by a few studies (e.g. [START_REF] Pickford | Morotochoerus from Uganda (17.5 Ma) and Kenyapotamus from Kenya (13-11 Ma): implications for hippopotamid origins[END_REF][START_REF] Pickford | Encore Hippo-thèses: Head and neck posture in Brachyodus (Mammalia, Anthracotheriidae) and its bearing on hippopotamid origins[END_REF]). Our study shows that the M3 tends to erupt late in both taxa, although there are variations with eruption of M3 and P4 being more closely spaced in time in hippopotamids, and with greater differences in time lags between them in suoids, like anthracotheres. These variations may suggest that convergence yielded the similarity in patterns. Such a convergence is suggested by similar ratios in relative timing of eruption of M1 (compared to mandibular growth; figure 4) found in some geologically old suoid, Palaeochoerus (i.e. Oligocene), and in young hippos (Hexaprotodon and Hippopotamus), but excludes other hippopotamids. Interestingly, the ratios for other extinct to extant hippopotamids (i.e. Archeopotamus, aff. Hippopotamus, Choeropsis) are closer to some anthracotheres (figure 4), including Bothriogenys and Brachyodus, which are frequently considered as an offshoot of hippopotamids (e.g. [START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF][START_REF] Boisserie | Basal hippopotamines from the upper Miocene of Chorora, Ethiopia[END_REF]). While these values from dental ontogenetic series might support phylogenetic ties between some anthracotheres and early diverging hippos [START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF][START_REF] Boisserie | Basal hippopotamines from the upper Miocene of Chorora, Ethiopia[END_REF], all the evidence needs to be considered to investigate this further.

A unique dental eruption pattern emerges in early cetartiodactyls: the disto-mesial eruption of permanent premolars, only observed in Entelodontidae, Raoellidae, and Archaeoceti during the Paleogene. This pattern is not rare in mammals, because it occurs for instance in some geologically younger cetartiodactyls (see suoids in figure 2), as well as in some lipotyphlans, macroscelidids, chrysochlorids, carnivores or most non-anthropoids and platyrrhine primates [START_REF] Smith | Schultz's rule" and the evolution of tooth emergence and replacement patterns in primates and ungulates[END_REF][START_REF] Henderson | Platyrrhine Dental Eruption Sequences[END_REF]. This pattern has been recognized as a secondarily derived condition in eutherians [START_REF] Luo | Evolution of dental replacement in mammals[END_REF]. Nonetheless, it could characterize a clade including Entelodontidae, Raoellidae, and Cetacea, partly consistent with findings of previous studies [START_REF] Naylor | Are the Fossil Data Really at Odds with the Molecular Data? Morphological Evidence for Cetartiodactyla Phylogeny Reexamined[END_REF][START_REF] Spaulding | Relationships of Cetacea (Artiodactyla) Among Mammals: Increased Taxon Sampling Alters Interpretations of Key Fossils and Character Evolution[END_REF][START_REF] Gatesy | A phylogenetic blueprint for a modern whale[END_REF][START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF][START_REF] Thewissen | Whales originated from aquatic artiodactyls in the Eocene epoch of India[END_REF] (figures 2 and 3). This disto-mesial eruption pattern of premolars might give more supports for an origin of cetaceans close to raoellids [START_REF] Thewissen | Whales originated from aquatic artiodactyls in the Eocene epoch of India[END_REF][START_REF] Thewissen | From Land to Water: the Origin of Whales, Dolphins, and Porpoises[END_REF]. These different examples of character evolution show the interest of considering dental eruption for further phylogenetic investigations, as previously suggested [START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF][START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF][START_REF] Monson | Breaking the rules: Phylogeny, not life history, explains dental eruption sequence in primates[END_REF].

(c) Intricate biological and ecological significance of dental eruption patterns

Changes towards eruption of M3 after the premolars evolved in different extant and extinct cetartiodactyls taxa (e.g. anthracotheres, Choeropotamidae, Gobiohyus, Hippopotamidae, Suoidea, Camelidae, Caprinae). Interestingly, the appearance of this derived eruption state is generally associated with increases in body mass, especially in Hippopotamoidea (figure 3). Such association between late eruption of molars and large size also occurs in other mammals, such as rhinos, elephants, the extinct pantodont Coryphodon (table S1), but also in primates [START_REF] Smith | Schultz's rule" and the evolution of tooth emergence and replacement patterns in primates and ungulates[END_REF][START_REF] Monson | Breaking the rules: Phylogeny, not life history, explains dental eruption sequence in primates[END_REF][START_REF] Mcgee | A Paleopopulation of Coryphodon lobatus (Mammalia: Pantodonta) from Deardorff Hill Coryphodon Quarry, Piceance Creek Basin, Colorado[END_REF][START_REF] Böhmer | Dental Eruption Series and Replacement Pattern in Miocene Prosantorhinus (Rhinocerotidae) as Revealed by Macroscopy and X-ray: Implications for Ontogeny and Mortality Profile[END_REF]. It suggests that most extinct hippopotamoids underwent a relatively slow pace of growth, like extant relatives and analogues (e.g. rhinos, elephants; [START_REF] Smith | Schultz's rule" and the evolution of tooth emergence and replacement patterns in primates and ungulates[END_REF][START_REF] Sibly | Effects of body size and lifestyle on evolution of mammal life histories[END_REF]). However, anthracotheres (except Brachyodus) have a more rapid eruption of M1 relative to skull growth than most hippopotamids (figure 4). In this regard, the dental eruption sequence of many anthracotheres is similar to extant suoids. This supports the view that life history traits of anthracotheres and close-sized suoids are similar (e.g. Bothriogenys and Sus, [START_REF] Sallam | Deciduous dentition and dental eruption sequence of Bothriogenys fraasi (Anthracotheriidae, Artiodactyla) from the Fayum Depression, Egypt[END_REF]). The same may be true for early known hippopotamids.

In most ruminants, M3 erupts before permanent premolars, but M1 erupts late with respect to skull growth (figure 4), unlike extinct South American ungulates that display the same eruption pattern [START_REF] Rodrigues | Ontogenetic and life history trait changes associated with convergent ecological specializations in extinct ungulate mammals[END_REF]. This relative eruption timing might be due to the rapid and precocious development of the skull of cetartiodactyls, especially jaw bones, compared to other mammals [START_REF] Koyabu | Phylogenetic patterns and diversity of embryonic skeletal ossification in Cetartiodactyla[END_REF]. The development of the jaw is accelerated in ruminants compared to other modern cetartiodactyls, and it might allow a more rapid eruption and setting of the entire dentition. More generally, the plesiomorphic pattern of early eruption of molars compared to permanent premolars in ruminants might be originally influenced by the faster skeletal growth of early mammals [START_REF] Schultz | Eruption and decay of the permanent teeth in primates[END_REF][START_REF] Henderson | Platyrrhine Dental Eruption Sequences[END_REF][START_REF] Kemp | The origin and evolution of Mammals[END_REF]. This limits the number of teeth within the jaw, but allows an earlier eruption of the molars before the permanent premolars. This rapid setting of the entire dentition in ruminants might serve their herbivorous specialization and might represent a condition that likely contributes to enhanced food processing in precocial young individuals and not an adaptation from their ancestors. This functional explanation has also been proposed for late diverging South American ungulates (i.e. notoungulates), which convergently acquired eruption of molars before premolars, and this has also been suggested to be related to environmental changes [START_REF] Rodrigues | Ontogenetic and life history trait changes associated with convergent ecological specializations in extinct ungulate mammals[END_REF]. However, this pattern does not occur in caprine ruminants where the last molar erupts well after the other teeth, and M1 erupts later compared to skull growth. This has been assumed to be related to erratic resource availability at high elevation habitats, rather than to life history traits [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF].

In archeocetes, which show the same ontogenetic patterns as most ruminants, dental eruption is also sped up in derived Basilosauridae (i.e. Chrysocetus). This may be a transitional state toward the loss of dental replacement and polydonty in post-Eocene cetaceans [START_REF] Uhen | New genus of Dorudontine archaeocete (Cetacea) from the Middle-to-Late Eocene of South Carolina[END_REF].

