Isma Bennabi

Isabelle Quéguiner

Agnieszka Kolano

Thomas Boudier

Philippe Mailly

Marie-Hélène Verlhac
email: marie-helene.verlhac@college-de-france.fr

Marie-Emilie Terret
email: marie-emilie.terret@college-de-france.fr

Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment

Keywords: chromosome misalignment, HSET, meiosis, mitosis, spindle morphogenesis Subject Category Cell Cycle

Mitotic spindles assemble from two centrosomes, which are major microtubule-organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an "inside-out" mechanism, ending with establishment of the poles. We used HSET (kinesin-14) as a tool to shift meiotic spindle assembly toward a mitotic "outside-in" mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic-like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique "inside-out" mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes.

Introduction

Animal cells generally assemble mitotic spindles using an "outside-in" mechanism that relies on centrosomes acting as dominant microtubule-nucleating centers (MTOCs). The two centrosomes define the spindle poles and thus the spindle axis along which chromosome segregation will take place at anaphase [START_REF] Toso | Kinetochore-generated pushing forces separate centrosomes during bipolar spindle assembly[END_REF][START_REF] Tanenbaum | Mechanisms of centrosome separation and bipolar spindle assembly[END_REF]. Oocytes however lack canonical centrosomes, the centrioles being lost before the meiotic divisions occur [START_REF] Sathananthan | Mitosis in the human embryo: the vital role of the sperm centrosome (centriole)[END_REF][START_REF] Szollosi | Absence of centrioles in the first and second meiotic spindles of mouse oocytes[END_REF]. Interestingly, it was shown in Drosophila that maintaining functional supernumerary centrioles during female meiotic divisions leads to abnormal meiosis and aborted embryonic development [START_REF] Pimenta-Marques | A mechanism for the elimination of the female gamete centrosome in Drosophila melanogaster[END_REF], highlighting the fact that centriole loss is essential for successful sexual reproduction. In mouse oocytes, microtubules are nucleated from chromatin and multiple acentriolar microtubule-organizing centers (aMTOCs) composed of pericentriolar material [START_REF] Maro | Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes[END_REF][START_REF] Gueth-Hallonet | gamma-Tubulin is present in acentriolar MTOCs during early mouse development[END_REF][START_REF] Palacios | Gamma-tubulin reorganization during mouse fertilization and early development[END_REF][START_REF] Carabatsos | Sorting and reorganization of centrosomes during oocyte maturation in the mouse[END_REF]. These aMTOCs are perinuclear before meiotic divisions and fragment at NEBD (nuclear envelope breakdown) to become evenly distributed around chromatin [START_REF] Luksza | Rebuilding MTOCs upon centriole loss during mouse oogenesis[END_REF]11]. Following NEBD, microtubules become nucleated and stabilized first around chromatin, forming a microtubule ball, and then organized into a stable central array via microtubule motors and microtubule-associated proteins, which sort and orient the microtubules [START_REF] Dumont | A centriole-and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes[END_REF][START_REF] Schuh | Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes[END_REF][START_REF] Brunet | Meiotic regulation of TPX2 protein levels governs cell cycle progression in mouse oocytes[END_REF][START_REF] Fitzharris | A shift from kinesin 5-dependent metaphase spindle function during preimplantation development in mouse[END_REF][START_REF] Breuer | HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells[END_REF][START_REF] Kolano | Errorprone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension[END_REF]. aMTOCs are then progressively sorted along this central array [START_REF] Breuer | HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells[END_REF]. Following spindle bipolarity setup, the aMTOCs become clustered to establish the spindle poles [START_REF] Kolano | Errorprone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension[END_REF]. Meiotic spindles in oocytes are thus assembled "inside-out".

Spindle assembly in oocytes is a very slow process. Spindle bipolarization is achieved by 4 h in mice [START_REF] Dumont | A centriole-and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes[END_REF][START_REF] Schuh | Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes[END_REF] and by around 7 h in humans [START_REF] Holubcová | Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes[END_REF], thus occupying about half the transition time from NEBD to anaphase in these species. It mirrors the long duration of the first meiotic division, as meiosis I requires 8-12 h in mice and more than 20 h in humans [START_REF] Holubcová | Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes[END_REF]. In addition, whereas spindle poles are organized by two centrosomes in mitosis, pole formation is different in meiosis. In mouse oocytes, poles are organized by multiple aMTOCs. Thus, meiotic spindle poles are often less focused than mitotic ones, having this typical barrelshaped aspect. Are these unique "inside-out" spindle assembly and organization required for meiotic spindle function, that is, segregating chromosomes? To answer this question, we switched meiotic spindle assembly toward a more mitotic-like mode, with rapid bipolarity and focused pole assembly, and looked at chromosome alignment and segregation. To do so, deregulation of HSET levels was used as a tool to alter early stages of spindle morphogenesis. The kinesin-14 HSET is a minus-end-directed microtubule cross-linking motor important for regulating spindle assembly, spindle length, and pole organization [START_REF] Walczak | XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts[END_REF][START_REF] Mountain | The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle[END_REF][START_REF] Sharp | Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos[END_REF][START_REF] Goshima | Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins[END_REF][START_REF] Cai | Kinesin-14 family proteins HSET/XCTK2 control spindle length by crosslinking and sliding microtubules[END_REF][START_REF] Hepperla | Minusend-directed Kinesin-14 motors align antiparallel microtubules to control metaphase spindle length[END_REF][START_REF] Syrovatkina | Loss of kinesin-14 results in aneuploidy via kinesin-5-dependent microtubule protrusions leading to chromosome cut[END_REF]. During mitosis, HSET can slide anti-parallel microtubules apart and sort them into parallel bundles [START_REF] Fink | The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding[END_REF][START_REF] Braun | The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP dependent sorting mechanism[END_REF][START_REF] Hentrich | Microtubule organization by the antagonistic mitotic motors kinesin-5 and kinesin-14[END_REF]. In contrast, when the orientation of two opposing microtubules is parallel, HSET cross-links them and transports them to the poles [START_REF] Cai | Kinesin-14 family proteins HSET/XCTK2 control spindle length by crosslinking and sliding microtubules[END_REF][START_REF] Fink | The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding[END_REF]. We show here that a slight increase in HSET levels accelerates spindle formation, in particular spindle bipolarization and aMTOCs clustering.

Importantly, this leads to severe chromosome alignment abnormalities. In an unexpected manner, the unusual length of meiosis I (8 h) is not sufficient to correct early spindle morphogenesis defects, contributing to chromosome misalignment and mis-segregation. Thus, the unique "inside-out" spindle assembly and organization prevent aneuploidy in female gametes.

EMBO reports

Mitotic-like spindles induce aneuploidy Isma Bennabi et al

Results

Altering the timing of spindle bipolarization

To modify spindle morphogenesis, we developed an HSET gain-offunction approach. The localization of endogenous HSET was first analyzed in mouse oocytes, by performing immunofluorescence experiments on fixed oocytes. We found that endogenous HSET is localized on the spindle in meiosis I (Fig EV2A).

The gain-of-function analysis was complemented with an HSET loss-of-function approach. To do so, oocytes were treated with AZ82, a small molecule inhibitor of HSET [START_REF] Wu | Discovery and mechanistic study of a small molecule inhibitor for motor protein KIFC1[END_REF][START_REF] Yang | Discovery of potent KIFC1 inhibitors using a method of integrated high-throughput synthesis and screening[END_REF], and meiotic spindle assembly was followed using time-lapse spinning disk microscopy. Spindle bipolarization was delayed in HSET-inhibited oocytes (HSET Inh) compared to controls (Ctrl, Fig 1C), requiring 6 h and 55 min in these oocytes (Figs 1D andEV2A). The delay of spindle bipolarization observed with AZ82 could be phenocopied using another allosteric inhibitor of HSET, CW069 (Fig EV2C andD), structurally unrelated to AZ82 [START_REF] Watts | Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes[END_REF]. Taken together, and in contrast to previously published observations [START_REF] Mountain | The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle[END_REF], these results suggest that HSET levels modulate the timing of meiotic spindle bipolarity in meiosis I.

To understand how HSET drastically impacts the timing of spindle bipolarization, we took advantage of a GFP-HSET mutant N593K (HSET N593K) that can cross-link but does not slide microtubules [START_REF] Cai | Kinesin-14 family proteins HSET/XCTK2 control spindle length by crosslinking and sliding microtubules[END_REF]. GFP-HSET N593K localized on the spindle ( 1E andF, andEV2B). This suggests that, for the most part, changes in the timing of spindle bipolarization require microtubule sliding by HSET.

Accelerating spindle pole assembly

Because spindle bipolarization occurs precociously in oocytes overexpressing HSET WT, we next analyzed the consequences of its overexpression on sorting of the aMTOCs. This process occurs concomitant with spindle bipolarization and is followed by aMTOC clustering, which allows spindle pole focusing. To do this, the behavior of aMTOCs was followed by time-lapse microscopy, using mCherry-Plk4 (Polo-like kinase 4) as a marker [START_REF] Bury | Plk4 and Aurora A cooperate in the initiation of acentriolar spindle assembly in mammalian oocytes[END_REF]. We performed an automated 3D analysis of aMTOCs within the spindle. For that, we developed a Fiji plug-in that converts images obtained using live microcopy to binary images and in 3D finds the spindle poles and calculates the distance of each aMTOC to the closest pole (see Materials and Methods; Fig EV3A). In addition, this plug-in allows extraction of the number and distribution of the aMTOCs together with spindle measurements (length, central width, spindle pole width). The measurements were performed at three time points during meiosis I, spanning the critical steps of spindle morphogenesis in controls (Figs 2A and EV3C, middle panels).

