Maiko Kitaoka

Rebecca Heald
email: bheald@berkeley.edu

Romain Gibeaux
email: romain.gibeaux@univ-rennes1.fr

Spindle assembly in

Keywords: Xenopus borealis, egg extracts, meiotic spindle, mitosis, TPX2, Ran

come

INTRODUCTION

In all eukaryotes, cell division requires the spindle, a highly dynamic, self-organizing, microtubule-based apparatus that accurately segregates replicated chromosomes to daughter cells. Spindle microtubules assemble into a bipolar structure that attaches to chromosomes via their kinetochores and aligns them at the metaphase plate so that sister chromatids are oriented toward opposite spindle poles. Although the basic form and composition of the spindle is conserved, spindle structure adapts to changes in cell size and type, and varies dramatically both across and within species [START_REF] Wühr | Evidence for an Upper Limit to Mitotic Spindle Length[END_REF][START_REF] Crowder | A comparative analysis of spindle morphometrics across metazoans[END_REF]. While many of the molecules and mechanisms that orchestrate spindle assembly have been elucidated, it remains unclear how specific spindle architectures are established, and how this variation impacts spindle function.

Unfertilized eggs of the African clawed frog Xenopus laevis arrested in metaphase of meiosis II by cytostatic factor (CSF) [START_REF] Masui | Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes[END_REF]) can be obtained in large quantities. Their fractionation by centrifugation yields a crude cytoplasmic extract that maintains the metaphase arrest and supports spindle assembly around sperm nuclei or chromatin-coated beads [START_REF] Sawin | Mitotic Spindle Assembly by Two Different Pathways in Vitro[END_REF][START_REF] Heald | Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts[END_REF]. Remarkably, spindles formed in egg extracts of the smaller, related frog Xenopus tropicalis are shorter in length independent of DNA source, and mixing the two extracts produced spindles of intermediate size [START_REF] Brown | Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle[END_REF].

It was found that compared to X. laevis, X. tropicalis possess smaller spindles due to (i) elevated activity of the hexameric AAA-ATPase microtubule severing enzyme katanin [START_REF] Loughlin | A computational model predicts Xenopus meiotic spindle organization[END_REF][START_REF] Loughlin | Katanin contributes to interspecies spindle length scaling in Xenopus[END_REF], and (ii) a higher concentration of the spindle assembly factor, TPX2 (targeting factor for Xklp2), which promotes association of the cross-linking spindle motor Eg5 and a shift of microtubule density from antiparallel overlap in the center of the spindle to the poles [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF]. X. tropicalis spindles were also observed to possess distinct morphological features, including more robust kinetochore fibers and a more circular shape [START_REF] Loughlin | Katanin contributes to interspecies spindle length scaling in Xenopus[END_REF][START_REF] Grenfell | A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles[END_REF]. Furthermore, whereas spindle assembly in X. laevis extracts required a chromatin-generated gradient of RanGTP [START_REF] Kalab | Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts[END_REF][START_REF] Cavazza | The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond[END_REF], X. tropicalis spindle assembly was not affected by disruption of this pathway [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF]. Therefore, not only spindle size, but also spindle architecture, morphology, and assembly mechanisms differ between these two species.

To further address the conservation of spindle morphology, assembly, and scaling mechanisms across evolution, we decided to characterize a third Xenopus species, the Marsabit clawed frog Xenopus borealis. Whereas allotetraploid X. laevis (36 chromosomes) and diploid X. tropicalis (20 chromosomes) diverged 48 million years ago, X. borealis is more closely related to X. laevis, having diverged 17 million years ago [START_REF] Session | Genome evolution in the allotetraploid frog Xenopus laevis[END_REF]. Like X. laevis, X. borealis possesses an allotetraploid genome of 36 chromosomes. Interestingly, X. borealis was used extensively in the 1970s to analyze the conservation of ribosomal nucleic acids compared to X. laevis [START_REF] Brown | A comparison of the ribosomal DNA's of Xenopus laevis and Xenopus mulleri: the evolution of tandem genes[END_REF][START_REF] Brown | The structure and evolution of ribosomal and 5S DNAs in Xenopus laevis and Xenopus mulleri[END_REF][START_REF] Griswold | An analysis of the degree of homology between 28S rRNA from Xenopus laevis and Xenopus mulleri[END_REF][START_REF] Leister | Mitochondrial ribosomal proteins in Xenopus laevis/X. mulleri interspecific hybrids[END_REF][START_REF] Wellauer | A comparison of the structural organization of amplified ribosomal DNA from Xenopus mulleri and Xenopus laevis[END_REF][START_REF] Ford | Sequences of 5S ribosomal RNA from Xenopus mulleri and the evolution of 5S gene-coding sequences[END_REF] and was misidentified as Xenopus mulleri until 1977 [START_REF] Brown | Xenopus borealis misidentified as Xenopus mulleri[END_REF]. Revisiting this species and applying the egg extract methodology allowed us to further evaluate spindle morphometric variation and scaling in Xenopus. We show that X. borealis spindles are intermediate in size and display properties of both X. laevis and X. tropicalis, resulting in distinct features. These findings highlight the evolutionary plasticity of spindle morphology and assembly mechanisms, and introduce X. borealis as a third Xenopus species amenable to in vitro assays to investigate the molecular basis of spindle variation.