Variation in dental ontogenetic sequences in suoids and camelids makes it difficult to assess evolutionary sequences. Nonetheless, the strong ecological specialization in camelids and some suids, as in ruminants, suggests that the eruption pattern might be related to functional aspects of food processing rather than to life history traits. The inconsistencies between the timing of M1 eruption and of the entire dentition compared to life history traits might be explained by the fact that cetartiodactyls have a precocial skull development combined to prolonged growth compared to most mammals [START_REF] Koyabu | Phylogenetic patterns and diversity of embryonic skeletal ossification in Cetartiodactyla[END_REF][START_REF] Asher | Dental Ontogeny in Macroscelides proboscideus (Afrotheria) and Erinaceus europaeus (Lipotyphla)[END_REF]. More absolute data with precise skull growth and eruption timing are needed to determine the relation between age at sexual maturity and M1 eruption, and the relation between the eruption of the entire dentition and the putative prolonged body growth of extant cetartiodactyls.

(d) Inter-and intraspecific variation and evolutionary interest of dental eruption patterns

Inferences on life history from dental ontogeny or body sizes are not straightforward in mammals, especially because of the high level of inter-and intraspecific variation [START_REF] Godfrey | Schultz's Unruly Rule: Dental Developmental Sequences and Schedules in Small-Bodied, Folivorous Lemurs[END_REF][START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF][START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF].

Changes in size and life history have occurred in parallel during evolution, with various skeletal or ontogenetic modifications [START_REF] Kolb | Growth in fossil and extant deer and implications for body size and life history evolution[END_REF]. However, some taxa are exceptions to these observations, and ecological factors may play a role [START_REF] Sibly | Effects of body size and lifestyle on evolution of mammal life histories[END_REF][START_REF] Western | Size, life history and ecology in mammals[END_REF]. While molars erupt early in most ruminants, the sequence of premolar replacement can vary in closely related species (figure 2), but also within a species [START_REF] Veitschegger | Tooth Eruption Sequences in Cervids and the Effect of Morphology, Life History, and Phylogeny[END_REF]. As a result the exact sequence of dental eruption is not a good predictor of life history traits, as shown in primates [START_REF] Godfrey | Schultz's Unruly Rule: Dental Developmental Sequences and Schedules in Small-Bodied, Folivorous Lemurs[END_REF][START_REF] Monson | Breaking the rules: Phylogeny, not life history, explains dental eruption sequence in primates[END_REF]. Based on a large sample of Ovis, Monson and Hlusko [START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF] proposed that determination of dental eruption sequence based on at least two observations is fairly accurate, which is obvious in most cases, but variations do occur, especially between upper and lower dentitions, as exemplified in hippopotamids in our study.

Past studies have shown that hippopotamids frequently show that the last lower molars erupt after the last premolars [START_REF] Smith | Schultz's rule" and the evolution of tooth emergence and replacement patterns in primates and ungulates[END_REF][START_REF] Monson | The Evolution of Dental Eruption Sequence in Artiodactyls[END_REF][START_REF] Laws | Dentition and ageing of the hippopotamus[END_REF]. However, the opposite pattern, or intermediate patterns, occur more frequently in upper cheek teeth, which has never been reported (table S1).

Interspecifically, the M2 erupts after P2-P3 in Hippopotamus, unlike extinct hippopotamids and the pygmy hippo (table S1). This could be related to a higher tooth crown for Hippopotamus compared to other genera [START_REF] Boisserie | Correlating the success of Hippopotaminae with the C4 grass expansion in Africa: Relationship and diet of early Pliocene hippopotamids from Langebaanweg, South Africa[END_REF]. Indeed, convergent trends related to M2 eruption have been reported in high-crowned mammals, such as extinct rhinocerotids and extant hyraxes [START_REF] Asher | Dental eruption and growth in Hyracoidea (Mammalia, Afrotheria)[END_REF][START_REF] Böhmer | Dental Eruption Series and Replacement Pattern in Miocene Prosantorhinus (Rhinocerotidae) as Revealed by Macroscopy and X-ray: Implications for Ontogeny and Mortality Profile[END_REF].

It has been proposed that this pattern is related to grazing which would cause severe wear on deciduous teeth. These teeth would then be more rapidly replaced, and the permanent premolars would erupt before most molars. This may be the case for Hippopotamus too.

Similarly, canines erupt early in ontogeny in hippopotamids and suoids, and late in the anthracothere Libycosaurus and in archaeocetes. Hippos and suoids use their evergrowing canines for fighting and/or food gathering early in life, while in Libycosaurus the canines are deeply rooted in males and they are assumed to be involved in sexual displays, and probably less in fights [START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the Late Miocene of Toros-Menalla, northern Chad[END_REF]. These constraints on canine function tend to be relaxed in the evolution of cetaceans and ruminants, as suggested by the small size or even absence of these teeth in most extant species. It is possible that specialized ways to process food affected the evolution of canines. However, this is not the case of camelid, tragulid and moschid males, which generally exhibit larger functional canines used in sexual displays or fighting.

These examples added to the current knowledge on mammals show that fine-tuning of specific changes in the dental eruption sequence are largely related to ecological factors, social behaviour, structural adjustments, and relaxed or neutral selections; and that life history traits affect them to a lesser degree. Histological studies may provide data on life history traits [START_REF] Hogg | Life-History Correlates of Enamel Microstructure in Cebidae (Platyrrhini, Primates)[END_REF][START_REF] Jordana | First Fossil Evidence for the Advance of Replacement Teeth Coupled with Life History Evolution along an Anagenetic Mammalian Lineage[END_REF], but in the absence of such data, inferences of pace of life from dental ontogeny should be limited to higher macroevolutionary events (e.g. origin and diversification of high-level taxa).

In that context, major modifications in the dental eruption sequence more likely highlight important biological changes, or pinpoint pivotal events during the evolutionary history of some families related to their diversification or specialization. less than five specimens). + represents "M3 erupting at the same time as P4" state, and * represents "M3 erupting last" state (hypothesized reconstructed state for Anthracotherium). In violet: early diverging cetartiodactyls; in blue: Ruminantia; in grey: Cetacea; in orange: "Anthracotheriidae"; in red: Hippopotamidae; in yellow: Suoidea; and in green: Camelidae.

Figure 1

 1 Figure 1 Main variations of dental eruption observed in 3D microtomographic reconstruction

Figure 2

 2 Figure 2 (a) Phylogenetic tree of extant and extinct cetartiodactyls with stratigraphic range

Figure 3

 3 Figure 3 Phylogenetic tree of extinct cetartiodactyls with a focus on Hippopotamoidea

Figure 4

 4 Figure 4 Ontogenetic data corresponding to ratio of juvenile mandibular (Md) size (as

Museum of Natural History of the town of Geneva, Switzerland; Ranga Rao collection, Dehra Dun, India) and from previously published studies (table

	two Phenacodontidae: Phenacodus,
	Meniscotherium; and one Leptictidae: Leptictis). Data on dental eruption sequences were
	obtained from material housed in different institutions (MNHN: Muséum National d'Histoire
	Naturelle, Paris; MNHT: Muséum d'Histoire Naturelle, Toulouse; University of Montpellier;
	University of Lyon; Confluences Museum, Lyon; University of Poitiers; MHNO, Muséum
	d'Histoire Naturelle, Orléans; Museum Crozatier of Le Puy-en-Velay, France; Peabody
	Museum, Harvard; AMNH: American Museum of Natural History, New York, USA; CNRD:
	Centre National de Recherche pour le Développement, N'djamena, Chad; National Museum of
	Ethiopia/Authority for Research and Conservation of the Cultural Heritage, Addis Ababa,
	Ethiopia; NMK: National Museums of Kenya, Nairobi; Forschungsinstitut und Naturmuseum
	Senckenberg, Frankfurt; Museum für Naturkunde, Berlin, SMNS: Staatliches Museum für
	Natürkunde, Stuttgart, Germany; RMCA: Royal Museum of Central Africa, Tervuren,
	Belgium; NHM: Natural History Museum, London, United Kingdom; Museum of Natural
	History, Bern;