At NEBD+1h30, microtubules form a ball, with aMTOCs dispersed around it [START_REF] Schuh | Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes[END_REF]. At NEBD+4h30, spindle bipolarization is achieved and a robust central array of microtubules allows the progressive sorting of aMTOCs to the poles [START_REF] Breuer | HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells[END_REF]. At NEBD+6h30, ▸ A Spinning disk confocal microscopy images showing spindle region magnifications of oocytes overexpressing HSET WT, controls and oocytes inhibited for HSET at NEBD+1h30, +4h30, and +6h30. All oocytes express GFP-EB3 (green) and mCherry-Plk4 (red). Scale bar 10 lm. B aMTOC sorting in oocytes overexpressing HSET WT (blue dots) and controls (gray dots) at NEBD+1h30, +4h30, and +6h30. The dot plot represents the standard deviation of the repartition of aMTOCs along the axis of the spindle for each oocyte analyzed. Each dot represents an oocyte; the number of oocytes analyzed is written in parentheses. Statistical significance of differences is assessed with a t-test with Welch correction where needed: *P-value = 0.018, **P-value = 0.002. As shown on the scheme, when aMTOCs are not sorted, the standard deviation is high; in contrast, when aMTOCs are sorted to the poles, the standard deviation is low. C aMTOCs clustering in oocytes overexpressing HSET WT (blue dots) and controls (gray dots) at NEBD+1h30, +4h30, and +6h30. The dot plot represents the number of aMTOCs per oocyte. Each dot represents an oocyte; the number of oocytes analyzed for each condition is written in parentheses. Statistical significance of differences is assessed with a t-test with Welch correction: *P-value = 0.011, **P-value = 0.003, ***P-value < 0.0001. D Super resolution images of aMTOCs using SIM, in fixed controls and HSET WT expressing oocytes (pericentrin antibody: gray). Scale bar 5 and 2 lm. E Quantification of aMTOCs volume from SIM super-resolution images. Control oocytes gray dots and HSET WT expressing oocytes blue dots. Statistical significance of differences is assessed with a t-test with Welch correction: *P-value = 0.0453. F FRAP analysis of SiR-Tubulin in controls (gray) and in oocytes overexpressing HSET WT (blue) at NEBD+6h30. SiR-Tubulin was photobleached at spindle poles, and its fluorescence recovery was followed. The SiR-Tubulin fluorescence intensity was normalized so that 1 corresponds to the prebleached value and 0 corresponds to the value at the first time point after bleaching. For a single exponential recovery model, the halftime to fluorescence recovery in controls oocytes is t 1/2 = 62 s compared to t 1/2 = 55 s for oocytes overexpressing HSET WT. Data are represented as mean AE SD. Statistical significance of differences for the t 1/2 is assessed with a Mann-Whitney test: P-value = 0.87. Interestingly, aMTOCs were also more compact in oocytes overexpressing HSET WT compared to controls (Fig 2A andD). First, their organization was different: in controls, aMTOCs formed a typical O-shaped structure circumscribing the poles [START_REF] Carabatsos | Sorting and reorganization of centrosomes during oocyte maturation in the mouse[END_REF], whereas in oocytes overexpressing HSET WT, they formed a single round entity (Fig 2A andD). Second, they occupied a smaller volume as quantified from the N-SIM super-resolution images (Fig 2E). This suggests that HSET may play a role in the spacing of aMTOCs at spindle poles.

We next assessed whether microtubule dynamics was altered in the hyper-clustered spindle poles of oocytes overexpressing HSET WT. To compare microtubule dynamics, we performed FRAP of SiR-Tubulin at spindle poles at NEBD+6h30 (Fig 2F). Essentially identical recovery curves were observed in oocytes overexpressing HSET WT and controls, indicating that microtubule dynamics at spindle poles was similar in the two groups. This strongly suggests that changes in microtubule nucleation or stability are not the root of the difference in spindle pole focusing. These results thus show that the timing of spindle morphogenesis is accelerated in oocytes overexpressing HSET WT: Spindle bipolarization is established precociously together with more efficient sorting and clustering of aMTOCs, markers of spindle pole assembly. We then analyzed the impact of accelerated kinetics on spindle shape.

▸

Shifting meiotic spindle morphology toward mitotic-like morphology

To determine whether accelerating bipolarization and spindle pole formation affected global spindle shape, spindle length, central spindle width, and spindle pole width were measured at the same time points used to analyze aMTOC behavior. In oocytes overexpressing HSET WT, the spindle was already bipolar at NEBD+1h30. Strikingly, the spindle at this stage was extraordinarily long (Fig 3A, live microscopy), with a mean length of 36 lm and reaching a maximum of 54 lm (Fig 3B). As previously shown, this effect of HSET overexpression on spindle length required microtubule sliding [START_REF] Cai | Kinesin-14 family proteins HSET/XCTK2 control spindle length by crosslinking and sliding microtubules[END_REF], as HSET N593K expressing oocytes displayed spindle lengths similar to controls (Fig EV4A). However, in oocytes overexpressing HSET WT, the spindles progressively shortened (Fig 3B ) to reach a size comparable to controls by NEBD+7h (Fig 3D ).

Even though spindles in oocytes overexpressing HSET WT did recover in length (Fig 3D ), we observed significant alterations in their shapes-both the poles and the central region were thinner, and the poles were pointed in contrast to the typical barrel-shape of meiotic spindle poles (Fig 3A andC). To confirm these observations, we measured spindle pole and central spindle widths at NEBD+1h30 and +7h. Oocytes overexpressing HSET WT displayed significantly reduced pole (Fig 3E) and central spindle widths (Fig 3F) compared to controls. In contrast, the spindle pole width of oocytes expressing HSET N593K was the same as controls (Fig EV4B). Thus, HSET levels, likely due to its microtubule-sliding activity, must be tightly regulated for proper spindle architecture in meiosis I.

HSET level must be tightly gated during early spindle morphogenesis

In an effort to understand how minor deregulation of HSET level impacts the entire process of spindle morphogenesis, endogenous HSET expression levels were measured before, throughout and after meiosis. Levels of HSET were low in Prophase I (1.6 times less than at NEBD+1h, Fig 4A andB). Endogenous HSET levels increased 1.7fold over the course of 7 h during meiosis I (Fig 4A andB). Interestingly, HSET levels were 1.28 times higher after meiosis (in activated oocytes, mimicking fertilization) compared to Prophase I-arrested oocytes (Fig 4A andB). In addition, HSET was strongly enriched in the female pronucleus after parthenogenetic activation (Fig 4A). Thus, after meiosis, the zygote, comparable in size to the oocyte and similarly devoid of centrioles in rodents, enters the first mitotic division with more HSET than Prophase I-arrested oocytes. Consistently, spindle shape in the zygote is more mitotic-like: elongated, with focused poles [START_REF] Louvet-Vallée | Mitotic spindles and cleavage planes are oriented randomly in the two-cell mouse embryo[END_REF][START_REF] Chaigne | F-actin mechanics control spindle centring in the mouse zygote[END_REF].

Next, we tested whether the alteration of spindle shape observed after HSET WT overexpression was a direct consequence of impairing early spindle morphogenesis via modifying HSET levels prior spindle morphogenesis or was due to the continuous perturbation of HSET levels throughout the first meiotic division. To discriminate between these two hypotheses, HSET levels were modified after early stages of spindle morphogenesis had occurred, namely at NEBD+4h once the spindle was already bipolar (Fig 4C, late HSET perturbations). Spindles were further observed at NEBD+6h30. Spindle shape was comparable to controls-both displaying a typical barrel-shape (Fig 4D, F and G)-following this late HSET increase. This was not due to lower expression levels reached in late versus early perturbations of HSET. Indeed, late injection of GFP-HSET WT allowed the production of levels of HSET that were 1.4 times higher than early injections (Fig 4E, compare gray and blue bars). Thus, the alteration of spindle shape observed after HSET WT overexpression is a direct consequence of impairing early stages of spindle morphogenesis, rather than due to continuous perturbation of HSET levels later during the first meiotic division.

Mitotic-like spindles display defects in chromosome alignment and segregation

We next asked what were the consequences of forcing a mitotic-like mode of spindle morphogenesis on chromosome alignment and segregation. To answer this question, chromosome behavior was followed in living oocytes. Whereas at NEBD+1h30 in controls, the chromosomes were inside the microtubule ball ( We then analyzed chromosome alignment in oocytes overexpressing HSET N593K, which in contrast to the HSET WT overexpressing spindles did not bipolarize quickly. In HSET N593K overexpressing oocytes, spindles did not skip the microtubule ball stage (Fig 1E, lower panel) and did not display the mitotic-like spindle shape of oocytes overexpressing HSET WT (Figs 1E and5D). In addition, the chromosomes were properly aligned on the metaphase plate before anaphase occurred (Fig 5D , right panel andE). These observations further re-enforce the view that chromosome alignment/segregation defects displayed by oocytes overexpressing HSET WT are not a consequence of HSET WT overexpression per se but rather a consequence of a shift toward a mitotic mode of spindle morphogenesis.

Discussion

We have shown that HSET levels must be tightly gated during meiosis I and that deregulation of HSET amount can be used as a tool to force spindle morphogenesis to be more mitotic-like in several aspects: accelerated kinetics of spindle bipolarization and spindle pole assembly coupled with focused poles. Interestingly, this mild HSET overexpression may be comparable to the physiological transition from meiosis I to meiosis II and beyond, that is, first zygotic mitosis. This shift toward mitotic-like spindle morphogenesis is sufficient to severely impair chromosome alignment.

Importantly, late HSET perturbations (overexpression or inhibition) have no effect on spindle shape. This strongly argues that the mitotic-like spindle shape observed after perturbing HSET levels early on are not due to a late and cumulative effect of HSET overexpression throughout meiosis I, but are strictly attributable to increasing HSET levels during early stages of meiosis I. In addition, we did not observe similar defects in spindle bipolarization, pole assembly or chromosome alignment in oocytes overexpressing HSET N593K that can cross-link but not slide microtubules [START_REF] Cai | Kinesin-14 family proteins HSET/XCTK2 control spindle length by crosslinking and sliding microtubules[END_REF]. Interestingly, mouse zygotes also lack centrioles, yet assemble spindles with mitotic characteristics: rapid bipolarization and focused pole formation [START_REF] Louvet-Vallée | Mitotic spindles and cleavage planes are oriented randomly in the two-cell mouse embryo[END_REF][START_REF] Chaigne | F-actin mechanics control spindle centring in the mouse zygote[END_REF]. Accordingly, we show that zygotes enter mitosis with more HSET than oocytes when they resume meiosis. This mitotic-like mode of spindle morphogenesis in the absence of centrioles does not create chromosome alignment abnormalities in zygotes, as it does in oocytes. However, chromosome properties are different between meiosis and mitosis. Meiosis I is peculiar since homologous chromosomes linked by chiasmata progressively align and biorient on the metaphase plate, instead of single chromosomes as in mitosis. Thus, the volume, shape, and occupancy of the objects (the chromosomes) moving toward the metaphase plate are strikingly different in meiosis, and chromosomes are active participants in meiotic spindle assembly [START_REF] Radford | The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes[END_REF]. We propose that early overexpression of HSET WT accelerates spindle bipolarization through increased microtubule sliding, skipping the microtubule ball stage, and thus scattering the chromosomes along the spindle axis. Although the spindle recovers in length as meiosis I progresses, its shape remains distorted, harboring mitotic-like pointed poles instead of the classical barrel-shaped meiotic spindle. The microtubule ball stage could serve as a chromosome shepherd to avoid precocious chromosome scattering. This strategy is used by starfish oocytes where an actin fishnet that forms at meiosis resumption gathers the chromosomes, which dispersed throughout the volume of the large nucleus [START_REF] Lénárt | A contractile nuclear actin network drives chromosome congression in oocytes[END_REF]. It is likely that for meiotic spindles assembled "insideout", the initial steps have to be precisely controlled in order to prevent chromosome defects that could persist throughout meiosis I.