RESULTS AND DISCUSSION

In this study, we characterized X. borealis and spindle assembly in its egg extracts, using the two well-characterized Xenopus species, X. laevis and X. tropicalis, for comparison.

X. borealis frogs, eggs, and sperm are morphologically distinct

At ~7 cm body length, adult X. borealis female frogs were intermediate in size between X. laevis (~10 cm) and X. tropicalis (~4 cm) and also displayed distinct physical traits including googly eyes and a pear-like body shape (Figure 1A). Measuring the size of X. borealis gametes revealed that X. borealis eggs averaged 1.2 ± 0.02 mm in diameter, slightly smaller than X. laevis (1.3 ± 0.05 mm) and larger than X. tropicalis (0.75 ± 0.02 mm) (Figure 1B). Interestingly, X. borealis sperm cells were significantly longer (average 22 ± 2.8 µm) than those of both X. laevis (19 ± 2.3 µm) and X. tropicalis (13.5 ± 2.1 µm) (Figure 1C), and similar size differences were observed comparing isolated sperm nuclei (Supplementary Figure S1) [START_REF] Grainger | Xenopus tropicalis as a Model Organism for Genetics and Genomics: Past, Present and Future[END_REF]. In vitro fertilization and live imaging revealed that the rate of early development of X. borealis and X. laevis embryos were similar (Figure 1D and Video 1) [START_REF] Nieuwkoop | Normal table of Xenopus laevis (Daudin)[END_REF]. Nuclei isolated from stage 8 (5 hpf) embryos of X. borealis (average 2214 ± 1016 µm 2 ) were comparable in size to those from X. laevis (2010 ± 1081 µm 2 ), while X. tropicalis nuclei (405 ± 116 µm 2 ) were significantly smaller (Figure 1E). In summary, although X. borealis display distinct traits, our measurements show that like X. laevis and X. tropicalis, X. borealis egg size scales with adult animal size, and that X. borealis development and nuclear size is very similar to that of X. laevis. These findings are consistent with the well-characterized phenomenon that genome size scales with nuclear and cell size among amphibians [START_REF] Levy | Biological scaling problems and solutions in amphibians[END_REF].

X. borealis egg extract recapitulates cell cycle events

To evaluate the utility of X. borealis eggs for in vitro assays, we adapted published ovulation procedures to reproducibly obtain metaphase II, CSF-arrested eggs in sufficient amounts to prepare egg extracts (see Material and Methods). X. borealis eggs were dejellied and fractionated using standard protocols [START_REF] Hannak | Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts[END_REF][START_REF] Maresca | Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts[END_REF][START_REF] Brown | Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle[END_REF], and produced similar yields of cytoplasmic extract compared to X. laevis despite their smaller body and egg size. X. borealis egg extract was then combined with X. borealis sperm nuclei and assayed for spindle assembly. When added directly to CSF extract, sperm nuclei induced the formation of asters (t = 5'), followed by half-spindles (t = 15') and bipolar CSF spindles (t = 30 -45') (Figure 2A,B). Addition of calcium together with sperm nuclei induced exit from metaphase arrest and formation of interphase nuclei within 45 min.

Following the addition of fresh CSF extract, bipolar "cycled" spindles assembled from 75 min to 90 min (Figure 2A,C). We noted that X. borealis extracts formed spindles more quickly than X. laevis, almost as rapidly as X. tropicalis (unpublished data). These experiments show that, as for the other Xenopus species, X. borealis egg extracts are fully capable of recapitulating the cell cycle and assembling spindles in vitro.

Xenopus species possess distinct spindle morphometrics

We next examined some parameters of X. borealis spindle size and microtubule organization and compared them to in vivo meiosis II spindles as well as to spindles assembled in X. laevis and X. tropicalis egg extracts. X. borealis spindles assembled in vitro were similar in length from poleto-pole and width at the metaphase plate to meiotic spindles imaged in metaphase II-arrested eggs (length/width average 43.9 ± 5.5 µm/21.7 ± 3.6 µm in vivo vs. 44.4 ± 5.5 µm/23.4 ± 5.9 µm in extract) (Figure 3A). X. borealis extract was also capable of inducing bipolar spindle assembly around stage 8 X. borealis embryo nuclei and 10 µm diameter chromatin-coated beads [START_REF] Halpin | Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts[END_REF]. Embryo nuclei spindles were slightly smaller than those formed around sperm nuclei (average length 39.7 ± 4.4 µm, width 25.1 ± 4.7 µm) (Figure 3B), while spindles assembled around DNA-coated beads were considerably shorter (average 22.4 ± 3.9 µm) (Figure 3C). These measurements demonstrate that like X. laevis and X. tropicalis, spindles formed in X. borealis egg extract mimic the size and morphology of the meiotic egg spindle, and that spindle assembly in vitro occurs in the presence or absence of centrosomes.