Acknowledgements. We are grateful to the curators and collection managers G. Billet, P. Tassy, J. Lesur, A. Verguin, F. Renoult, J. Cuisin and C. Denys (MNHN Paris), E. Robert (University of Lyon), M. Philippe and F. Vigouroux (Confluences Museum, Lyon), Y. Laurent (MNHT, Toulouse), B. Marandat and S. Jiquel (University of Montpellier), G. Garcia (University of Poitiers), M. Binon, C. Mansuy, and L. Danilo (MNHO, Orléans), F. Saragoza and A. Bonnet (Le Puy-en-Velay), J. Barry (Peabody Museum, Harvard), R. O'Leary and D. Diveley (AMNH, New York), B. Mallah and C. Nekoulnang (CNRD, N'djamena), M. Bitew and T. Getachew (National Museum of Ethiopia/Authority for Research and Conservation of the Cultural Heritage, Addis Ababa), M. Leakey, E. M'Bua and F. K . Manthi (NMK, Nairobi), G. Storch (Forschungsinstitut und Naturmuseum Senckenberg, Frankfurt), W.-D. Heinrich and M. Ade (Museum für Naturkunde, Berlin), R. Ziegler (SMNS, Stuttgart), W. Wendelen and W. Van Neer (RMCA, Tervuren), P. Jenkins and R. Sabin (NHM, London), P. Schmid (Museum of Natural History, Bern), F. Baud (Museum of Natural History of the town of Geneva) who allowed access of paleontological and zoological collections, as well as to research programs that found specimens included in this study, notably the Mission Paléoanthropologique Franco-

Data accessibility. Supporting data are accessible in electronic supplementary material, tables S1, S2 and S3.


Kalpana Mandal

Katarzyna Pogoda

Satabdi Nandi

Samuel Mathieu

Amal Kasri

Eric Klein

François Radvanyi

Bruno Goud

Paul A Janmey

Jean- Baptiste Manneville
email: baptiste.manneville@curie.fr

Role of a kinesin motor in cancer cell mechanics

Keywords: KIF20A, microrheology, optical tweezers, AFM, bladder cancer, power law, cell migration

Molecular motors play important roles in force generation, migration and intracellular trafficking.

Changes in specific motor activities are altered in numerous diseases. KIF20A, a motor protein of the kinesin-6 family, is overexpressed in bladder cancer, and KIF20A levels correlate negatively with clinical outcome. We report here a new role for the KIF20A kinesin motor protein in intracellular mechanics. Using optical tweezers to probe intracellular mechanics and surface AFM to probe cortical mechanics, we first confirm that bladder cells soften with increasing cancer grade. We then show that inhibiting KIF20A makes the intracellular environment softer for both high and low grade bladder cancer cells. Upon inhibition of KIF20A cortical stiffness also decreases in lower grade cells, while it surprisingly increases in higher grade malignant cells. Changes in cortical stiffness correlate with the interaction of KIF20A with myosin IIA. Moreover, KIF20A inhibition negatively regulates bladder cancer cell motility irrespective of the underlying substrate stiffness. Our results reveal a central role for a microtubule motor in cell mechanics and migration in the context of bladder cancer.

Introduction

Molecular motors convert chemical energy into mechanical work to achieve active cellular processes.

The physical properties of molecular motors have been extensively studied in vitro by single molecular approaches [START_REF] Rai | Molecular Adaptations Allow Dynein to Generate Large Collective Forces inside Cells[END_REF][START_REF] Neuman | Single-Molecule Force Spectroscopy: Optical Tweezers, Magnetic Tweezers and Atomic Force Microscopy[END_REF][START_REF] Xiao | Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron[END_REF] . In cells, assemblies of molecular motors and collective active processes generate random fluctuating forces in the cytoplasm, which have recently been measured using force spectrum microscopy [START_REF] Guo | Cell Volume Change through Water Efflux Impacts Cell Stiffness and Stem Cell Fate[END_REF] . However, the individual contribution of a given type of molecular motor to cell mechanics is still underexplored. Kinesins form a superfamily of molecular motors that interact with microtubules and regulate key cell functions including mitosis, cell migration and organelle transport [START_REF] Vale | The Molecular Motor Toolbox for Intracellular Transport[END_REF][START_REF] Vershinin | Multiple-Motor Based Transport and Its Regulation by Tau[END_REF][START_REF] Exertier | Impaired Angiogenesis and Tumor Development by Inhibition of the Mitotic Kinesin Eg5[END_REF][START_REF] Wang | 30 Years Later: Recent Insights from Structural Studies[END_REF][START_REF] Hirokawa | Kinesin Superfamily Motor Proteins and Intracellular Transport[END_REF] . We focus here on the role of the KIF20A kinesin (also called MKLP2 for mitotic kinesin-like protein 2 or Rabkinesin-6) in the mechanics of bladder cancer cells. KIF20A is a microtubule plus-end directed motor of the kinesin-6 family [START_REF] Verhey | Traffic Control: Regulation of Kinesin Motors[END_REF] . KIF20A was initially shown to bind the small G protein RAB6 and to regulate retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) during interphase [START_REF] Echard | Interaction of a Golgi-Associated Kinesin-like Protein with Rab6[END_REF] . In mitosis, KIF20A localizes to the central spindle, and its phosphorylation is required for cytokinesis [START_REF] Neef | Phosphorylation of Mitotic Kinesin-like Protein 2 by Polo-like Kinase 1 Is Required for Cytokinesis[END_REF] . A recent study shows that KIF20A and myosin II interact at Golgi hotspots to regulate the intracellular trafficking of RAB6-positive vesicles during interphase [START_REF] Miserey-Lenkei | Coupling Fission and Exit of RAB6 Vesicles at Golgi Hotspots through Kinesin-Myosin Interactions[END_REF] . KIF20A is involved in the fission of transport intermediates from the Golgi apparatus and serves to anchor RAB6 on Golgi and trans-Golgi network (TGN) membranes near microtubule nucleating sites [START_REF] Echard | Interaction of a Golgi-Associated Kinesin-like Protein with Rab6[END_REF][START_REF] Miserey-Lenkei | Coupling Fission and Exit of RAB6 Vesicles at Golgi Hotspots through Kinesin-Myosin Interactions[END_REF][START_REF] Miserey-Lenkei | Rab and Actomyosin-Dependent Fission of Transport Vesicles at the Golgi Complex[END_REF] . The regulation of myosin II might also be affected by KIF20A dynamics and thereby affect force generation and intracellular microrheological properties [START_REF] Chan | Myosin II Activity Softens Cells in Suspension[END_REF][START_REF] Koenderink | An Active Biopolymer Network Controlled by Molecular Motors[END_REF][START_REF] Mizuno | Nonequilibrium Mechanics of Active Cytoskeletal Networks[END_REF] . It was recently reported that, during cortical neurogenesis, knockout of KIF20A causes the loss of neural progenitor cells and neurons due to early cell cycle exit and neuronal differentiation [START_REF] Geng | KIF20A/MKLP2 Regulates the Division Modes of Neural Progenitor Cells during Cortical Development[END_REF] .