We were surprised that these early spindle defects were not fully rescued over the extremely long duration of meiosis I. In particular, more highly focused poles were shown to be associated with fewer chromosome alignment defects in meiosis when the microtubule ball stage was not skipped [START_REF] Kyogoku | Large cytoplasm is linked to the error-Prone nature of oocytes[END_REF]. One possibility is that, when bypassing the microtubule ball stage, chromosomes are quickly scattered over a long distance and those located near the poles never become aligned on the metaphase plate. Indeed, polar chromosomes can be found occasionally in unmanipulated oocytes and usually are not able to gather on the metaphase plate before anaphase [START_REF] Kitajima | Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes[END_REF][START_REF] Gui | Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes[END_REF]. Microtubule dynamics is the same at hyperfocused (HSET WT OE) and normal barrel-shaped spindle poles, and the total amount or the density of microtubules might be reduced at poles (spindle pole width is significantly reduced in HSET WT overexpressing oocytes compared to controls). Taken together, this might impair the efficiency of capturing and aligning chromosomes that have been lost early on at the poles, a phenomenon that occurs more often in HSET WT overexpressing oocytes because of the early chromosome scattering.

In conclusion, forcing meiosis I spindle morphogenesis to be more mitotic-like leads to chromosome alignment abnormalities that cannot be fully reversed. In an unexpected manner, the unusual length of meiosis I (8 h) is not sufficient to correct early spindle morphogenesis defects, contributing to chromosome misalignment and segregation. This could be relevant to other systems as well, spindle formation being even slower in human oocytes, taking ~15 h [START_REF] Holubcová | Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes[END_REF]. Avoiding a mitotic-like mode of spindle morphogenesis could be one reason why most oocytes lose canonical centrosomes. It is thus possible that mouse oocytes, and maybe also human oocytes, eliminated canonical centrosomes to prevent a mitotic-like mode of spindle assembly during meiosis I, thereby to safeguarding against further increases in aneuploidy levels, already high during this specific division in these species [START_REF] Hassold | The origin of human aneuploidy: where we have been, where we are going[END_REF].

Materials and Methods

Oocyte collection and culture

Ovaries were collected from 11-week-old OF1 (wt) female mice. Fully-grown oocytes were extracted by shredding the ovaries [START_REF] Verlhac | Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes[END_REF] and then releasing the germ cells in M2 + BSA medium supplemented with 1 lM milrinone to block and synchronize them in Prophase I of meiosis [START_REF] Reis | APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division[END_REF]. Meiosis resumption was triggered by transferring oocytes into milrinone-free M2 + BSA medium. All liveculture and imaging were carried out under oil at 37°C.

Oocyte activation

Oocytes in metaphase of meiosis II were incubated 2 h in M2 + BSA medium lacking CaCl 2 and supplemented with 10 mM SrCl 2 . Activated oocytes were then cultured in M2 + BSA medium for 6 h until pronuclear formation.

Constructs

hHSET WT and hHSET N593K were subcloned from plasmids provided by Claire E. Walczak (Indiana University, USA) into a pRN3 plasmid suitable for in vitro cRNA transcription. The hHSET WT and N593K expressing plasmids were amplified using One shot Top 10 competent bacteria (Invitrogen), subsequently extracted and purified using mini and midi prep kits (Qiagen).

We used the following constructs: pRN3-GFP-hHSET, pRN3-hHSET, pSpe3-GFP-hHSET-N593K, pRN3-GFP-EB3 [START_REF] Breuer | HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells[END_REF][START_REF] Kolano | Errorprone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension[END_REF], pRN3-Histone(H2B)-RFP [START_REF] Breuer | HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells[END_REF][START_REF] Kolano | Errorprone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension[END_REF], pCS2-mCherry-Plk4 [START_REF] Marthiens | Centrosome amplification causes microcephaly[END_REF].

In vitro transcription of cRNAs and microinjection

Plasmids were linearized using appropriate restriction enzymes. cRNAs were synthesized with the mMessage mMachine kit (Ambion) and subsequently purified using the RNAeasy kit (Qiagen). Their concentration was measured using NanoDrop 2000 from ThermoScientific. cRNAs were centrifuged at 4°C during 45 min prior to microinjection into the cytoplasm of oocytes blocked in Prophase I in M2 + BSA medium supplemented with 1 lM milrinone at 37°C. cRNAs were microinjected using an Eppendorf Femtojet microinjector [START_REF] Verlhac | Asymmetric division in mouse oocytes: with or without Mos[END_REF]. After microinjection, cRNA translation was allowed for 1 h, and oocytes were then transferred into milrinonefree M2 + BSA medium to allow meiosis resumption and meiotic divisions. For Fig 4C-G, late HSET OE oocytes were microinjected at NEBD+4h.

HSET WT and HSET N593K overexpression experiments

Oocytes were microinjected with 150 ng/ll of hHSET WT cRNAs or 250 ng/ll of GFP-hHSET WT cRNAs. We have observed that this is the optimal concentration to detect interpretable phenotypes whereas lower or higher concentrations gave, respectively, no phenotypes or induced spindle collapse and mono-aster formation. Oocytes were microinjected with 250 ng/ll of GFP-hHSET N593K. After microinjection, cRNA translation was allowed for 1 h and oocytes were transferred into milrinone-free M2 + BSA medium to allow meiosis resumption.

Drug treatment

The AZ82 inhibitor of HSET was a gift from AstraZeneca (USA) [START_REF] Wu | Discovery and mechanistic study of a small molecule inhibitor for motor protein KIFC1[END_REF][START_REF] Yang | Discovery of potent KIFC1 inhibitors using a method of integrated high-throughput synthesis and screening[END_REF]. AZ82 was stored diluted in DMSO at 100 lM and further diluted in M2 medium to a final concentration of 10 lM. Control experiments were done in M2 + BSA medium supplemented with equivalent concentrations of DMSO.

The CW069 inhibitor of HSET was a gift from Fanni Gergely (Cancer Research UK Cambridge Institute, UK) [START_REF] Watts | Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes[END_REF]. CW069 was stored diluted in DMSO at 100 lM and then diluted in M2 medium at a final concentration of 25 lM. Control experiments were done in M2 + BSA medium with equivalent concentrations of DMSO. It had been previously shown that a concentration of 1/100 of DMSO in M2 medium does not perturb oocyte maturation [START_REF] Terret | The meiosis I-to-meiosis II transition in mouse oocytes requires separase activity[END_REF].

Live imaging and SIM super-resolution microscopy

Spinning disk movies were acquired using a Plan-APO 40×/1.25NA objective on a Leica DMI6000B microscope enclosed in a thermostatic chamber (Life Imaging Service) equipped with a CoolSnap HQ2/CCD camera coupled to a Sutter filter wheel (Roper Scientific) and a Yokogawa CSU-X1-M1 spinning disk. Metamorph Software (Universal Imaging) was used to collect data.

For SIM super-resolution microscopy of aMTOCs, image acquisition was performed in 3D SIM mode, with a N-SIM Nikon microscope (Nikon Imaging Centre @ Institut Curie-CNRS) before image reconstruction using the NISElements software [START_REF] Gustafsson | Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination[END_REF]. The system is equipped with an APO TIRF 100× 1.49NA Oil Immersion, a laser illumination (488 nm at 200 mW and 561 nm at 100 mW), and an EMCCD DU-897 Andor camera.

Immunofluorescence

After in vitro culture of oocytes, their zona pellucida was removed by incubation in acid Tyrode's medium (pH = 2.3). Prophase I-arrested oocytes were incubated in M2 + BSA medium supplemented with 0.4% pronase to remove the zona pellucida.

To visualize aMTOCs by SIM, oocytes were fixed 30 min at 30°C in 4% formaldehyde at NEBD+6h30 on coverslips treated with gelatin and polylysine. Permeabilization was achieved by incubating oocytes in 0.5% Triton X-100 in PBS for 10 min at room temperature. Mouse anti-pericentrin antibody (BD Transduction Laboratories) was used at 1:2,000. As secondary antibody, anti-mouse Cy3 (Molecular Probes) was used at 1:600. Slides were mounted in ProLong Gold.

To visualize endogenous HSET and exogenous microinjected hHSET, oocytes were fixed 30 min at 30°C in 3.7% formaldehyde and permeabilized 10 min at room temperature in 0.25% Tween-20-PBS. The HSET antibody was a gift from Renata Basto (Curie Institute, Paris, France). As secondary antibody, anti-rabbit Cy2 (Molecular Probes) was used at 1:200. Chromosomes were stained with Prolong-DAPI (10 lg/ml final DAPI).

FRAP analysis

Images were acquired using a Plan-APO 60×/1.4NA objective on a Ti Nikon microscope enclosed in a thermostatic chamber (Life Imaging Service) equipped with a Flash4.0 V2 CMOS camera (Hamamatsu) coupled to a Yokogawa CSU-X1 spinning disk. Metamorph Software (Universal Imaging) was used to collect data. All oocytes expressed SiR-Tubulin (from Spirochrome reference SC002, used at 0.1 lM). For all oocytes, an identical region of interest (diameter of 5 lm) was bleached at spindle poles. Images were acquired every 5 s for 125 s. One image was taken before bleaching. The SiR-Tubulin fluorescence intensity quantification was performed using the Metamorph software (Universal Imaging). Normalization of the measured fluorescence intensities was performed using the Microsoft Excel software. As the expression levels of SiR-Tubulin vary from one experiment to another, the signal intensity was normalized so that the prebleached value was 1 and the value at the first time point after bleaching was 0.