Despite their smaller egg size, we found that cycled X. borealis spindles were similar in length to X. laevis (average 41.9 ± 4.1 µm vs. 41.6 ± 6.0 µm), but significantly narrower (average 19.7 ± 3.7 µm vs. 22.4 ± 3.6 µm), thereby reducing spindle area and therefore total microtubule content. X. borealis spindles were both longer and wider than X. tropicalis spindles (average 23.2 ± 3.0 µm in length, 10.4 ± 2.6 µm in width) (Figure 4A). Thus, X. borealis spindles scale with egg size and are intermediate in size between those of X. laevis and X. tropicalis. Interestingly, the three species each displayed unique spindle microtubule distributions across the spindle. As previously reported, X. laevis spindles showed a consistent plateau of tubulin intensity along the length of the spindle indicating the presence of an overlapping, tiled array of microtubules [START_REF] Loughlin | A computational model predicts Xenopus meiotic spindle organization[END_REF]. In contrast, X. tropicalis tubulin intensity was greater at the poles and reduced in the center of the spindle at the metaphase plate [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF].

Interestingly, X. borealis spindles displayed a similar increase in microtubule density at the poles as X. tropicalis, a similar density at the metaphase plate as X. laevis, and a unique dip in tubulin intensity between the poles and metaphase plate (Figure 4B). While similar microtubule distribution patterns were observed in CSF spindles, they were less distinct than in cycled spindles, potentially due to the absence of sister kinetochores and associated microtubules [START_REF] Grenfell | A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles[END_REF]. X. borealis CSF spindles were also longer and wider than both X. laevis and X. tropicalis (Supplementary Figure S2). A difference in spindle morphology between spindles with replicated (cycled) and unreplicated (CSF) chromosomes has been observed previously and emphasizes the fact that spindle morphology can vary in different cellular contexts [START_REF] Grenfell | A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles[END_REF][START_REF] Levy | Biological scaling problems and solutions in amphibians[END_REF]. We decided to focus on cycled spindle assembly for subsequent experiments, as these form around duplicated chromosomes with assembled kinetochores and thus are more physiologically relevant. Therefore, X. borealis fits into the Xenopus interspecies scaling regime, as the animals, eggs and meiotic spindles are all intermediate in size between the other two species. However, X. borealis microtubule arrays share some similarity with both species, leading to a unique spindle architecture.

X. borealis spindles are sensitive to perturbation of the RanGTP gradient

To compare spindle assembly mechanisms among the three species, we examined the role of the RanGTP gradient, which acts to release nuclear localization signal (NLS)-containing spindle assembly factors from importins near mitotic chromatin where the guanine nucleotide exchange factor RCC1 (Regulator of Chromosome Condensation 1) is localized, thereby promoting microtubule nucleation, stabilization, and crosslinking [START_REF] Cavazza | The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond[END_REF]. Perturbing the RanGTP gradient by the addition of Ran mutants, such as a dominant negative (T24N) or constitutively active, GTP-bound (Q69L) form, is detrimental to X. laevis spindle assembly, abolishing it completely or increasing ectopic microtubule nucleation and formation of multipolar spindles, respectively [START_REF] Kalab | The Ran GTPase regulates mitotic spindle assembly[END_REF][START_REF] Kalab | Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts[END_REF]. In contrast, we showed previously that X. tropicalis spindles are much less affected by perturbation of the RanGTP gradient [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF]. To test the role of this pathway in X. borealis, we added the Ran mutants at the onset of spindle assembly. Upon addition of RanT24N, spindle assembly was strongly impaired, with dramatically reduced microtubule nucleation around sperm nuclei, while RanQ69L increased the number of multipolar spindle structures (Figure 5 andSupplementary Table 1). Thus, like X. laevis, X. borealis bipolar spindle assembly depends on the RanGTP gradient.

Common spindle scaling mechanisms operate among Xenopus species

It has been shown that hybrid embryos can be generated by fertilization of X. borealis eggs with sperm from either X. laevis and X. tropicalis [START_REF] De Robertis | Hybrids of Xenopus laevis and Xenopus borealis express proteins from both parents[END_REF][START_REF] Gibeaux | Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus[END_REF], indicating compatibility between the X. borealis cytoplasm and the chromosomes of both species. Addition of either X. laevis or X. tropicalis sperm nuclei to X. borealis extract did not alter spindle microtubule distribution (Supplementary Figure S3A), consistent with previous studies indicating that egg cytoplasm composition is the primary determinant of spindle morphology [START_REF] Brown | Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle[END_REF]. To determine whether maternal cytoplasmic factors affect spindle size similarly among the three Xenopus species, we mixed X. borealis egg extract with either X. laevis or X. tropicalis extract in the presence of X. laevis or X. tropicalis sperm nuclei, respectively. Whereas titration of X. borealis extract with X. laevis did not affect spindle dimensions (Figure 6A), X. tropicalis extract caused X. borealis spindles to shrink proportionately with the amount added (Figure 6B). Analysis of spindle microtubule distributions in mixed extracts revealed that these were also affected proportionately, favoring the tubulin distribution of the more abundant extract (Supplementary Figure S3B andC).