KIF20A was previously reported to be highly upregulated in many cancer cell types such as pancreatic cancer, melanoma, bladder cancer, liver cancer and breast cancer [START_REF] Ho | Deregulation of Rab and Rab Effector Genes in Bladder Cancer[END_REF][START_REF] Zaman | Migration of Tumor Cells in 3D Matrices Is Governed by Matrix Stiffness along with Cell-Matrix Adhesion and Proteolysis[END_REF][START_REF] Yu | The Role of Kinesin Family Proteins in Tumorigenesis and Progression Potential Biomarkers and Molecular Targets for Cancer Therapy[END_REF][START_REF] Liu | Overexpression of Kinesin Family Member 20A Correlates with Disease Progression and Poor Prognosis in Human Nasopharyngeal Cancer: A Retrospective Analysis of 105 Patients[END_REF][START_REF] Imai | Identification of HLA-A2-Restricted CTL Epitopes of a Novel Tumour-Associated Antigen, KIF20A, Overexpressed in Pancreatic Cancer[END_REF][START_REF] Gasnereau | KIF20A MRNA and Its Product MKlp2 Are Increased During Hepatocyte Proliferation and Hepatocarcinogenesis[END_REF][START_REF] Lu | Cross-Species Comparison of Orthologous Gene Expression in Human Bladder Cancer and Carcinogen-Induced Rodent Models[END_REF] . Overexpression of KIF20A is associated with increased proliferation and tumor progression, poor prognosis and increased drug resistance in many cancers [START_REF] Duan | Positive Expression of KIF20A Indicates Poor Prognosis of Glioma Patients[END_REF][START_REF] Lan | Cross-Linked Hyaluronic Acid Gel Inhibits Metastasis and Growth of Gastric and Hepatic Cancer Cells: In Vitro and in Vivo Studies[END_REF] . In contrast, downregulation of KIF20A reduces cell proliferation in pancreatic cancer and glioma cells due to cytokinesis failure, appearance of binucleated cells, and apoptosis [START_REF] Duan | Positive Expression of KIF20A Indicates Poor Prognosis of Glioma Patients[END_REF][START_REF] Saito | Functional Analysis of KIF20A, a Potential Immunotherapeutic Target for Glioma[END_REF] . However it has also been proposed that KIF20A could play antagonistic roles by both activating and inhibiting tumor progression [START_REF] Ho | Deregulation of Rab and Rab Effector Genes in Bladder Cancer[END_REF] . Peptides derived from KIF20A have been used to activate the immune system to kill cancer cells, which endogenously express the KIF20A antigen [START_REF] Tomita | Identification of Promiscuous KIF20A Long Peptides Bearing Both CD4+ and CD8+ T-Cell Epitopes: KIF20A-Specific CD4+ T-Cell Immunity in Patients with Malignant Tumor[END_REF] . Hence, KIF20A has become an immunotherapeutic target for several cancers, such as pancreatic or breast cancers [START_REF] Tomita | Identification of Promiscuous KIF20A Long Peptides Bearing Both CD4+ and CD8+ T-Cell Epitopes: KIF20A-Specific CD4+ T-Cell Immunity in Patients with Malignant Tumor[END_REF][START_REF] Suzuki | A Phase I Clinical Trial of Vaccination With KIF20A-Derived Peptide in Combination With Gemcitabine For Patients With Advanced Pancreatic Cancer[END_REF][START_REF] Khongkow | Paclitaxel Targets FOXM1 to Regulate KIF20A in Mitotic Catastrophe and Breast Cancer Paclitaxel Resistance[END_REF] .

Cell stiffness is often altered in pathological situations including fibrosis and cancer [START_REF] Chin | Mechanotransduction in Cancer[END_REF][START_REF] Reid | Tumor Matrix Stiffness Promotes Metastatic Cancer Cell Interaction with the Endothelium[END_REF][START_REF] Levental | A Simple Indentation Device for Measuring Micrometer-Scale Tissue Stiffness[END_REF][START_REF] Lee | Regulation of Breast Cancer Progression by Extracellular Matrix Mechanics: Insights from 3D Culture Models[END_REF][START_REF] Alibert | Are Cancer Cells Really Softer than Normal Cells?[END_REF] . In most cancers, isolated cancer cells have been shown to be softer than their healthy counterparts 36,37 . 4 Cancer cell softening is thought to be a key event during the metastatic process because more deformable cells should migrate more efficiently through confined environments to form metastases at a distant site from the primary tumor. Since actomyosin contractility has been shown to be crucial for maintaining cell stiffness and for force generation [START_REF] Tojkander | Actin Stress Fibers -Assembly, Dynamics and Biological Roles[END_REF][START_REF] Mackintosh | Elasticity of Semiflexible Biopolymer Networks[END_REF] , a large body of research has been dedicated to study the role of actomyosin in cell mechanics [START_REF] Pelham | Cell Locomotion and Focal Adhesions Are Regulated by Substrate Flexibility[END_REF][START_REF] Mandal | Cell Dipole Behaviour Revealed by ECM Sub-Cellular Geometry[END_REF][START_REF] Murrell | Forcing Cells into Shape: The Mechanics of Actomyosin Contractility[END_REF] . In contrast, the role of the microtubule cytoskeleton and its associated motors in cell stiffness has been much less studied. In particular, how microtubule motors participate to determine cell stiffness and how deregulation of kinesins in disease correlates with cell mechanics are still open questions.

In this study, we investigate the mechanical role of KIF20A in bladder cancer cells using a combination of active intracellular microrheological measurements by optical tweezers and cell surface atomic force microscopy (AFM) indentation experiments. We focus here on the role of KIF20A in regulating intracellular and cortical mechanics and correlate the mechanical effects of KIF20A with its effects on cell migration. Our results suggest that the interaction of KIF20A with myosin II differently regulates intracellular and cortical mechanics. The role of KIF20A in cell mechanics and cell migration points to KIF20A as a potential therapeutic target for bladder cancer.

Results

Bladder cancer cells are softer than normal urothelial cells

We first compared the viscoelastic properties of primary normal human urothelial (NHU) cells and two bladder cancer cell lines, RT112 cells and KU19-19 (KU) cells using intracellular active microrheology and AFM indentation experiments (Fig. 1). RT112 cells were derived from the transitional cell carcinoma (histological grade II) excised from a woman of unknown age, while KU cells were established from the invasive transitional cell carcinoma of the bladder (grade III) of a 76-year-old man. RT112 cells are non-invasive and tumorigenic, while KU cells are invasive and metastatic. In addition, RT112 cells are more differentiated and do not express the epithelial-mesenchymal transition (EMT) marker vimentin in contrast to KU cells. In the following we refer to RT112 cells as low grade (grade II) cells and to KU cells as high grade (grade III) cells [START_REF] Marshall | Markers of Neoplastic Transformation in Epithelial Cell Lines Derived from Human Carcinomas[END_REF][START_REF] Tachibana | Autocrine Growth of Transitional Cell Carcinoma of the Bladder Induced by Granulocyte-Colony Stimulating Factor[END_REF] . Cells were plated on crossbow shaped adhesive micropatterns to normalize their shape and intracellular organization.

Internalized 2 µm diameter beads were displaced in an oscillatory fashion using optical tweezersbased rheology to measure the intracellular shear modulus (Fig. 1A, see Methods). We found that the intracellular shear modulus was almost twice as high in normal NHU cells than in bladder cancer cells and was significantly reduced in grade III cells compared to grade II cells (Fig. 1A). Intracellular relaxation experiments of beads trapped by optical tweezers (Fig. 1B) allowed us to measure two phenomenological parameters, the rigidity index and the bead step amplitude, and to extract the complex shear modulus using a model based on power law rheology (see Methods). These measurements confirmed that high grade (KU, grade III) cells are softer than low grade (RT112, grade III) cells [START_REF] Alibert | Are Cancer Cells Really Softer than Normal Cells?[END_REF][START_REF] Abidine | Mechanosensitivity of Cancer Cells in Contact with Soft Substrates Using AFM[END_REF] (Fig. 1B) at the subcellular scale. AFM indentation experiments showed that cortical stiffness is also lower in grade III cells (Fig. 1C). Note that the stiffness measured by AFM indentation experiments was larger by two orders of magnitude than the intracellular stiffness measured either by oscillation or relaxation experiments, suggesting that AFM measurements, but not optical trapping measurements of internalized beads, are dominated by cortical stiffness. Stiffness maps obtained on micropatterned cells show a strong spatial dispersion of stiffness values (Fig. 1D) with higher values in the perinuclear region in both RT112 and KU cells, as observed using intracellular rheology [START_REF] Mandal | Mapping Intracellular Mechanics on Micropatterned Substrates[END_REF] . Despite the spatial variability of the elasticity measurements, the perinuclear region appears to be softer in grade III cells than in grade II cells (Fig. 1.D), consistent with the spatially averaged data (Fig. 1C).