Quantifications

Metamorph (Universal Imaging), Imaris (Oxford Instruments), and Fiji (NIH) software were used to analyze and process data. A homemade plug-in was developed for ImageJ/Fiji software to analyze aMTOCs position within the spindle. This 3D_Spindle_Analysis plug-in is available at https://github.c om/pmailly/3D_Spindle_Analysis. Oocyte boundaries were first extracted with variance filter and triangle method for thresholding from the bright-field channel and used to crop the image in the other two channels. Spindle (GFP channel) was first filtered using 3D Gaussian filter (radius = 2) to reduce noise and then thresholded using MaxEntropy method. aMTOCs (RFP channel) were first filtered using difference of Gaussians (GDSC libraries from Alex Herbert, University of Sussex) to increase spot-like signals, then thresholded using MaxEntropy method. For each channel, 3D objects were segmented using the 3D ImageJ suite [START_REF] Ollion | TANGO: a generic tool for high-throughput 3D image analysis for studying nuclear organization[END_REF]. The spindle poles positions were computed as the extremities of the larger diameter of the object (Feret diameter). For each aMTOCs, minimum distances to poles, and border distances to the spindle were computed. (vi) The spindle length, central spindle width, and spindle pole measurements were performed in 3D using Imaris software (see Figs 3 andEV3D). The spindle poles positions were considered as being the extremities of the larger diameter of the spindle, and spindle length was measured as the distance between poles. (vii) The aMTOCs volume measurements were performed in 3D using Imaris software (see Fig 2D andE). The input data consist of SIM super-resolution microscopy acquisitions performed in 3D SIM mode, and the total volume of aMTOCs per oocyte was measured.

Statistical analysis

Experiments were repeated at least three times, and a sample of sufficient size was used. The statistical analysis was performed using GraphPad Prism version 7.00 for MacOS, GraphPad Software, La Jolla, California, USA, www.graphpad.com. For comparisons between two groups, the normality of the variables was checked (D'agostino-Pearson normality test) and parametric Student's t-tests (with Welch correction when indicated) or nonparametric comparison tests were performed with a confidence interval of 95%. For chromosome alignment experiments, repartitions were analyzed for statistical significance using Fisher's test used with a confidence interval of 95%. All error bars are expressed as standard deviation (SD). Values of P < 0.05 were considered significant. In all figures, * corresponds to a P-value < 0.05, ** to a P-value < 0.005, *** to a P-value < 0.0001. n.s.: not statistically significant.

Expanded View for this article is available online.

Figure 1 .

 1 Figure 1. Modification of the timing of spindle bipolarization. A Time-lapse spinning disk confocal microscopy of oocytes expressing GFP-EB3 (gray) alone (Ctrl, upper panel) or together with HSET WT (lower panel). Spindle bipolarization is advanced in HSET WT expressing oocytes compared to controls. Scale bar 10 lm. B Graph showing the kinetics of spindle bipolarization in controls (gray squares) vs. HSET WT oocytes (blue dots). The kinetics of bipolarization is accelerated in oocytes overexpressing HSET WT compared to controls. C Time-lapse spinning disk confocal microscopy of oocytes expressing GFP-EB3 (gray) treated (HSET Inh, lower panel) or not (Ctrl, upper panel) with the HSET inhibitor AZ82. Spindle bipolarization is delayed in oocytes inhibited for HSET. Scale bar 10 lm. D Graph showing the kinetics of spindle bipolarization in controls (gray squares) vs. oocytes inhibited for HSET (purple diamonds). The kinetics of bipolarization is delayed in oocytes inhibited for HSET compared to controls. E Time-lapse spinning disk confocal microscopy of control oocytes (Ctrl, upper panel) and oocytes expressing HSET N593K (HSET N593K, lower panel). Spindle bipolarization is slightly advanced in oocytes overexpressing HSET N593K compared to controls. All oocytes were incubated with SiR-Tubulin (gray). Scale bar 10 lm. F Graph showing the kinetics of spindle bipolarization in controls (gray squares) vs. HSET N593K oocytes (dark blue dots). The kinetics of bipolarization is modestly affected in oocytes overexpressing HSET N593K compared to controls.

  Fig EV1A, right panel, immunofluorescence) and had similar distribution along the spindle as GFP-HSET WT (compare Fig EV1B and EV1E, live microscopy). It reached even higher expression levels at NEBD+7h (Fig EV1F, quantification of live microscopy). The timing of spindle bipolarization was only slightly advanced in oocytes overexpressing HSET N593K compared to controls (Figs

Figure 2 .

 2 Figure 2. Acceleration of aMTOCs sorting and clustering.

Figure 3 .

 3 Figure 2.

  the spindle poles begin to focus following clustering of the aMTOCs. In oocytes inhibited for HSET by treatment with AZ82, the spindle was not yet bipolar at NEBD+6h30 (Figs 2A and EV3C, right panels). Instead, these spindles remained in a ball-shape, as quantified in Fig EV3D. The diameter of the microtubule mass even decreased slightly between the first and last time points in the HSET-inhibited oocytes (Fig EV3D, purple dots, 25 lm at NEBD+1h30 vs. 22 lm at NEBD+6h30), whereas in control oocytes, the spindle elongated (Fig EV3D, gray dots, 26 lm at NEBD+1h30 vs. 33 lm at NEBD+6h30). Therefore, measurements of aMTOCs sorting and clustering were not relevant in oocytes inhibited for HSET and we focused our analysis on oocytes overexpressing HSET WT where spindle bipolarization is advanced.We first analyzed aMTOC sorting in controls and HSET WT oocytes (Figs 2A and EV3C, middle and left panels). To do so, the distribution of the aMTOCs was measured in 3D along the long axis of the spindle at the time points where the spindle is bipolar (Fig EV3B, each dot corresponds to one aMTOC, the horizontal axis represents an hemi-spindle from the central spindle to the pole, the distance of aMTOCs to the nearest spindle pole is normalized by the spindle length, and no measurements were conducted at NEBD+1h30 in controls since at that stage spindles are not yet bipolar). In controls, the spindle was bipolar at NEBD+4h30 and the aMTOCs were scattered along the spindle's long axis (Fig EV3B, upper panel, all the gray dots are homogeneously distributed along the hemi-spindle). At NEBD+6h30, the aMTOCs were partially sorted and began to accumulate at spindle poles (Fig EV3B, upper panel, gray dots). We also plotted the standard deviation of the distribution of aMTOCs along the axis of the spindle for each oocyte analyzed (Fig 2B, each dot represents one oocyte). Before aMTOCs are sorted, the standard deviation is high; in contrast, once they are sent to the poles, the standard deviation is low (Fig 2B, scheme). In controls, the difference between NEBD+4h30 and +6h30 was small, highlighting the fact that aMTOC sorting is a long and progressive process (Fig 2B, gray dots). In oocytes overexpressing HSET, the spindle was already bipolar at NEBD+1h30 and aMTOCs were scattered along its long axis (Fig EV3B, lower panel, blue dots are homogeneously distributed along the hemi-spindle), resembling the NEBD+4h30 time point in controls. At NEBD+4h30, the aMTOCs were partially sorted as indicated by their substantial accumulation at spindle poles (Fig EV3B, lower panel, blue dots), resembling the NEBD+6h30 time point in controls. By NEBD+6h30, aMTOCs were further sorted (Fig EV3B, lower panel, blue dots). The standard deviation of the distribution of aMTOCs along the axis of the spindle for each oocyte showed the same behavior (Fig 2B, blue dots): The standard deviation at NEBD+1h30 in oocytes overexpressing HSET WT was comparable to the standard deviation at NEBD+4h30 in the controls, and at NEBD+4h30 and 6h30, it was smaller than in the controls. Altogether, these results show that aMTOC sorting takes place precociously in oocytes overexpressing HSET WT. We then analyzed aMTOC clustering in controls and oocytes overexpressing HSET WT (Figs 2A and EV3C, middle and left panels). To do so, the number of aMTOCs per oocyte was counted in 3D (Fig 2C, each dot represents one oocyte). In controls, the number of aMTOCs diminished in parallel with meiosis I progression (Fig 2C, gray dots). This shows that aMTOCs tend to fuse and cluster during meiosis I. In oocytes overexpressing HSET WT, this process started earlier when the spindle bipolarized around NEBD+1h30, as evidenced by a reduced number of aMTOCs (Fig 2C, compare blue and gray dots). Later during meiosis I, the clustering of aMTOCs continued to be enhanced compared to controls (Fig 2C, compare blue and gray dots).

Figure 4 .Figure 4 .