These results are consistent with cytoplasmic mixing of X. laevis with X. tropicalis extracts, which also caused spindle shrinkage in a dose-dependent manner [START_REF] Brown | Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle[END_REF].

Altogether, these data indicate that common scaling and microtubule organization mechanisms operate among the three Xenopus species.

X. borealis spindles combine scaling factor features of both X. laevis and X. tropicalis

To investigate the molecular basis of X. borealis spindle size and morphology, we compared the localization, abundance, and sequence of katanin and TPX2, two spindle scaling factors that contribute to spindle size differences between X. laevis and X. tropicalis, as well as the microtubule polymerase XMAP215, levels of which have been shown to regulate spindle length in Xenopus [START_REF] Reber | XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules[END_REF]. XMAP215 levels and localization were similar for all three Xenopus species (Supplementary Figure S4). Katanin, a microtubule severing AAA-ATPase, has higher activity in X. tropicalis due to the absence of an inhibitory phosphorylation site (Serine 131) found in the X. laevis protein [START_REF] Loughlin | Katanin contributes to interspecies spindle length scaling in Xenopus[END_REF]. Immunofluorescence and line scan analysis of spindles revealed that katanin localization on the X. borealis spindle displays a small but distinct increase at the metaphase plate as well as the spindle poles, while the localization on X. laevis spindles is more uniform and X. tropicalis shows katanin enrichment solely at the poles (Figure 7A). By Western blot, katanin levels did not differ significantly across the three species (Figure 7B). Although the amino acid sequence of X. borealis katanin is over 97% identical to that of both X. tropicalis and X. laevis, it contains the key inhibitory serine residue that regulates katanin activity in X. laevis (Supplementary Figure S5). It remains to be determined whether katanin activity is similarly regulated in X. borealis. TPX2 is a Ranregulated microtubule-associated protein that is found at three-fold higher concentrations in X. tropicalis compared to X. laevis. Increasing the level of TPX2 in X. laevis extracts to that of X. tropicalis was previously shown to reduce spindle length through its increased recruitment of the spindle motor Eg5 to spindle poles [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF]. Interestingly, X. borealis spindles displayed an overall TPX2 localization more similar to X. tropicalis, with higher intensity at the spindle poles, but also possessed a central smaller peak of TPX2 reminiscent of X. laevis (Figure 7C). Levels of TPX2 in X. borealis egg extracts were two-fold higher than in X. laevis, but not elevated to X. tropicalis levels. (Figure 7D). Notably, the amino acid sequence of X. borealis TPX2 is more similar to X. laevis compared to X. tropicalis and contains the seven amino acid residues thought to reduce microtubule nucleation activity of X. laevis TPX2 (Supplementary Figure S6) [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF][START_REF] Grenfell | A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles[END_REF]. Altogether, these results suggest that X. borealis spindle length results from its lower katanin activity similar to X. laevis, while its microtubule distribution is likely affected by increased levels of TPX2, which is more reminiscent of X. tropicalis.

Overall, these observations reveal that X. borealis meiotic spindles, although more similar in size and RanGTP-dependence to X. laevis, also possess some structural and molecular features characteristic of X. tropicalis. These findings support the idea that katanin and TPX2 are important spindle length control and assembly factors in Xenopus. Katanin-mediated microtubule severing activity has been shown to regulate spindle size in several organisms, including Caenorhabditis elegans, Xenopus, and human cells [START_REF] Mcnally | Katanin controls mitotic and meiotic spindle length[END_REF][START_REF] Loughlin | Katanin contributes to interspecies spindle length scaling in Xenopus[END_REF][START_REF] Joly | Microtubule-severing Page 21 of 38 Cytoskeleton activity of the AAA+ ATPase Katanin is essential for female meiotic spindle assembly[END_REF][START_REF] Jiang | Microtubule minus-end regulation at spindle poles by an ASPM-katanin complex[END_REF], which is consistent with results from meiotic spindle assembly simulations showing that altering microtubule depolymerization rates robustly scales spindle length [START_REF] Loughlin | A computational model predicts Xenopus meiotic spindle organization[END_REF].