Inhibiting KIF20A softens the cell cytoplasm

Out-of-equilibrium active cellular processes impact strongly on cell rheology [START_REF] Ahmed | Active Mechanics Reveal Molecular-Scale Force Kinetics in Living Oocytes[END_REF] . While acto-myosin contractility is well-known to play a major role in the mechanical properties of cells and in force generation, the role of the microtubule-associated kinesin motors in cell mechanics is much less studied. Because the expression levels of several molecular motors including KIF20A have been shown to be deregulated in bladder cancer [START_REF] Ho | Deregulation of Rab and Rab Effector Genes in Bladder Cancer[END_REF] , we asked whether the kinesin KIF20A could participate in bladder cancer cell mechanics. We inhibited KIF20A by treating RT112 and KU bladder cancer cells plated on glass coverslip with 50 µM paprotrain, a specific KIF20A inhibitor, for 1 hr [START_REF] Tcherniuk | Relocation of Aurora B and Survivin from Centromeres to the Central Spindle Impaired by a Kinesin-Specific MKLP-2 Inhibitor[END_REF] . Cells were treated with DMSO in the corresponding control experiments. Viscoelastic relaxation experiments showed an intracellular softening of both RT112 and KU cells upon inhibition of KIF20A (Fig. 2). The rigidity index, the bead step amplitude, and the storage modulus decrease significantly upon treatment with paprotrain for both cell lines, while the loss modulus decreases significantly only for RT112 cells (Fig. 2B-C). Interestingly, in RT112 cells, the decrease in loss modulus was larger than that of the storage modulus (Fig. 2B).

KIF20A regulates cortical stiffness and cell motility on both soft and stiff substrates

To correlate intracellular rheology with the mechanical properties of the cell cortex, we measured cortical stiffness in RT112 and KU cells by AFM indentation experiments. Surprisingly, while grade II RT112 cells became softer when KIF20A was inhibited, grade III KU cells became stiffer (Fig. 3 A,B).

The effects of KIF20A on bladder cancer cell rheology suggest that KIF20A impacts cellular functions that depend on cell mechanics such as cell migration. We studied individual 2D cell motility by plating RT112 and KU bladder cancer cells on soft (G=500 Pa) polyacrylamide hydrogels or on stiff (glass) substrates (Fig. 4). On both substrates, the higher-grade KU cells moved about twice as fast as the lower grade RT122 cells (Fig. 4 A, B and supplementary movies 1 and 2). When KIF20A was inhibited, the motility of RT112 and KU cells decreased. The speed of KU cells decreased about 3-fold and 4-fold on stiff and soft substrates respectively. In contrast, KIF20A inhibition had less effect on RT112 cell motility with only a 1.5-fold and 1.1-fold decrease in cell speed on stiff and soft substrates respectively (Fig. 4A, B and supplementary movies 3 and 4). Interestingly, both RT112 and KU bladder cancer cell lines are more motile on soft substrates than on stiff substrate, contrary to other cancer cell types such as gliomas [START_REF] Ulrich | The Mechanical Rigidity of the Extracellular Matrix Regulates the Structure, Motility, and Proliferation of Glioma Cells[END_REF] . Migration velocity as a function of substrate stiffness is known to exhibit a maximal value at an optimal stiffness which depends on the number of active molecular motors and clutches [START_REF] Bangasser | Shifting the Optimal Stiffness for Cell Migration[END_REF] . In the case of bladder cancer cells, the optimal stiffness may be shifted towards lower values.

Inhibiting KIF20A affects the subcellular localization of cortical actin and myosin II

We have shown so far that the effects of KIF20A inhibition are different in grade II RT112 cells than in grade III KU cells. First, KIF20A inhibition decreases cortical stiffness in RT112 cells, whereas it increases cortical stiffness in KU cells (Fig. 3). Second, KIF20A has stronger effects on single cell motility in KU cells than in RT112 cells, especially on soft substrates (Fig. 4). To explain these differences, we have first compared the expression levels and localization of KIF20A in both cell lines. RT112 cells express higher levels of KIF20A than KU cells (Fig. 5A). Since RT112 and KU have different cell cycle durations, we also measured the KIF20A expression levels in synchronized cells and found a similar trend as in non-synchronized cells, but with higher amounts of KIF20A (Fig. 5A and S1A).

Because KIF20A interacts with myosin II [START_REF] Miserey-Lenkei | Coupling Fission and Exit of RAB6 Vesicles at Golgi Hotspots through Kinesin-Myosin Interactions[END_REF] , inhibition of KIF20A may have an effect on myosin II localization and/or cell contractility and hence cortical stiffness and cell motility. We first checked that KIF20A inhibition does not alter the expression levels of KIF20A and myosin II (Fig. 5B andS1B) and that the previously reported KIF20A/myosin II interaction also occurs in bladder cancer cells (Fig. 5C andS1C). The interaction between KIF20A and myosin II was comparable in RT112 and KU cells but was specifically reduced in KU cells when KIF20A was inhibited (Fig. 5C andS1C). We then asked whether KIF20A inhibition could affect KIF20A localization and the distribution of cytoskeletal fibers. While KIF20A inhibition did not affect the colocalization of KIF20A with microtubules and myosin in the perinuclear region (Fig. 5D andS2), the distribution of actin and myosin II was clearly affected in KU cells, but not in RT112 cells, with actin and myosin accumulating at the cell periphery specifically in KU cells (Fig. 5D andS2). Such enrichment in peripheral contractile acto-myosin upon KIF20A inhibition in KU cells could be responsible for the increase in cortical stiffness and the effects on migration observed specifically in this cell line.

Discussion

We report here that the KIF20A kinesin motor plays a major role in bladder cancer cell mechanics.

We found that a low grade (RT112, grade II) and a high grade (KU, grade III) bladder cancer lines are softer than normal urinary human (NHU) cells at the intracellular scale (Figure 1A), consistent with previously published results [START_REF] Alibert | Are Cancer Cells Really Softer than Normal Cells?[END_REF][START_REF] Abidine | Mechanosensitivity of Cancer Cells in Contact with Soft Substrates Using AFM[END_REF] . When KIF20A was inhibited, intracellular rigidity decreased in both cell lines (Fig. 2). While a cortical softening concomitant with intracellular softening occurred in low grade RT112 cells, high grade KU cells exhibited a stiffer cortex upon KIF20A inhibition (Fig. 3). To our knowledge, this is the first observation of an effect of a microtubule motor on cortical mechanics.

The mechanical effects of KIF20A are mediated by its interaction with myosin II and subsequent regulation of the subcellular localization of actin and myosin II. The KIF20A/myosin II interaction was originally described to occur through Rab6 at the Golgi apparatus [START_REF] Miserey-Lenkei | Coupling Fission and Exit of RAB6 Vesicles at Golgi Hotspots through Kinesin-Myosin Interactions[END_REF] . We have focused here on the effects of KIF20A inhibition at the cell cortex, but it is likely that inhibition of KIF20A also perturbs post-Golgi trafficking and more generally intracellular transport. We propose that two pools of myosin II interact with KIF20A, a first pool localized at the Golgi apparatus and a second pool localized at the cell cortex (Fig. S2,S3). Inhibition of KIF20A may affect the Golgi pool in both RT112 and KU cells and reduce intracellular stiffness by softening internal membranes [START_REF] Mandal | Mapping Intracellular Mechanics on Micropatterned Substrates[END_REF] such as Golgi membranes where KIF20A and myosin interact.