 44 Figure 4. HSET levels must be tightly gated during early stages of spindle morphogenesis. A Immunofluorescence on fixed control oocytes showing that endogenous HSET (HSET antibody: gray) is present in mouse oocytes from Prophase I and localized on the spindle at NEBD+1h, NEBD+5h, NEBD+8h, metaphase of meiosis II (MII), and in the nucleus 6 h after parthenogenetic activation. Scale bar 10 lm. B Endogenous HSET levels progressively increase throughout meiosis I. Endogenous HSET intensity measured for fixed control oocytes in Prophase I, at NEBD+1h, NEBD+5h, NEBD+8h, metaphase of meiosis II (MII), and after activation. Data are represented as mean AE SD. Statistical significance of differences is assessed with a Mann-Whitney test: *P-value = 0.017, **P-value = 0.0017, ***P-value < 0.0001. The ratio of HSET expression between NEBD+8h and NEBD+1h is 1.7, the ratio of HSET expression between NEBD+1h and Prophase I is 1.6, and the ratio of HSET expression between activated and Prophase I oocytes is 1.28. C Scheme of the experimental setup for early and late HSET perturbations. DNA is in blue, microtubules in green, aMTOCs in red, NEBD stands for nuclear envelope breakdown. D Spinning disk confocal microscopy images showing spindle region magnifications of oocytes expressing HSET WT (late HSET OE, cRNA injected at NEBD+4h, left panel), controls (middle panel) and oocytes inhibited for HSET at NEBD+4h (late HSET Inh, right panel), all imaged at NEBD+6h30. All oocytes express mCherry-Plk4 (red), Ctrl and late HSET Inh oocytes express GFP-EB3 (green), and late HSET OE oocytes express GFP-HSET WT (green). Scale bar 10 lm. E GFP-HSET WT total fluorescence intensity measured in the whole cell was assessed after cRNA injection in early overexpression oocytes (cRNA injected in Prophase I, gray bars) and late overexpression oocytes (cRNA injected at NEBD+4h, blue bar). The number of oocytes analyzed is written in parentheses. The total GFP-HSET WT fluorescence intensity for early HSET overexpression oocytes at NEBD+1h is 2.77 AE 1.47 arbitrary units (a.u.) and 2.40 AE 0.97 a.u. at NEBD+7h compared to 3.95 AE 1.70 a.u. for late HSET overexpression oocytes. Standard deviation is plotted on each bar. Statistical significance of differences is assessed with a Mann-Whitney test: not significant (n.s.) P-value = 0.408, *P-value = 0.048, ***P-value < 0.0001. F, G Quantification of the spindle length (F) and spindle pole width (G) in late HSET WT overexpression oocytes (cRNA injected at NEBD+4h) and controls atNEBD+6h30. Statistical significance of differences is assessed with a t-test: for (F), not significant (n.s.) P-value = 0.06; for (G), not significant (n.s.) P-value = 0.96. Each dot represents an oocyte, and the number of oocytes analyzed is written in parentheses.

Figure 5 .Figure 5 .

 55 Figure 5. Early spindle morphogenesis defects induce errors in chromosome alignment and segregation. A Spinning disk confocal microscopy images showing spindle region magnifications of controls (left panel) and oocytes overexpressing HSET WT (right panel) at NEBD+1h30 and +6h30. All oocytes express GFP-EB3 (green) and Histone-RFP (blue). The white asterisk marks a chromosome outside of the metaphase plate. Scale bar 10 lm. B Spinning disk confocal microscopy images showing spindle region magnifications of controls (left panel) and oocytes overexpressing HSET WT (right panel) before and after anaphase. Oocytes express GFP-EB3 (green, left panel) or GFP-HSET WT (green, right panel) and Histone-RFP (blue). The white asterisk marks a lagging chromosome in the oocyte before and after anaphase. Scale bar 10 lm. C Graph representing the percentage of oocytes with aligned (gray) and not aligned (black) chromosomes before anaphase, quantified for controls (left bar), and oocytes expressing HSET WT (right bar). Statistical significance of differences is assessed with a Fisher test: **P-value = 0.006. D Spinning disk confocal microscopy images showing spindle region magnifications of controls (left panel) and oocytes overexpressing HSET N593K (right panel) before and after anaphase. Oocytes express GFP-EB3 (green, left panel) or GFP-HSET N593K (green, right panel) and Histone-RFP (blue). Scale bar 10 lm. E Graph representing the percentage of oocytes with aligned (gray) and not aligned (black) chromosomes before anaphase, quantified for controls (left bar) and HSET N593K oocytes (right bar). Statistical significance of differences is assessed with a Fisher test: not significant (n.s.) P-value > 0.99.

  (i) The timing of spindle bipolarization was measured on oocytes expressing GFP-EB3 or SiR-Tubulin (from Spirochrome reference SC002, used at 0.1 lM) using Metamorph software; bipolarity was scored when two poles were distinguishable (see Fig 1A-F). (ii) For endogenous and exogenous HSET intensity measurements on fixed samples, HSET intensity was measured inside a circle of a fixed size having the mean diameter of all oocytes (see Fig EV1C and D). (iii) The GFP-HSET WT intensity measurements 3 h after cRNA injection (see Fig 4E) were performed using Metamorph software. After background subtraction, the total fluorescence intensity was measured inside a circle of a fixed size having the mean diameter of all oocytes. (iv) Chromosome alignment before anaphase was measured on oocytes expressing Histone-RFP using Metamorph software (see Fig 5). (v) For the aMTOCs 3D analysis (see Fig 2A-C), the input data consist of multichannel Z-stack images from spinning disk microscopy, containing bright-field, GFP and RFP channels.

Published online:January 12, 2018

Acknowledgements

We thank AstraZeneca (USA) for the gift of AZ82, Fanni Gergely (Cancer Research UK Cambridge Institute, UK) for the gift of CW069, and Claire E.

Walczak (Indiana University, USA) for the gift of plasmids. We thank Renata Basto (Curie Institute, Paris, France) for sharing the unpublished HSET antibody. We thank Lucie Sengmanivong for her precious help using the N-SIM Nikon microscope from the Nikon Imaging Centre @ Institut Curie-CNRS, member of the France-BioImaging national research infrastructure for confocal microscopy. We thank Christophe Klein (Centre de Recherche des Cordeliers-INSERM, Paris, France) for his help with the FRAP analysis. We also thank Hugh J. Clarke (McGill University, Montreal, Canada) for critical reading of the manuscript, the members of the Verlhac/Terret team for helpful discussions, and Anne Dumas (Collège de France, Paris, France) for her help with MTA writing. We thank the "Fondation Bettencourt Schueller". This work was supported by grants from the Fondation pour la Recherche Médicale (FRM label DEQ20150331758 to MHV), from the Fondation ARC (PJA20131200412 to MET), and from Inca (PLBIO 2016-270-TRAN). This work has received support under the program "Investissements d'Avenir" launched by the French Government and implemented by the ANR, with the references: ANR-14-CE11/DIVACEN/MHV, ANR-10-LABX-54 MEMO LIFE, ANR-11-IDEX-0001-02 PSL* Research University.

Author contributions

IB, AK, M-HV, and M-ET designed the experiments, interpreted the results, and wrote the manuscript. IB, IQ, AK, and M-ET carried out the experiments. TB and PM designed the Fiji plug-in. M-ET and M-HV supervised the project.

Conflict of interest

The authors declare that they have no conflict of interest.


Laëtitia Nowacki

Pascale Vigneron

Laura Rotellini

Hélène Cazzola

Franck Merlier

Elise Prost

Robert Ralanairina

Jean-Pierre Gadonna

Claire Rossi
email: claire.rossi@utc.fr

Muriel Vayssade
email: muriel.vayssade@utc.fr

Betanin-enriched red beetroot (Beta vulgaris

Keywords: betanin concentrate, isobetanin, betalains, anticancer activity, 2D and 3D cultures

INTRODUCTION

With surgery and radiotherapy, chemotherapy is a major treatment for cancer. Chemical agents act on cancer cells at different levels and trigger cell destruction via the induction of an apoptotic program. However classic chemotherapeutic agents often target rapidly dividing cancer and normal cells, leading to side effects for the patients [START_REF] Rao | P53-based cyclotherapy: exploiting the 'guardian of the genome' to protect normal cells from cytotoxic therapy[END_REF]. As the cancer incidence in worldwide tends to increase, the development of new, safer and efficient anticancer agents is a major challenge.

Certain plant-derived compounds exhibit anticancer activities and have attracted interest in preventing and treating cancers with over 60% of currently used anticancer agents derived from natural sources [START_REF] Cragg | Plants as a source of anti-cancer agents[END_REF][START_REF] Gupta | Phytonutrients as therapeutic agents[END_REF].

Beetroots contain both red (betacyanins) and yellow pigments (betaxanthins) known collectively as betalains, which constitute a class of highly bioavailable natural antioxidant pigments [START_REF] Kanner | Betalains-a new class of dietary cationized antioxidants[END_REF][START_REF] Tesoriere | Absorption, excretion, and distribution of dietary antioxidant betalains in LDLs: potential health effects of betalains in humans[END_REF]2013). In the red beetroot species, the predominant betacyanins are betanin (Figure 1A), which represents between 75 and 90 % of the total pigments of red beetroot [START_REF] Henry | Natural Food Colours[END_REF], and isobetanin, its C15 stereoisomer. In addition to the powerful antioxidant properties of betalains, studies have pointed out their preventive role against cancers [START_REF] Kapadia | Chemoprevention of lung and skin cancer by Beta vulgaris (beet) root extract[END_REF][START_REF] Kapadia | Chemoprevention of DMBAinduced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot[END_REF][START_REF] Lechner | Drinking water with red beetroot food color antagonizes esophageal carcinogenesis in N-nitrosomethylbenzylamine-treated rats[END_REF]. Nevertheless these studies were based on animal diet with red food coloring (E162) and other standard beetroot extracts, whose betanin content does not exceed 1.2%. Antitumoral activity of betalains was also evaluated in vitro on several cancer cell lines, but data obtained by the authors were disparate according to the cell lines and the cytotoxicity assays used [START_REF] Reddy | Relative inhibition of lipid peroxidation, cyclooxygenase enzymes, and human tumor cell proliferation by natural food colors[END_REF][START_REF] Sreekanth | Betanin a betacyanin pigment purified from fruits of Opunita ficus-indica induces apoptosis in human chronic myeloid leukemia cell line K562[END_REF][START_REF] Paluszczak | The effect of dietary polyphenols on the epigenetic regulation of gene expression in MCF7 breast cancer cells[END_REF]. Moreover, the betanin content or the purity degree of beetroot extracts used have never been precisely defined. In order to dispel any ambiguity, the characterization of the betanin effect on tumor cells requires a highly purified betanin concentrate with a precisely known composition.

During preclinical testing, in vitro analyses are performed to select compounds with potential antitumor activity before in vivo studies in relevant animal models. Cell culture approach is therefore of the highest importance for the initial screening of molecules. However cell lines used are conventionally cultured as monolayers (2D culture) and do not mimic the phenotype of cancer tissues due to the lack of cell-cell and cell-extracellular matrix interactions. 3D models (cell spheroids or aggregates) are obtained by preventing cells from attaching to the culture substrata. Based on their ability to mimic tissue-like structures more effectively than 2D cell cultures, the use of 3D in vitro models is believed to be a requisite mean into anticancer drug development [START_REF] Breslin | Three-dimensional cell culture: the missing link in drug discovery[END_REF].