The role of TPX2 in spindle assembly and size control is more complicated. Depletion of the TPX2 ortholog D-TPX2 from Drosophila melanogaster syncytial embryos and S2 cells resulted in spindle shortening [START_REF] Goshima | Identification of a TPX2-like microtubule-associated protein in Drosophila[END_REF][START_REF] Hayward | Chromatin-mediated microtubule nucleation in Drosophila syncytial embryos[END_REF], and human cells require phosphorylation of TPX2 by Aurora A for normal spindle length [START_REF] Bird | Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A[END_REF][START_REF] Fu | TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux[END_REF]. In contrast, addition of recombinant TPX2 to X. laevis extracts decreased spindle length [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF], and we have observed similar effects on X. borealis spindles (unpublished data). Spindle-shrinking effects in Xenopus did not depend on Aurora A, but rather on a change in spindle microtubule distribution caused by increased recruitment of the cross-linking Eg5 motor to spindle poles [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF]. TPX2 also regulates microtubule nucleation in the spindle [START_REF] Petry | Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2[END_REF][START_REF] Scrofani | Microtubule nucleation in mitosis by a RanGTP-dependent protein complex[END_REF][START_REF] Petry | Mechanisms of Mitotic Spindle Assembly[END_REF][START_REF] Alfaro-Aco | Structural analysis of the role of TPX2 in branching microtubule nucleation[END_REF]. TPX2 has thus emerged as a central player of spindle assembly and organization in a wide variety of cell types [START_REF] Ma | TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle[END_REF][START_REF] Helmke | Interplay between spindle architecture and function[END_REF][START_REF] Fu | TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux[END_REF][START_REF] Levy | Biological scaling problems and solutions in amphibians[END_REF] and is overexpressed in many cancers [START_REF] Chang | The TPX2 gene is a promising diagnostic and therapeutic target for cervical cancer[END_REF][START_REF] Neumayer | TPX2: Of spindle assembly, DNA damage response, and cancer[END_REF]. Our results highlight the importance of TPX2 levels and localization as determinants of meiotic spindle microtubule architecture. TPX2 localization (Figure 7C) correlates well with the distribution of tubulin intensity across all three species (Figure 4B), particularly at the spindle poles [START_REF] Ma | TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle[END_REF][START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF]. Despite having higher TPX2 levels, the greater sequence similarity of X. borealis TPX2 to the X. laevis protein (Supplementary Figure S6) may explain the increased sensitivity to Ran inhibition in X. borealis (Figure 5C), since a 7 amino acid sequence shown to decrease microtubule nucleation activity relative to X. tropicalis is present in both X. borealis and X. laevis TPX2 (Figure 7D) [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF]. Altogether, our results provide clues to how TPX2 levels may tune meiotic spindle architecture and assembly pathways by altering microtubule nucleation and organization.

In summary, Xenopus borealis provides a robust in vitro egg extract that adds to the characterized X. laevis and X. tropicalis systems, enabling further investigation of spindle assembly and interspecies scaling. Our findings highlight that egg meiosis II spindle architecture varies across Xenopus species and suggest that X. borealis spindles assemble with a combination of X. laevis as well as X. tropicalis features.

MATERIALS AND METHODS

Chemicals

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich, St. Louis, MO.

Frog care

All animal experimentation is this study was performed according to our Animal Use Protocol approved by the UC Berkeley Animal Care and Use Committee. Mature X. laevis, X. tropicalis, and X. borealis frogs were obtained from NASCO, WI. X. laevis, X. tropicalis, and X. borealis females were ovulated with no harm to the animals with a 6-, 3-, and 4-month rest interval, respectively. To obtain testes, males were euthanized by over-anesthesia through immersion in ddH 2 O containing 0.15% MS222 (Tricaine) neutralized with 5 mM sodium bicarbonate prior to dissection, and then frozen at -20°C.

Sperm nuclei purification

X. laevis and X. tropicalis sperm nuclei were prepared and purified as previously described [START_REF] Grainger | Xenopus tropicalis as a Model Organism for Genetics and Genomics: Past, Present and Future[END_REF]. The following modifications were made for X. borealis sperm nuclei purification. X. borealis males were primed with 40 U of PMSG 3 days prior to dissection and boosted with 100 U hCG 12-24 h before dissection. Testes were removed and cleaned from all blood vessels and fat with forceps. Testes were then washed 3X in cold 1X MMR (1X MMR: 100 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 and 5 mM HEPES-NaOH pH 7.6, 0.1 mM EDTA) and 3X in cold Nuclear Preparation Buffer (NPB) (2X NPB: 500 mM sucrose, 30 mM HEPES, 1 mM spermidine trihydrochloride, 0.4 mM spermine tetrahydrochloride, 2 mM dithiothreitol, 2 mM EDTA) and placed in 1 mL of cold NPB for homogenization with scissors and a pestle. Samples were briefly spun to pellet the remaining tissue before transferring the supernatant to a new tube for centrifugation at 1500 g for 10 min at 4°C to pellet sperm. The pellet was resuspended in 1 mL NPB supplemented with 50 µL of 10 mg/mL lysolecithin and incubated for 5 min at RT. The resuspended pellet was then added to 4 mL of cold NPB supplemented with 3% BSA and spun at 3,000 RPM for 10 min at 4°C. The resulting pellet was resuspended in 2 mL of cold NPB with 0.3% bovine serum albumin (BSA) and 30% filtered glycerol. Finally, the pellet was resuspended in 500 µL of cold NPB with 0.3% BSA and 30% filtered glycerol. Sperm nuclei were adjusted to a concentration of 10 8 nuclei per mL using cold NPB with 0.3% BSA and 30% glycerol to make a 200X stock, aliquoted, frozen in liquid nitrogen, and stored at -80°C.

In vitro fertilization X. borealis males were injected with 300 U of human chorionic gonadotropin hormone (hCG) 12-24 h before dissection and testes were collected in Leibovitz L-15 Medium (Gibco -Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum (FBS; Gibco) for immediate use. X. laevis males were injected with 500 U of hCG 12-24 h before dissection and testes were stored at 4°C in 1X MR (100 mM NaCl, 1.8 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 and 5 mM HEPES-NaOH pH 7.6) for 1-2 weeks.