By disrupting the interaction between KIF20A and myosin II on Golgi membranes, KIF20A inhibition may also lead to an increase in myosin motor activity in the cytoplasm, which may be responsible for intracellular softening. Since the elastic storage modulus is larger than the loss 𝐺' modulus in both bladder cancer cell lines, as generally reported in rheological measurements of 𝐺'' cytoplasm, the elastic behavior dominates over viscous loss. However, we observed a decrease in loss modulus upon KIF20A inhibition that is larger than the decrease in storage modulus in RT112 𝐺'' cells (Fig. 2B). The loss modulus is related to energy dissipation in the cytoplasm and thus to the 𝐺'' effective viscosity of the cytoplasm when active non-equilibrium forces are present. We and others have shown previously that non-equilibrium forces soften the cytoplasm by adding non Brownian fluctuating noise to the motion of intracellular particles [START_REF] Mandal | Mapping Intracellular Mechanics on Micropatterned Substrates[END_REF][START_REF] Guo | Probing the Stochastic, Motor-Driven Properties of the Cytoplasm Using Force Spectrum Microscopy[END_REF] . Our results are thus consistent with an increased activity of myosin motors in the cytoplasm upon KIF20A inhibition. Additional effects of KIF20A inhibition on intracellular transport or microtubule dynamics, especially those of Golginucleated microtubules, may also contribute to intracellular softening.

In KU cells which display lower basal levels of KIF20A, the KIF20A/myosin II interaction is decreased when KIF20A is inhibited, which may release myosin II from Golgi membranes and allow enrichment of acto-myosin at the cortex. An increase in cortical acto-myosin density may lead to an increase in contractility and a stiffening of the cortex and to the reduced cell motility observed specifically in KU cells (Fig. 4). Because RT112 cells have a higher basal level of KIF20A and inhibition of KIF20A does not impact its interaction with myosin II in these cells (Fig. 5 and see discussion below), KIF20A inhibition could perturb the cortical pool of myosin II to a lesser extent and have a lower effect on cell motility in RT112 cells. Traction force measurements could help clarify the effects of KIF20A inhibition on cell-substrate adhesion and on cell motility [START_REF] Mandal | Cell Dipole Behaviour Revealed by ECM Sub-Cellular Geometry[END_REF][START_REF] Mandal | Soft Hyaluronic Gels Promote Cell Spreading, Stress Fibers, Focal Adhesion, and Membrane Tension by Phosphoinositide Signaling, Not Traction Force[END_REF][START_REF] Roca-Cusachs | Quantifying Forces in Cell Biology[END_REF] .

The different effects of KIF20A inhibition in grade II RT112 and grade III KU cells can likely be explained by the difference in KIF20A expression levels (Fig. 5A) and by the different strength of the previously reported KIF20A/myosin II interaction [START_REF] Miserey-Lenkei | Coupling Fission and Exit of RAB6 Vesicles at Golgi Hotspots through Kinesin-Myosin Interactions[END_REF] (Fig. 5B-C). We found that inhibiting KIF20A did not affect the levels of KIF20A or myosin II in either cell line (Fig. 5B) but strongly reduced the KIF20A/myosin II interaction in KU cells but not in RT112 cells (Fig. 5C). In parallel with the reduced KIF20A/myosin II inhibition, actin and myosin reorganized at the cortex of KU cells (Fig. 5D and Fig. S3). A circular acto-myosin cortical rim appeared at the periphery of KU cells treated with the KIF20A inhibitor replacing the lamellipodial protrusions observed in control KU cells. This finding may explain both the loss of motility of individual KU cells and the increased cortical rigidity upon KIF20A inhibition. Similarly, KIF20A was reported to participate in actin rearrangement and protrusion formation in pancreatic cancer cells [START_REF] Taniuchi | KIF20A-Mediated RNA Granule Transport System Promotes the Invasiveness of Pancreatic Cancer Cells[END_REF] . In contrast no significant change in cortical acto-myosin organization was observed in RT112 cells, which correlates with the lower effects of KIF20A inhibition on RT112 cell motility.

Because microtubules play a central role in cell division, they have been early targets of cancer drug treatments. For instance, taxol (Paclitaxel) blocks mitosis by stabilizing microtubules and is a widely used chemotherapy drug. Similarly, microtubule-associated molecular motors are potential targets for cancer therapy [START_REF] Chandrasekaran | Hitting the Brakes: Targeting Microtubule Motors in Cancer[END_REF] . Kinesins and dynein play a crucial role in many cellular functions such as mitosis and cell proliferation and, not surprisingly, the expression levels of kinesins are often modified during tumor progression. Here we have used the specific KIF20A inhibitor paprotrain to study the effects of KIF20A inhibition on mechanical phenotypes in RT112 and KU bladder cancer cells. Our results point to KIF20A as a potential target for bladder cancer therapy. KIF20A inhibition ACS Paragon Plus Environment Nano Letters had stronger effects on single cell motility in KU cells than in RT112 cells (Fig. 4). The migration velocity decreased by a factor greater than two in the case of KU cells on stiff or soft substrates (Fig. 4B). Thus, KIF20A is involved in two hallmarks of malignant transformation, hyper-proliferation and, as we show here through its role in cell mechanics, increased cell motility. We can thus speculate that a KIF20A inhibitor could be of considerable therapeutic interest as it would not only target cancer cell proliferation but also motility and probably invasion.

Material and Methods

Cell

Adhesive micropatterns

Crossbow-shaped micropatterns were printed on PEG-coated glass coverslips by deep UV photolithography, and then coated with 50 μg/mL fibronectin and 20 μg/mL Alexa 546-fibrinogen-red (Sigma). Cells were seeded on freshly prepared protein-coated micropatterns and allowed to spread for at least 2-3 hrs before experiment started [START_REF] Mandal | Thermoresponsive Micropatterned Substrates for Single Cell Studies[END_REF] . Non-adherent cells were washed off by rinsing with culture medium.

Optical tweezers-based intracellular microrheology

The setup combining optical tweezers and fast confocal microscopy was described in detail previously [START_REF] Mandal | Mapping Intracellular Mechanics on Micropatterned Substrates[END_REF] . Briefly, a single fixed optical trap was built on an inverted Eclipse microscope (Nikon) equipped with a resonant laser confocal A1R scanner (Nikon), a 37 °C incubator, and a nanometric piezo stage (Mad City Labs). Coverslips with micropatterned or non-patterned cells containing typically one to three internalized beads were mounted in a Ludin chamber or glass-bottom dishes (MatTek Corporation) were used. Trapped beads were subjected either to an automated oscillatory displacement of 0.5-μm amplitude (intracellular oscillations experiments) or to a 0.5 µm step displacement (intracellular relaxation experiments). Images were recorded at 120 or 15 frames per second in the resonant mode of the confocal scanner for 40 s using the NIS Nikon software. The bead position was tracked using a homemade Matlab single particle tracking routine [START_REF] Mandal | Mapping Intracellular Mechanics on Micropatterned Substrates[END_REF] .

Intracellular relaxation experiments

We assume that the creep function behaves as a power law with an exponent  and a prefactor

𝑱(𝒕)

A, as described in [START_REF] Mandal | Mapping Intracellular Mechanics on Micropatterned Substrates[END_REF] (see methods). The bead position was fitted using: 𝑱(𝒕) = 𝑨𝒕 𝜶 𝑥 𝑏 (𝑡)

(Eq. 1) 𝑥 𝑏 (𝑡) = 𝑋 𝑠 𝑡 𝜏 1 ∑ ∞ 𝑛 = 0 [ -Γ(1 + 𝛼)𝜎 * 𝐴𝑡 𝛼 ] 𝑛 Γ(2 + 𝑛𝛼) - 𝑡 -𝜏 1 𝜏𝜏 1 ∑ ∞ 𝑛 = 0 [ -Γ(1 + 𝛼)𝜎 * 𝐴(𝑡 -𝜏 1 ) 𝛼 ] 𝑛 Γ(2 + 𝑛𝛼)
We use the first four terms of Eq.1 to fit the first ten seconds of the relaxation of the bead position 𝑥 𝑏 The characteristic length was chosen so that the generalized Stokes relation is verified 𝐿 𝑐 17,50,56,57 : , where is the shear modulus and the apparent complex stiffness of the bead 𝐾 = 6𝜋𝑅𝐺 𝐺 𝐾 microenvironment. In our case, , where is the applied stress with and the 𝐺 = 𝜎/𝜖 𝜎 = 𝐾(𝑥 𝑠 -𝑥 𝑏 )

4𝜋𝑅 2
𝑥 𝑏 𝑥 𝑠 bead and the stage displacements respectively, and is the strain of the 𝜀 = (𝑥 𝑠 -𝑥 𝑏 )/𝐿 𝑐 microenvironment Thus, , and taking into account the generalized Stokes relation, .