This study was carried out to clarify the antitumor effect of a betanin-enriched beetroot dried extract. We used both 2D and 3D cultures to assess the cytotoxic activity of betanin on cancer and normal cells. Our results showed that the betanin concentrate induces cancer cell growth inhibition, associated with an apoptotic cell death and an autophagic activity increase in MCF-7 cells, but has no obvious effect towards normal cells.

MATERIALS AND METHODS

Betanin extraction and purification

Grinded red beetroots (local market) were extracted into ethanol/water (80/20 v/v) solvent at a solid/liquid ratio of 1/3 (g/mL) for 1 h under continuous mechanical stirring.

The solid material was separated from the macerate by centrifugation at 12 000 g for 15 min at 4°C followed by a filtration on a polypropylene membrane filter (0.2 µm, Pall Corporation). The filtrate was concentrated by ethanol evaporation under reduced pressure at 30°C and then enriched in betanin and isobetanin by adsorption chromatography [START_REF] Stintzing | Identification of betalains from yellow beet (Beta vulgaris L.) and cactus pear [Opuntia ficus-indica (L.) Mill.] by high-performance liquid chromatography-electrospray ionization mass spectrometry[END_REF]. A glass column was packed with 1 L of activated polymeric adsorbent (Amberlite XAD-16, Sigma-Aldrich, France). The pH of beetroot extract was adjusted to 3 with hydrochloric acid (HCl). 100 mL of extract was passed through the resin column at 1 Bed Volume (BV) per hour. After adsorption of the betacyanins, the other extract constituents were eluted using 3 BV of deionized water (resistivity > 18.2 mΩ.cm -1 , MilliQ plus unit, Merk-Millipore) acidified to pH 3 at 2 BV/h. Betanin and isobetanin were eluted with ethanol at 0.5 BV/h. The collected fraction was concentrated by ethanol evaporation under vacuum at 30°C and freezedried. The obtained betanin/isobetanin-enriched powder was stored at -80°C.

Extract characterization

Betanin quantification. All betacyanin/betaxanthin quantifications were performed using the multi-component photometric method described by J.H. von [START_REF] Elbe | Betalains[END_REF]. The pigment concentration calculations were based upon the absorptivity values: A 1% 1120 for betanin and isobetanin (at 538 nm) and 750 for betaxanthins (at 477 nm).

Protein and total carbohydrate quantifications. Total carbohydrate amount present in extracts was determined using the DuBois method [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF]. Glucose was used as a standard. The protein quantification was performed using the Lowry method [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF]. The absence of pigment inferences with these two colorimetric methods was controlled.

HPLC/ESI-MS analysis. The HPLC system (Infinity 1290, Agilent Technologies, France) was equipped with diode array detector coupled with a Q-TOF micro hybrid quadrupole time of flight mass spectrometer (Agilent 6538, Agilent technologies, France). HLPC analyses were performed using an analytical scale (150 mm x 2.1 mm i.d.) hypersil gold C18 reversed phase column with a particle size of 3 µm (Thermo Scientific, France). Eluents A and B (LC-MS grade) consisted of 100% water with 0.2 % (v/v) formic acid and 100 % acetonitrile respectively. Betalains separation was achieved within 40 min at 20°C at a flow rate of 0.3 mL/min. The elution profile was: 0-3 min 100% A, 3-21 min 0-13% B in A (linear gradient), 21-24 min 13 % B, 24-30 min 13-50% B (linear gradient), 30-33 min 50-100 % B, 33-40 min 100 % B. Detection wavelengths were 477 nm (betaxanthins and betacyanins) and 538 nm (betacyanins only). Positive ion electrospray mass spectra were acquired by scan mode, consisted of scanning from m/z 50 to 1700 at electrospray voltage 3800 V, fragmentor voltage 140 V. Nitrogen was nebulized at 12.0 L/min under 45.0 psi and heated at 350°C. Betanin and isobetanin structural identities were confirmed by tandem mass (relative collision energy of 15 eV) spectroscopy and NMR studies. The 1 H NMR spectrum of betanin was recorded in D 2 O (data not shown) and corresponds to the previously published data (Stinzting et al., 2004).

Cell culture and treatment

The mouse melanoma cell line B16F10 was a generous gift from Dr L. Larue (Institut Curie, France). Human breast cancer lines (MCF-7 ATCC ® HTB-22, MDA-MB-231 ATCC ® HTB-26), human colorectal cells (HT-29 ATCC ® HTB-38) and normal human fibroblasts (MRC-5 ATCC ® CCL-171) were obtained from the American Type Culture Collection. Human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell.

Cells were cultured as monolayers in RPMI 1640 (B16F10), MEM (MCF-7, MDA-MB-231, MRC-5), DMEM (HT-29) or M199 (HUVEC). All cell culture media (Gibco, Invitrogen, France) were supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen), 2 mM L-glutamine (Gibco, Invitrogen), penicillin (100 µg/mL, Gibco, Invitrogen) and streptomycin (100 µg/mL, Gibco, Invitrogen). All cell lines were maintained at 37°C in an air atmosphere of 10% CO 2 (B16F10) or 5% CO 2 (MCF-7, MDA-MB-231, HT-29, HUVEC, MRC-5).

Nunclon® polystyrene plates (tPS) were used for 2D cultures.

Polyhydroxyethylmethacrylate (polyHEMA, Sigma Aldrich) coated polystyrene plates were prepared as previously described [START_REF] Velzenberger | Study of cell behaviour on a cellulose anti-adhesive substratum[END_REF] and used for 3D cultures. Cells were seeded at 10000 cells/cm 2 without (controls) or with increasing concentrations of betanin/isobetanin (from 10 to 40 µM), and cultured for 24 or 48 hours.

Proliferation assay and cell cycle analysis

In 2D cultures, cells were removed by incubation for 5 min at 37°C in 0.25% trypsin + 1 mM EDTA solution (Gibco, Invitrogen). In 3D cultures, aggregates were collected by centrifugation (5 min, 200g) and dissociated using trypsin-EDTA solution. Trypsin reaction was stopped by adding half a volume of FBS, and cells were counted in a

Malassez hemocytometer using trypan blue exclusion assay. The proliferative index was estimated as the ratio between viable cell counts 48 hour post-seeding and number of seeded cells. Each proliferative index was normalized to this one obtained for control cells and data were expressed as percentages.

For cell cycle analysis, harvested cells were washed twice in PBS containing 5 mM EDTA, and fixed for 45 min at 4°C in 1 mL 75% ethanol in PBS with 5 mM EDTA.

Cell lines were washed twice and suspended in PBS, 5 mM EDTA containing 0.1% TritonX100 (Sigma Aldrich) mixed with 40 µg RNase A (Sigma Aldrich) and 25 µg propidium iodide (PI, Sigma Aldrich) and incubated for 15 min protected from light.

The stained samples were analysed in Gallios flow cytometer (Beckman Coulter, France). Histograms were analysed using Wincycle software (PHOENIX flow systems, USA).

Apoptosis detection and quantification

To assess early stage of apoptosis, cells cultured in 2D and 3D conditions were treated with 30 µM betanin/isobetanin mix for 24 hours and labeled with Annexin-V-FITC (Beckman Coulter). For each cell line, a positive control for apoptosis was tested (cells treated with 1 µM staurosporine). The cytometer was adjusted according to the Annexin-V positive cells of these controls. According to the manufacturer's instructions, cells were washed with PBS and suspended in the binding buffer at 5.10 5 to 5.10 6 cells/mL. 1 µL of Annexin-V FITC solution and 5 µL of PI solution were added to 100 µL of cell suspension. Cells were incubated for 15 min on ice in the dark, and then diluted by adding 400 µL of binding solution. The Annexin-V positive/IP negative cell populations were detected with a Gallios flow cytometer and were representative of apoptotic cells.

To detect DNA fragmentation, an Apostain binding assay (AbCys) was used. MCF-7

and HUVEC cells were treated with betanin/isobetanin (30 µM) or staurosporine (1 µM) for 48 hours, fixed with ice-cold methanol 80% (v/v) for 30 min, washed and suspended in 250 µL formamide for 10 min at 75°C and for 5 min at room temperature (RT). Cells were then incubated for 45 min at RT with a mouse monoclonal antibody to single-stranded DNA (ssDNA, diluted 1:10 in PBS containing 5% FBS). Bound specific antibodies were revealed by incubation with Cy3-conjugated anti-mouse antibody (diluted 1:200 in PBS containing 1% milk, Jackson ImmunoResearch) for 30 min at RT.

Each sample was counterstained with DAPI (4',6'-diamidino-2-phénylindole, 1 µg/mL, Sigma-Aldrich). The cells were then cytocentrifuged (Shandon Cytospin®) and each slide was mounted in mowiol and examined by epifluorescence microscopy (Leica DMI6000). Ten fields were randomly chosen from each slide and the percentage of apoptotic cells in each picture was calculated as the ratio of the labeling cells to the total number of cells.

Apoptotic signaling pathway analysis

Human Apoptosis arrays (Proteome Profiler TM Array, R&D Systems) were used to simultaneously detect the relative levels of expression of 35 apoptosis-related proteins.

MCF-7, MDA-MB-231 and HUVEC cells were treated with betanin/isobetaninenriched extract for 48 hours, rinsed with PBS and lysed for 30 min at 4°C using lysis buffer provided by the manufacturer. Lysates were then centrifuged at 14 000 g for 5 min and supernatants were harvested. Protein concentrations for each sample were determined using the Bradford method (Coomassie Protein Assay Reagent, Pierce, Interchim). 400 µg of cell lysates were incubated on each array overnight at 4°C. Arrays were incubated for 1h at RT with a reconstituted detection antibody cocktail, and then with a streptavidin-HRP solution for 30 min. Arrays were revealed with a Chemireagent Mix using ChemiDoc (Biorad), and each spot was quantified with Quantity One software (Biorad).

To detect change in the mitochondrial membrane potential, a MitoCapture TM dye (BioVision) was used. MCF-7 cells were treated with betanin/isobetanin mix (15 and 30 µM) for 24 hours, harvested and then resuspended in 1 mL diluted MitoCapture TM reagent provided by the manufacturer. Cells were incubated for 20 min at 37°C in a 5% CO 2 incubator. After centrifugation (500 g), the supernatant was discarded, the cells were suspended in 1 mL of pre-warmed incubation buffer and analyzed by epifluorescence microscopy.