X. borealis and X. laevis females were primed with 60 U and 100 U, respectively, of pregnant mare serum gonadotrophin (PMSG, National Hormone and Peptide Program, Torrance, CA) at least 48 h before use and boosted with 300 U and 500 U, respectively, of hCG 12-24 h before the experiment. X. borealis and X. laevis frogs were kept at 16°C in 0.5X and 1X MMR, respectively. X. borealis eggs were picked from the tub and deposited onto petri dishes coated with 1.5% agarose in 1/10X MMR. X. laevis females were squeezed gently to deposit eggs onto petri dishes coated with 1.5% agarose in 1/10X MMR. Two X. borealis testes or 2/3 of an X. laevis testis were collected and homogenized using scissors and a pestle in 1 mL of L-15 and 10% FBS. Any excess liquid in the petri dishes was removed, and the eggs were fertilized with 500 µL of sperm solution per dish. Eggs were swirled in the solution to individualize eggs as much as possible and incubated for 5 min. Dishes were flooded with ddH 2 O, swirled and incubated for 10 min, followed by buffer exchange to 1/10X MMR and incubation for 10 min.

The embryo jelly coats were then removed with a 3% cysteine solution in ddH 2 O-NaOH, pH 7.8.

After extensive washing (>4X) with 1/10X MMR, embryos were incubated at 23°C. At stage 2-3, fertilized embryos were sorted and placed in fresh 1/10X MMR in new petri dishes coated with 1.5% agarose in 1/10X MMR.

All embryos were staged according to Nieuwkoop and Faber [START_REF] Nieuwkoop | Normal table of Xenopus laevis (Daudin)[END_REF].

Embryo video imaging

Page 13 of 38 Cytoskeleton

Imaging dishes were prepared using a homemade PDMS mold designed to print a pattern of 1 mm large wells in agarose that allowed us to image 4 X. borealis or X. laevis embryos simultaneously within the 3X4 mm camera field of view for each condition. Embryos were imaged from stage 2-3. Videos were taken simultaneously using two AmScope MD200 USB cameras (AmScope, Irvine, CA) each mounted on an AmScope SE305R stereoscope. Time lapse movies were acquired at a frequency of 1 frame every 10 s for 20 h and saved as Motion JPEG using a MATLAB (The MathWorks, Inc., Natick, MA) script. Movie post-processing (cropping, concatenation, resizing, addition of scale bar) was done using MATLAB and Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. All MATLAB scripts written for this study are available upon request. Two of the scripts used here were obtained through the MATLAB Central File Exchange: "videoMultiCrop"

and "concatVideo2D" by Nikolay S.

Embryo nuclei purification

Following in vitro fertilization, X. borealis embryos were arrested at stage 8 (~5 hpf) in late interphase using 150 µg/mL cycloheximide in 1/10X MMR for 60 min. Embryos were washed several times in Egg Lysis Buffer (ELB; 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl 2 , and 10 mM HEPES pH 7.8) supplemented with LPC (10 µg/mL each leupeptin, pepstatin, chymostatin), cytochalasin D (100 µg/mL), and cycloheximide (100 µg/mL), packed in a tabletop centrifuge at 200 g for 1 min, crushed with a pestle, and centrifuged at 10,000 g for 10 min at 16°C. The cytoplasmic extract containing endogenous embryonic nuclei was collected, supplemented with 8% glycerol, aliquoted, frozen in liquid nitrogen, and stored at -80°C.

X. borealis egg extract

X. borealis metaphase-arrested egg extracts were prepared similarly to X. laevis and X. tropicalis [START_REF] Maresca | Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts[END_REF][START_REF] Brown | Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle[END_REF], with the following modifications. 65.8 µg/uL stock) for spindle assembly reactions. Sperm nuclei, embryo nuclei, or 10 µm chromatin-coated beads, prepared as previously described [START_REF] Halpin | Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts[END_REF], were used as a source of DNA for spindle assembly.

X. laevis and X. tropicalis egg extracts and mixing experiments

X. laevis and X. tropicalis metaphase arrested egg extracts were prepared and spindle reactions conducted as previously described [START_REF] Maresca | Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts[END_REF][START_REF] Brown | Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle[END_REF]. X. laevis or X. tropicalis extracts were mixed in different proportions to X. borealis extract and supplemented with either X. laevis or X. tropicalis sperm nuclei, respectively.

Extract treatments

To examine sensitivity to perturbations of the RanGTP gradient, X. laevis, X. tropicalis, and X.

borealis sperm nuclei were added to their respective egg extracts and cycled through interphase.

Ran T24N (final concentration 1 µM) and Q69L (final concentration 5 µM) mutant proteins, purified as previously described [START_REF] Helmke | TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts[END_REF], were added to the interphase extract reaction when fresh CSF extract was added to induce the extract to cycle back to mitosis.