𝐺 = 𝐾𝐿 𝑐 /(4𝜋𝑅 2 )

, we find and . 𝐺 = 𝐾/6𝜋𝑅 𝐿 𝑐 = 2𝑅/3 = 0.67 µm 𝜎 * = 12.7 ± 0.5 Pa

The prefactor A and the exponent  are the only two fit parameters in Eq. 1. Because bead tracking was noisier at longer timescales due to intracellular dynamics, the data was fitted using the following weights: 0.9, 0.09 and 0.01 for 0<t<2s, 2<t<5s and 5<t<10s respectively. The complex shear modulus G = G' + iG'' was deduced from A and  using:

𝐺 ′ = (2𝜋) 𝛼 cos ( 𝛼 𝜋 2 ) /(𝐴 Γ(1 + 𝛼))
Page 10 of 26

ACS Paragon Plus Environment

(2)

𝐺 ′′ = (2𝜋) 𝛼 sin ( 𝛼 𝜋 2 ) /(𝐴 Γ(1 + 𝛼))
As in [START_REF] Mandal | Mapping Intracellular Mechanics on Micropatterned Substrates[END_REF] , we also used a phenomenological index to quantify intracellular rigidity which we called the 'rigidity index' (RI) and defined as:

. RI varies between 0 and 1. The bead step amplitude 𝑅𝐼 = ∫ 𝑇 0 𝑥 𝑏 (𝑡) 𝑑𝑡 𝑋 𝑠 𝑇 X b corresponds to the initial displacement of the bead following the step displacement of the stage (X s =0.5 µm > X b ).

Intracellular oscillations experiments

The bead is initially trapped at the center of the optical tweezers at time t =0 s. An oscillating displacement of the stage was applied at a frequency =1 Hz for 10 sec and 𝑥 𝑠 (𝑡) = 𝑋 𝑠 sin (𝜔𝑡) 𝜔 amplitude =0.5 μm 58 , with and the phase lag due to the viscoelastic nature of the 𝑋 𝑠 𝑋 𝑏 < 𝑋 𝑠 ∆𝜃 cytoplasm.

The force applied on the bead trapped in the optical tweezers of stiffness is 𝑘 𝑡𝑟𝑎𝑝 = 214 pN 𝐹(𝑡) = 𝐹 0 , with , and the corresponding bead displacement is , sin (𝜔𝑡)

𝐹 0 = 𝑘 𝑡𝑟𝑎𝑝 𝑋 𝑠 𝑥 𝑏 (𝑡) = 𝑋 𝑏 sin (𝜔𝑡 -∆𝜃)
where the phase lag is 0 for a purely elastic material and /2 for a purely viscous material. The ∆𝜃 complex shear modulus is given by

𝐺 = 𝐺 ′ + 𝑖 𝐺 ′′ = 𝐹 0 6𝜋𝑅𝑋 𝑏 [cos ∆𝜃 + 𝑖 sin ∆𝜃]
Where, is the storage modulus and

𝐺 ′ = 𝐹 0 6𝜋𝑅𝑋 𝑏 cos ∆𝜃 is the loss modulus. 𝐺 ′′ = 𝐹 0 6𝜋𝑅𝑋 𝑏 sin ∆𝜃
The frequency was fixed to =1 Hz and the amplitude of the stage displacement was . The 𝑋 𝑠 = 0.5 µ𝑚 amplitude of the bead displacement and the phase lag were determined using sinusoidal fitting 𝑋 𝑏 ∆𝜃 with a homemade Matlab routine after tracking the bead position as described above.

AFM-based rheology

Atomic force microscopy (AFM)-based microindentation was performed using a Dimension Icon AFM (ORC Bruker Nano) and a quadratic pyramid tip (Opening angle = 36°) with stiffness value . At each indentation location, the probe tip was programmed to indent the cell at a 15 𝑘 = 0.1 𝑁/𝑚 μm/s constant z-piezo displacement rate (equal rate of retraction) to an indentation depth 0.5 μm with a maximum indentation force of 2 nN. For each cell, indentation was performed on relatively flat regions (avoiding cell edges) of a 5 μm × 5 μm cell surface area in contact mode surface scans. Force-distance indentation curves were collected from 25 different locations for each cell. For each indentation curve, the cantilever deflection (in volts) and z-piezo displacement (in μm) were converted to an indentation force (in nN) and depth (in μm) through calibrating the cantilever deflection sensitivity (nm/V) by indenting on a hard mica substrate and a spring constant (nN/nm) via thermal vibration. The loading portion of the curve at each location was fitted to the elastic Hertz model via least-squares linear regression to calculate the effective indentation modulus at the given indentation rate [START_REF] Solon | Fibroblast Adaptation and Stiffness Matching to Soft Elastic Substrates[END_REF] . To determine the elasticity of the cell, every individual curve is fitted by the Hertz model. Whole cell stiffness is determined by the width of stiffness (E) distributions when fitted with a log normal distribution (Fig. 3B). Force

; where E* is the effective Young's modulus,

𝐹 = 𝐸 * 1 -Ʋ 2 𝑡𝑎𝑛𝛼 2 𝛿 2 1 𝐸 * ≈ ; = Poisson's ratio , = face angle ; = indentation depth 1 -µ 𝑠𝑎𝑚𝑝𝑙𝑒 𝐸 𝑠𝑎𝑚𝑝𝑙𝑒 Ʋ 𝛼 𝛿

Hydrogel preparation

For single cell random migration, cells were cultured either on glass (stiff substrate) or 500 Pa polyacrylamide hydrogels (soft substrate) using previously reported methods [START_REF] Mandal | Cell Dipole Behaviour Revealed by ECM Sub-Cellular Geometry[END_REF][START_REF] Mandal | Soft Hyaluronic Gels Promote Cell Spreading, Stress Fibers, Focal Adhesion, and Membrane Tension by Phosphoinositide Signaling, Not Traction Force[END_REF][START_REF] Solon | Fibroblast Adaptation and Stiffness Matching to Soft Elastic Substrates[END_REF] . Briefly, 500 Pa polyacrylamide hydrogels were prepared by mixing 7 % acrylamide and 0.28 % bis-acrylamide in water. APTMS and TEMED were added to initiate gel polymerization. A sulfo-SANPAH solution in water was added and the gel surface was activated by UV light. Gels were coated with 100 mg/ml collagen type I.

Single cell migration

RT112 and KU cells were plated at a density of 30,000 cells per gel or on bare glass 22x22 mm coverslips for 24 hrs at 37 °C with 5% (vol/vol) CO 2 . Cells were treated with DMSO (control) or 50 μM paprotrain before starting time-lapse acquisition. 5 hrs to 8 hrs time lapse sequences are acquired with a frame every 5mins. The Image J manual tracking plugin is used to find the cell displacement and velocity. Velocities between two time points are obtained and averaged over the total time period.

Western Blot

KU and RT112 cells were either non treated, treated with DMSO, or treated with nocodazole (0.4 mg/ml) for 18h to synchronize the cells. Cells were lysed in a buffer containing 150 mM NaCl, 50 mM

Tris-HCl pH 7.5, and 1% Nonidet-P40 (Sigma). Protein concentrations were determined by Quick Start™ Bradford 1x Dye Reagent (Bio-Rad). For Western blot analysis a nitrocellulose Protran BA 83 membrane (Life science) was used and the signal from HRP-conjugated secondary antibodies was detected with an enhanced chemiluminescence system (ChemiDoc Touch System, Bio-Rad).