Autophagy assay

Autophagy was detected using the Autophagy Detection Kit (Abcam). Briefly, MCF-7 cells were grown 24 hours on glass slides, and then were treated with betanin/isobetanin (15 and 30 µM) or with an autophagy inducer, rapamycin (500 nM provided by the manufacturer) for 24 hours. Medium was removed and cells were washed with a buffer supplied by the manufacturer. Cells were incubated with the dye (diluted at 1:500 in assay buffer) and DAPI for 20 min at 37°C. Cells were carefully washed and slides were examined by epifluorescence microscopy.

Statistical analysis

All statistical evaluations were performed using GraphPad InStat software. Continuous variables are expressed as means ± standard deviation. As most of the continuous values measured had a non-Gaussian distribution, non-parametric Dunn and Kruskal-Wallis tests were used for comparisons. A value of p<0.05 was taken as significant.

RESULTS

Betanin/isobetanin concentrate characterization

Figure 1B shows the chromatogram corresponding to the pigment-enriched red beetroot dried extract obtained after purification by adsorption chromatography. The HPLC profile monitored at 477 nm combined with electrospray mass spectrometry in positive mode allows the identification of the betaxanthin and betacyanin molecules contained in this final extract [START_REF] Stintzing | Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy[END_REF]. Only two betalains were detected and identified as betanin (t R = 13.05 min, [M+H] + = 551) and isobetanin, the 15R stereoisomer of betanin (t R = 14,51 min, [M+H] + = 551) [START_REF] Kujala | Betalain and phenolics composition of four beetroot (Beta vulgaris) cultivars[END_REF][START_REF] Stintzing | Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy[END_REF]. The All the betaxanthins were eluted during beetroot extract purification on polymeric resin while the betacyanins remained absorbed. Total carbohydrate quantification reveals that they are totally eliminated at this purification step, no residual complex or simple carbohydrates could be detected in the final betanin concentrate. As betanin and isobetanin differ only by the absolute configuration of their C15 chiral center, we did not distinguish them for determination of the total pigment amount in the purified extract. The total betanin/isobetanin content in the dried concentration was evaluated at 80% by photometric quantification. The remaining 20% was attributed to residual proteins by total protein content determination. Based on HPLC peak area values, the betacyanin composition was estimated at 64% of betanin and 36% of isobetanin. In the rest of the article, all the concentrations given correspond to the total betanin/isobetanin (Bet./IsoBet.) concentrations applied to the cells.

Effect of betanin/isobetanin on cell proliferation and morphology

We used both cancer (B16F10, MCF-7, MDA-MB-231, HT-29) and normal (HUVEC, MRC-5) cells to assess the biological effect of betanin extract. All cells were cultured as monolayer (2D) on tPS and as aggregates (3D) on anti-adhesive polyHEMA substratum, and then treated with betanin-enriched extract for 48 hours.

As shown in Figure 2A

Effect of betanin/isobetanin concentrate on cell cycle distribution

To better understand betanin/isobetanin concentrate effect, we focused on three human cell lines that represent three different and specific phenotypes: p53 wild-type cancer cell line MCF-7, p53-mutated cancer cell line MDA-MB-231 and normal HUVEC cells.

The cell cycle progression was studied 48 hour post-seeding without (control) or with 30 µM Bet./IsoBet. (Table 1). For each cell line, the cell cycles of control cultures on tPS and polyHEMA were significantly different: there were more cells in G1 phase and fewer in the S and G2/M phases in 3D cultures.

Betanin/isobetanin-enriched extract significantly decreased the percentage of MCF-7 cells in G1 phase (2D and 3D conditions) and increased percentage of cells in S phase (2D culture) or G2/M phase (3D culture). For MDA-MB-231 and HUVEC cell lines, betanin/isobetanin concentrate decreased cell number in S phase (2D). No obvious effect of Bet./IsoBet. was observed on the 3D MDA-MB-231 cell cycle repartition.

Betanin/isobetanin treatment decreased the 3D HUVEC cell number in G1 phase and increased the percentage of cells in S and G2/M phases.

Characterization of MCF-7 cell death induced by betanin/isobetanin concentrate

As MCF-7 cell viabilities strongly decreased during Bet./IsoBet. treatment, we analyzed the nature of cell death induced by the pigments. First we checked apoptosis induction and quantification using Annexin-V-FITC labeling and cell analysis by flow cytometry (Table 2, Figure 3A). Our results showed that betanin/isobetanin treatment significantly increased the percentage of Annexin-V positive/PI negative MCF-7 cells, as the positive control staurosporine. MCF-7 3D cell culture promoted phosphatidylserine externalization, and the percentage of Annexin-V positive cells was found to be increased with betanin/isobetanin or staurosporine. MDA-MB-231 cells cultured in 2D

or 3D with Bet./IsoBet. were not strongly labeled with Annexin-V-FITC probe (Table 2). Interestingly, HUVEC cells were found to be non-apoptotic when cultured in 2D and treated with Bet./IsoBet., whereas staurosporine treatment induced significantly apoptosis (Table 2, Figure 3A). Anchorage-independent culture of HUVEC cells triggered Annexin-V labeling and the percentage of apoptotic cells was slightly increased with betanin/isobetanin treatment. To confirm apoptosis induction in MCF-7 cells treated with betanin/isobetanin concentrate, we analyzed DNA fragmentation using an antibody recognizing single-stranded DNA. After culture for 48 hours with betanin/isobetanin (30 µM) or staurosporine (1 µM), about 80% of MCF-7 cells were labelled (Figure 3B). Only 1.84% of HUVEC cells treated with betanin-enriched extract were found to be positive (Figure 3B), whereas 29.03% were labeled with the positive control staurosporine.

Proteome profiler arrays allowed us to detect the expression levels of apoptosis-related proteins in MCF-7, MDA-MB-231 and HUVEC cells treated with 30 µM Bet./IsoBet.

Typical results of obtained arrays are shown in Figure 4A. The most important differences in protein expressions were found in sensitive MCF-7 cells: we observed a strong induction of Bad, TRAILR1/DR4, Fas/TNFRSF6/CD95 and phospho-p53 (S392) proteins in betanin-treated cells compared to control cells (Figure 4B, Table I supplementary data). No significant variation in protein expression was observed in MDA-MB-231 cells. In HUVEC cells, Bet./IsoBet. treatment induced a moderate overexpression of p21 and p27 proteins (Table I supplementary data).

The effect of betanin/isobetanin on the mitochondrial membrane potential was checked in MCF-7 cells using MitoCapture dye (Figure 5). Our data showed an increase in green fluorescence in betanin/isobetanin-treated cells, in a same extent than positive control (curcumin 50 µM).

To assess another cell death, autophagy, MCF-7 cells treated with Bet./IsoBet. or rapamycin (an autophagy inducer) were analyzed for lysosomal vacuole formation (Figure 6). We observed positive labeling in MCF-7-treated cells, indicating an autophagic activity increase.

DISCUSSION

We described that a highly betanin/isobetanin-enriched concentrate produced from red beetroots inhibits cancer cell proliferation and induces MCF-7 cell death, but has no obvious effect towards normal cells.

Betanin and isobetanin are the most predominant betalains in red beetroot. We were able to obtain a pigment-enriched dried extract from beetroots which contains only betanin/isobetanin as coloring agents. This concentrate consists of 80 % of betanin/isobetanin mixture (of which betanin accounts for 64% and isobetanin for 36%) and corresponds, to our knowledge, to the highest purified betanin extract used for studying its anticancer activities. To better evaluate the cytotoxicity of the betaninenriched extract, we used two different culture conditions: classical monolayer culture (2D) and cells cultured as aggregates on anti-adhesive substratum (3D) [START_REF] Velzenberger | Study of cell behaviour on a cellulose anti-adhesive substratum[END_REF]. These 3D culture models are now commonly used to study new anticancer agents because they are believed to bridge the gap between in vitro 2D assessment and animal models [START_REF] Breslin | Three-dimensional cell culture: the missing link in drug discovery[END_REF]. Cell cytotoxicity analysis revealed that p53 wild-type cancer cell lines (B16F10, MCF-7) were highly sensitive to 40 µM betanin/isobetanin mix (assessed by proliferation inhibition and low cell viabilities), whereas cancer cell lines expressing mutated p53 were less (MDA-MB-231) or not sensitive (HT-29) for the same concentration range. The betanin-enriched extract effect was similar whatever the culture conditions, 2D or 3D. This shows that betanin/isobetanin concentrate inhibits aggregated cancer cell proliferation, a cell structure known as more resistant to apoptosis induction [START_REF] Hirschhaeuser | Multicellular tumor spheroids: an underestimated tool is catching up again[END_REF]. In this way, B16F10 and MDA-MB-231 are metastatic cell lines, resistant to anoikis when cultured under anchorage-independent conditions because of the activated ERK signaling pathway [START_REF] Goundiam | Growth and survival signalling in B16F10 melanoma cells in 3D culture[END_REF][START_REF] Fukazawa | Mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitors restore anoikis sensitivity in human breast cancer cell lines with a constitutively activated extracellularregulated kinase (ERK) pathway[END_REF]. For these reasons, identifying an efficient molecule on anoikis resistant cancer cells is a very hopefully result. Cell proliferation inhibition by betanin/isobetanin was confirmed with affected cell cycle. In MCF-7 cells, betanin/isobetanin extract decreased the G1 and G2/M cell number and promoted S phase increase, a behavior previously described when MCF-7 cells were treated with cell growth inhibitors as resveratrol and riproximin [START_REF] Joe | Resveratrol induces growth inhibition, S-phase arrest, apoptosis, and changes in biomarker expression in several human cancer cell lines[END_REF][START_REF] Pervaiz | Riproximin modulates multiple signaling cascades leading to cytostatic and apoptotic effects in human breast cancer cells[END_REF]. Betanin/isobetanin extract may downregulate the expression of cell cycle regulators (cyclin B1, cyclin A2) resulting in G2 phase entry inhibition and S phase arrest, as act resveratrol and riproximin. 2D cultured MDA-MB-231 cells were blocked in G1 phase during treatment, but the molecules did not significantly affect the cell cycle repartition when cells were cultured as aggregates, probably because of the G1 arrest induced by the culture configuration. [START_REF] Kapadia | Cytotoxic effect of the red beetroot (Beta vulgaris L.) extract compared to doxorubicin (Adriamycin) in the human prostate (PC-3) and breast (MCF-7) cancer cell lines[END_REF]2013) have previously evaluated the cytotoxic effect of a red beetroot extract in MCF-7 cell line and the IC50 value they obtained was 600 µM (after 72 hours of exposure). [START_REF] Reddy | Relative inhibition of lipid peroxidation, cyclooxygenase enzymes, and human tumor cell proliferation by natural food colors[END_REF] also observed a growth inhibition of MCF-7 cells treated with a betanin concentrate for 48 hours (IC50 value was 294 µM). Our data clarify these previous studies. The betanin purification process we applied to the crude beetroot extract, allowed us to obtain a significant MCF-7 growth inhibition associated with cell death for very low concentrations (below 40 µM).