Spindle spin-down and immunofluorescence

Spindle reactions were prepared, spun-down, and processed for immunofluorescence as previously described [START_REF] Hannak | Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts[END_REF]. Briefly, the extract reactions were fixed for 5-10 minutes with 2.5% formaldehyde and spun down at 10,200 rpm for 16 min at 16°C. The coverslips were incubated for 30 s in cold methanol, washed in PBS+NP40, and blocked overnight in PBS + 5% BSA at 4°C. The anti-TPX2 (1:150 dilution, unpublished), anti-katanin p60 (1:500 dilution) [START_REF] Loughlin | Katanin contributes to interspecies spindle length scaling in Xenopus[END_REF], or anti-XMAP215 (1:200, Tony Hyman & Kazu Kinoshita) rabbit antibodies were added for 1 h in PBS + 5% BSA. After washing with PBS+NP40, the coverslips were incubated with 1:1000 anti-rabbit antibody coupled to Alexa

Fluor 488 (Invitrogen -Thermo Fisher Scientific, Waltham, MA) for 30 min and then with 1:1000 Hoechst for 5 min. The coverslips were then washed and mounted for imaging with Vectashield (Vector Laboratories, Burlingame, CA).

Egg whole mount immunofluorescence

Metaphase-arrested X. borealis eggs were fixed for 1-3 h at RT using MAD fixative (2 parts of methanol (Thermo Fisher Scientific, Waltham, MA), 2 parts of acetone (Thermo Fisher Scientific, Waltham, MA), 1 part of DMSO), and stored overnight at -20°C in fresh MAD fixative. Embryos were then processed as previously described [START_REF] Lee | Whole-Mount Fluorescence Immunocytochemistry on Xenopus Embryos[END_REF] with some modifications. Following gradual rehydration in 0.5X SSC (1X SSC: 150 mM NaCl, 15 mM Na citrate, pH 7.0), embryos were bleached with 2% H 2 O 2 (Thermo Fisher Scientific, Waltham, MA) in 0.5X SSC containing 5% formamide for 2-3 h under light, then washed in PBT, a PBS solution containing 0.1% Triton X-100 (Thermo Fisher Scientific, Waltham, MA) and 2 mg/ml BSA. Embryos were blocked in PBT supplemented with 10% goat serum (Gibco -Thermo Fisher Scientific, Waltham, MA) and 5% DMSO for 1-3 h and incubated overnight at 4°C in PBT supplemented with 10% goat serum and the primary antibodies. We used the following antibodies: 1:500 mouse anti-beta tubulin (E7; Developmental Studies Hybridoma Bank, Iowa City, IA), 1:350 rabbit anti-histone H3 (ab1791; Abcam, Cambridge, MA). Embryos were then washed 4 X 2 h in PBT and incubated overnight in PBT supplemented with 1:500 goat antimouse secondary antibody coupled to Alexa Fluor 488 and 1:500 goat anti-rabbit secondary antibody coupled to Alexa Fluor 568 (Invitrogen -Thermo Fisher Scientific, Waltham, MA).

Embryos were then washed 4 X 2 h in PBT and gradually dehydrated in methanol. Embryos were finally cleared in Murray's clearing medium (2 parts of Benzyl Benzoate, 1 part of Benzyl Alcohol). Embryos were placed in a chamber made using a flat nylon washer (Grainger, Lake Forest, IL) attached with nail polish (Sally Hansen, New York, NY) to a slide and covered by a coverslip, and filled with Murray's clearing medium for confocal microscopy.

Confocal microscopy

Multiple sequence alignments were performed using Clustal Omega (default parameters).

Sequence similarities were determined by pairwise alignments using EMBOSS Needle (default parameters).

Page 18 of 38 Cytoskeleton Comparison and quantification of egg size. Egg size (diameter in mm) is plotted as boxplots for X. laevis (green), X. tropicalis (blue), and X. borealis (purple). Each dot represents an individual egg (n = 89 for X. laevis, n = 132 for X. tropicalis, and n = 77 for X. borealis, from 3 females for each species). Representative images of eggs for each species are shown (right). Scale bar is 200 µm. (C) Comparison and quantification of sperm size. Sperm head length (µm) is plotted as boxplots for X. laevis (green), X. tropicalis (blue), and X. borealis (purple). Each dot represents an individual sperm cell (n = 288 for X. laevis, n = 278 for X. tropicalis, and n = 310 for X. borealis, from 3 males for each species). Representative images of sperm cells for each species are shown (right). Scale bar is 20 µm. (D) Early embryo development at room temperature for X. laevis and X. borealis. Embryos were staged according to [START_REF] Nieuwkoop | Normal table of Xenopus laevis (Daudin)[END_REF]. Data for X. laevis embryos (n = 12) are blotted in green and X. borealis to X. borealis (purple) (n = 341 for 100% X. borealis, 0% X. laevis; n = 350 for 75% X. borealis, 25% X. laevis; n = 331 for 50% X. borealis, 50% X. laevis; n = 313 for 25% X. borealis, 75% X. laevis; and n = 267 for 0% X. borealis, 100% X. laevis over 3 independent experiments). (B) X. borealis extract mixed with X. tropicalis extract. Spindle length (left panel) and width (right panel) are plotted as columns, with colors proportionate to the amount of X. tropicalis (blue) added to X. borealis (purple) (n = 295 for 100% X. borealis, 0% X. tropicalis; n = 306 for 75% X. borealis, 25% X. tropicalis; n = 295 for 50% X. borealis, 50% X. tropicalis; n = 337 for 25% X. borealis, 75% X. tropicalis; and n = 334 for 0% X. borealis, 100% X. tropicalis over 3 independent experiments). In A-B, spindle length and width were normalized to the length and width, respectively, of 100% X. borealis CSF spindles. Error bars represent the standard deviation. Representative images are shown with tubulin (red) and DNA (blue) signal. Scale bar is 20 µm. Statistical significance was determined by a two-tailed, two-sample unequal variance t-test.