Quantification of the mean band intensity relative to the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed using the Image Lab software.

  culture and reagents RT112 and KU19-19 (KU) cells were grown in RPMI medium supplemented with 10% (vol/vol) FBS and 1% Penicillin/Streptomycin at 37 °C with 5% (vol/vol) CO 2 . Primary normal human urothelium (NHU) (Southgate et al., 1994) cells were grown in KSFMC medium (Life Technologies) supplemented with 5% (vol/vol) horse serum. For intracellular rheology, cells were incubated overnight with 2-μm-diameter fluorescently labeled latex beads (660/690 fluorescence; Bangs Laboratories) before seeding them on the micropatterns. To inhibit KIF20A, cells were incubated for at least 1 hr with 50 μM Paprotrain (Biokinesis, gift from Stéphanie Miserey-Lenkei) before intracellular rheology and AFM measurements. 10µM Hepes was added before microrheology and AFM experiments. For immunofluorescence experiments, cells were fixed with 4% (vol/vol) paraformaldehyde. Primary antibodies were anti-β tubulin (Sigma; T4026), anti-myosin, and phalloidin (Invitrogen), DAPI (Sigma), Myosin II (Sigma), KIF20A (Tocris bioscience). Secondary antibodies were from Jackson Immuno Research Laboratories, E-cadherin (Cell Signaling), vinculin (Santa Cruz).

  as a function of time knowing the stage step displacement and the duration of the step (𝑡) 𝑋 𝑠 = 0.5 µ𝑚 (the stage step displacement is modelled by a linear increase from to 𝜏 1 = 40 ms 0 µ𝑚 𝑋 𝑠 = 0.5 µ𝑚 between and seconds), and is a geometrical factor involving the trap stiffness 𝑡 = 0 𝑡 = 𝜏 1 𝜎 * = 𝑘 𝑡𝑟𝑎𝑝 𝐿 𝑐 4𝜋𝑅 , the bead radius and a characteristic length 𝑘 𝑡𝑟𝑎𝑝 = 210 pN 𝑅 = 1 µ𝑚 𝐿 𝑐 .

  acknowledges funding from INSERM Plan Cancer 2009-2013 INSERM -CEA Tecsan (grant number PC201125).

Figure 1 :

 1 Figure 1: Bladder cancer cells are softer than normal urothelial cells and soften with increasing grade. (A) Intracellular oscillations experiments. (Left) Typical fluorescent and corresponding brightfield images of a normal human urothelial cell plated on a fibronectin-coated crossbow shape micropattern (cyan) with an internalized bead (red). The line shows the cell contour. Scale bars, 10 μm. (Middle) Cartoon showing the bead initially trapped at the center of the optical tweezers at time t =0 s, while the cell is moved in an oscillatory fashion. Typical oscillatory displacement of the bead and fitting of

Figure 2 :

 2 Figure 2: KIF20A inhibition softens the cytoplasm of both low and high grades bladder cancer cells. (A) Average intracellular relaxation curves of control RT112 (grade II, blue) and KU (grade III, orange) cells and for cells treated with the KIF20A inhibitor paprotrain (light blue, light orange). (B-C) Rigidity index, bead step displacement, storage modulus G' and loss modulus G'' in control and KIF20A inhibited RT112 cells (B) or KU cells (C). Data are from N=28 and 27 cells for control and

Figure 3 :

 3 Figure 3: KIF20A inhibition softens the cortex of low-grade bladder cancer cells but stiffens the cortex of high grade bladder cancer cells. (A) (Left) Distribution of the elasticity E measured by AFM indentation experiments for control grade II RT112 cells (black) and for RT112 cells treated with the KIF20A inhibitor (grey). (Right) Elasticity of control RT112 cells (blue) and RT112 cells treated with the KIF20A inhibitor (light blue). (B) Same as (A) for grade III KU cells. Data are from N=60 cells per condition and for each cell about 25 force vs. distance curves were acquired. Distributions are fitted with a log normal distribution. p values are determined from Student's t test for unpaired samples with respect to control cells (*** p<0.001).

Figure 4 :

 4 Figure 4: KIF20A inhibition reduces individual bladder cancer cell motility. (A) Rose plots of randomly migrating grade II RT112 cells (upper panels) and grade KU cells (lower panels) plated on stiff (glass, left) and soft (500 Pa polyacrylamide hydrogel, right) substrates in control conditions or in the presence of the KIF20A inhibitor (see supplementary movies S1-S4. (B) Cell speed measured from images acquired every 5 min for 5-8 hours. On stiff substrates, N=20 and 18 control and KIF20A inhibited RT112 cells respectively, and N=17 and 22 for control and KIF20A inhibited KU cells respectively. On soft substrates, data are from N=25 and 22 control and KIF20A inhibited RT112 cells respectively, and N=24 and 18 control and KIF20A inhibited KU cells respectively. The statistical differences between the groups are determined by a two-way ANOVA test and p-values are indicated (*** p<0.001; n.s. p>0.05).

Figure 5 :

 5 Figure 5: KIF20A interacts with myosin II in bladder cancer cells and its inhibition affects cortical acto-myosin organization specifically in high grade bladder cancer cells. (A) (Left) KIF20A expression in non-treated (NT), control (DMSO) or synchronized in mitosis (Sync) grade II RT112 and grade III KU cells (top). (Right) Quantification of the blots showing the normalized expression of KIF20A relative to the loading control (GAPDH). Error bars represent standard deviation of N=3 independent experiments (results from the individual experiments are shown in supplementary Fig. S3A). The statistical differences between the groups are determined by a twoway ANOVA test and p-values are indicated (*** p<0.001; * p<0.05; n.s. p>0.05 non significant). (B) (Left panel) Levels of myosin II and KIF20A in RT112 and KU cells treated with DMSO (control) or

ACS Paragon Plus EnvironmentNano Letters

Acknowledgements

We thank Stephanie Miserey-Lenkei for useful gift of Paprotrain and technical assistance of KIF20A western blots. We thank Atef Asnacios and Charlotte Alibert for technical support and discussions. KM was supported by a grant from MERSEC, Physical Science Oncology Center (PSOC) grant U54-CA193417 and Labex postdoctoral fellowship. KP was funded Physical Science Oncology Center (PSOC) grant U54-CA193417. SM was funded by a grant from Sorbonne Université UPMC University Paris 06 (Programme Doctoral "Interfaces Pour le Vivant"). AK was funded by a grant from the Agence Nationale de la Recherche (ANR). PAJ acknowledges NIH grant GM111942. This work was also supported by the Center for Engineering Mechanobiology through NSF STC 1548571. JBM ACS Paragon Plus Environment Nano Letters

Co-Immunoprecipitation

The previously reported interaction between myosin II and KIF20A was tested by coimmunoprecipitation in bladder cancer cells. RT112 and KU cells were treated with 2 mg/ml nocodazole for 16 hrs. Cells were washed and supplemented with fresh media to recover. Cells were either treated with DMSO or with 50 μM paprotrain for 1 hr, washed twice in PBS, and incubated on ice for 60 min in a lysis buffer (25mM Tris pH 7.5, 50 mM NaCl, 0.1% NP40, with freshly added protease and phosphatase inhibitor cocktails (Sigma). Cells were clarified by centrifuging at 10,000 x g for 10 minutes. 500 µg of cell lysates were processed for co-immunoprecipitation using 5 µg of KIF20A antibody coupled to dynabeads Protein A overnight at 4 °C in lysis buffer. Bead control was performed using 500 ug of cell lysate without KIF20A antibody. Beads were washed four times in 1 ml of lysis buffer followed by heating in 2x Laemmli sample buffer. The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting. The following primary antibodies were used: rabbit anti-MHC (Covance; 1:2000), rabbit anti-KIF20A (Bethyl or A17425; 1:1000). Secondary horseradish peroxidase (HRP)-coupled antibodies were from Jackson Laboratories.

Statistical analysis

Data