As MCF-7 cell viability strongly decreased when treated with betanin/isobetanin mix, the nature of cell death was analyzed. Using different methods, we showed that betanin treatment induced apoptosis in 2D MCF-7 cells. The expressions of apoptosis-related proteins (Bad, TRAILR4, FAS, phosphorylated p53) were strongly increased and the mitochondrial membrane potential was clearly altered. Taken together, all these data suggest both mitochondrial and death-receptor pathway involvement, and a p53dependent response in MCF-7 cells treated with the betanin/isobetanin concentrate.

Betanin extract (30 µM) only induced a moderate apoptosis in aggregated MCF-7 cells, a 3D configuration often promoting cell death resistance. Recently, Gong et al. (2015) have compared the cytotoxicity effect of the doxorubicin on MCF-7 cells cultured as monolayer or spheroids. Their data show that 3D cultured MCF-7 cells were less sensitive than their 2D counterpart. According to our data on cell proliferation and viability, we would probably observe an apoptosis induction on 3D MCF-7 cells with higher concentrations of betanin/isobetanin mix.

Intrinsic pathway activation upon betanin exposure was previously observed by [START_REF] Sreekanth | Betanin a betacyanin pigment purified from fruits of Opunita ficus-indica induces apoptosis in human chronic myeloid leukemia cell line K562[END_REF] in K562 cells with mitochondrial membrane potential decrease and cytochrome c release. A significant activation of caspase 9 and effectors (caspases 3 and 7) was also reported in lung cancer cells treated with 400 µM betanin for 48 hours [START_REF] Zhang | Beetroot red (betanin) inhibits vinyl carbamate-and benzo(a)pyrene-induced lung tumorigenesis through apoptosis[END_REF]. Our results confirm these previous studies about the mitochondrial pathway involvement. Moreover, we show that betanin/isobetanin mix induces upregulation of death receptors and p53 activation, as curcumin does on MCF-7 cells [START_REF] Choudhuri | Curcumin induces apoptosis in human breast cancer cells through p53-dependent Bax induction[END_REF][START_REF] Mohankumar | Mechanism of apoptotic induction in human breast cancer cell, MCF-7, by an analog of curcumin in comparison with curcumin--an in vitro and in silico approach[END_REF]. P53 is a well-known important actor in response to cellular stresses [START_REF] Goh | The role of mutant p53 in human cancer[END_REF]. The mutated p53 status in MDA-MB-231 and HT-29 cells [START_REF] Bartek | Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines[END_REF][START_REF] Rodrigues | p53 mutations in colorectal cancer[END_REF] As some natural-derived compounds have already been shown to induce cell death via an autophagic process [START_REF] Zhang | Plant natural compounds: targeting pathways of autophagy as anti-cancer therapeutic agents[END_REF], we investigated and revealed lysosomal vacuole formation in MCF-7 cells upon betanin treatment. We are the first to describe that betanin/isobetanin mix also induces an autophagic response in cells. The identification of molecules that target autophagy is believed to be a realistic prospect for cancer therapy [START_REF] Bincoletto | Interplay between apoptosis and autophagy, a challenging puzzle: new perspectives on antitumor chemotherapies[END_REF].

To fully characterize the biological effect of betanin, we also evaluated its cytotoxicity on normal cells. Importantly, normal cell lines (HUVEC, MRC-5) were found to be weakly or not sensitive to betanin C15 stereoisomer mix for the same concentration range used on cancer cells. Particularly, HUVEC endothelial cells slowed down their proliferation rate upon betanin exposure, remaining viable for higher concentrations than MCF-7 cells. When cultured as monolayer and treated with the betanin/isobetanin extract, HUVEC cells accumulated in the G1 phase and the percentage of cells in the DNA replicative phase decreased, confirming the cell proliferation data. 3D betanin treated-HUVEC cells progressively accumulated in the S and G2/M phases. Moreover the betanin/isobetanin treatment triggered an overexpression of p21 and p27 proteins, cyclin-dependent kinase inhibitors involved in cell cycle arrest. However, the HUVEC cell proliferation inhibition by the betanin concentrate was not associated with apoptosis or autophagy induction: this suggests that undesired side effects could be limited in therapies integrating these molecules. Indeed, the preliminary data we performed on the non-tumorigenic MCF-12F cell line confirmed the harmless interaction of the molecules on normal epithelial breast cells (data not shown).

In conclusion, we reported that a purified betanin/isobetanin concentrate produced from red beetroots is cytotoxic at low concentrations for cancer cells expressing functional p53, but have no or moderate effect on normal cells, suggesting limited in vivo side effects. In p53 wild-type cancer cells, betanin/isobetanin extract induces apoptosis (via extrinsic and intrinsic activation pathways) and autophagy. Moreover, betanin-enriched extract inhibits some p53 mutated cancer cell growth, without inducing apoptosis. Our data bring new insight to consider betanin/isobetanin as therapeutic anticancer molecules alone or in combination with classical chemotherapeutic drugs, especially in functional p53 tumors.

Tables

  betanin and isobetanin structures were confirmed by mass tandem spectroscopy. The positive ion spray mass spectrum (Figure 1C) shows the daughter ion produced by fragmentation of the parent ion of m/z of 551 [M+H] + = 551 assigned to betanin or isobetanin. The fragment ion at the mass charge (m/z) of 389 indicated that this ion is obtained by glucose loss and corresponds to the protonated aglycones [betanidin + H] + or [isobetanidin + H] + (Castellanos-Santiago and Yahia, 2008).

  Cell morphologies were examined (Figure2D): MCF-7 and HUVEC control cells cultured on tPS spread well and colonized the culture dishes, while cells on polyHEMA had a rounded aggregated morphology. Betanin-enriched extract reduced MCF-7 cell spreading on tPS and MCF-7 cell aggregation on polyHEMA, whereas a moderate effect of betanin/isobetanin concentrate was observed on HUVEC cells cultured in 2D and 3D conditions. No morphological change was observed for the HT-29 and MRC-5 cells treated with Bet.IsoBet. (data not shown).Thus we identified three groups of cell lines with different sensitivities to betanin/isobetanin: B16F10 and MCF-7 as highly sensitive (no viable cells with 40 µM Bet./IsoBet.); MDA-MB-231 and HUVEC as weakly sensitive (cell viabilities from 35% to 70% with 40 µM Bet./IsoBet.); HT-29 and MRC-5 as resistant (cell viabilities about 90% with 40 µM Bet./IsoBet.).

  could explain in part the weak betanin cytotoxicity for these cell lines. Particularly, HT-29 cells express a mutant p53 protein which gains function, promoting cell proliferation and chemoresistance[START_REF] Van Oijen | Gain-of-function mutations in the tumor suppressor gene p53[END_REF]. In this way,[START_REF] Arafa | Synthesis and in vitro antiproliferative effect of novel quinoline-based potential agents[END_REF] have recently compared HT-29 and MDA-MB-231 sensitivity to novel quinoline-based compounds. They found that the HT-29 cell line was more refractory to the cytotoxic activity of most compounds than MDA-MB-231 cells. In our work, HT-29 cells were also resistant to betanin/isobetanin effect, whereas molecules slowed down MDA-MB-231 cell proliferation without inducing apoptosis. Therefore low concentrations of betanin-enriched extract appear to be efficient on a wild-type p53 cancer cells to induce cell death (MCF-7) and inhibit a p53-mutated cancer cell proliferation (MDA-MB-231), but have no effect on another p53-mutated cell line (HT-29).

Table 1 : Cell cycle distribution of cells grown on tPS or polyHEMA in the presence or absence (Control) of betanin/isobetanin extract.

 1 Results are means ± SD of three experiments each performed in triplicate. Asterisks indicate significant differences in cell cycle distribution of treated and control cells (Dunn test): *, p<0.05; **, p<0.01; ***, p<0.001.

			Control	Bet./IsoBet. (30 µM)
	MCF7 2D	G1	54.7% ± 1.6	49.1% ± 4.2*
		S	26.4% ± 2.8	34.2% ± 9.8
		G2/M	18.8% ± 4.1	16.7% ± 5.5
	3D	G1	77.0% ± 2.6	68.2% ±1.8**
		S	12.4% ± 3.2	17.8% ± 2.2
		G2/M	10.6% ± 0.7	14.0% ± 0.7***
	MDA-MB231 2D	G1	62.3% ± 1.9	67.4% ± 1.2
		S	24.8% ± 1.6	19.8% ± 0.9*
		G2/M	12.9% ± 0.8	13.5% ± 2.2
	3D	G1	74.6% ± 10.0	72.4% ± 10.5
		S	22.9% ± 11.6	21.7% ± 13.2
		G2/M	2.5% ±2.9	5.8% ± 2.7
	HUVEC 2D	G1	51.2% ± 2.4	58.4% ± 1.9
		S	40.0% ± 1.5	30.2% ± 2.5*
		G2/M	8.8% ± 1.4	11.7% ± 1.6*
	3D	G1	90.1% ± 1.4	80.9% ± 4.5*
		S	5.5% ± 1.2	13.1% ± 4.6
		G2/M	4.3% ± 1.3	5.9% ± 1.2

Acknowledgments

We thank the European Union (co-funding of equipment within the CPER 2007-2013 and FEDER) and the Conseil Régional of Picardie for financial support (BetOX project).