SUPPLEMENTARY FIGURE S5: Protein sequence alignments of Xenopus katanin

Protein sequences of X. tropicalis, X. laevis, and X. borealis katanin were aligned using Clustal Omega (default parameters). The X. borealis sequence is 97.6% identical to that of X. tropicalis, and 97.0% identical to X. laevis. The red box indicates the key regulatory serine residue at position 131.

SUPPLEMENTARY FIGURE S6: Protein sequence alignments of Xenopus TPX2

Protein sequences of X. tropicalis, X. laevis, and X. borealis TPX2 were aligned using Clustal Omega (default parameters). The X. borealis sequence is 75.7% identical to X. tropicalis, and 85.8% and 91.3% identical to sequences from the two subgenomes of X. laevis, L and S, respectively. The red box indicates the key 7 amino acids at position 619-624 that are absent from X. tropicalis and regulate microtubule nucleation activity.

Page 30 of 38 Cytoskeleton

  X. borealisfemale frogs were primed with 60 U PMSG at least 48 hours before use and boosted with 300 U hCG 12-24 hours before the experiment. Frogs were kept at 16°C in 0.5X MMR. The eggs were dejellied in a 3% cysteine solution in 1X XB-salts (20X: 2M KCl, 20 mM MgCl 2 , 2 mM CaCl 2 ), pH 7.8, and washed in CSF-XB (100 mM KCl, 0.1 mM CaCl 2 , 3 mM MgCl 2 , 50 mM sucrose, 10 mM K-EGTA, 10 mM K-HEPES, pH 7.8). CSF-XB buffer was exchanged for CSF-XB supplemented with LPC (10 µg/mL) (CSF-XB+). 1 mL of CSF-XB+ supplemented with cytochalasin D (100 µg/mL) was added to the bottom of an ultracentrifuge tube (SW-55, Beckman) before loading eggs into the tube. Extract tubes were placed in adapter tubes and spun at 1600 g for 1 min to pack the eggs. The excess liquid was removed and the eggs crushed by centrifugation at 10,200 rpm for 16 min at 16°C. The cytoplasmic fraction was then removed and supplemented with LPC (1:1000 dilution; 10 mg/mL stock), cytochalasin D (1:500 dilution; 10 mg/mL stock), energy (1:50 dilution; 50X stock: 190 mM creatine phosphate, 25 mM adenosine triphosphate, 25 mM MgCl 2 , 2.5 mM K-EGTA pH 7.7), and rhodamine-tubulin (1:300 dilution;

FIGURE 1 :

 1 FIGURE 1: Characterization of the Marsabit clawed frog, X. borealis (A) Relative body size of X. borealis frogs compared to X. laevis and X. tropicalis. (B)

  purple. Error bars show the standard deviation. (E) Stage 8 embryo nuclei comparison and quantification. Representative images of nuclei isolated from stage 8 X. laevis, X. tropicalis, and X. borealis embryos are shown with DNA signal. Scale bar is 10 µm. Nuclear size (area in µm 2 )is plotted as boxplots for X. laevis (green), X. tropicalis (blue), and X. borealis (purple). Each dot represents an individual nucleus (n = 68 for X. laevis, n = 71 for X. tropicalis, and n = 84 for X. borealis). In B, C, and E, the thicker black line indicates the average, and the outlined gray box represents the standard deviation. Statistical significance was determined by a two-tailed, twosample unequal variance t-test.

FIGURE 2 :

 2 FIGURE 2: X. borealis egg extracts at a glance (A) Schematic of extract preparation and spindle assembly reactions. (B) Images of spindle assembly intermediates in CSF extract reactions. (C) Images of spindle assembly intermediates in cycled extract reactions. Images in B-C of representative structures are shown with tubulin (red) and DNA (blue) signal. Scale bar is 20 µm.

FIGURE 3 :

 3 FIGURE 3: X. borealis CSF egg extracts reconstitute spindle assembly

FIGURE 4 :

 4 FIGURE 4: Analysis of X. borealis cycled spindles (A) Quantification of X. borealis cycled spindle dimensions. Length (left), width (middle), and area approximated as an ellipse (right) are plotted (µm or µm 2 ) as boxplots for X. laevis (green), X. tropicalis (blue), and X. borealis (purple) spindles. Each dot represents an individual spindle measurement (n = 256 spindles for X. laevis, n = 241 for X. tropicalis, and n = 493 for X. borealis, from 3 independent egg extracts for each species), the thicker black line indicates the average, and the outlined gray box represents the standard deviation. Statistical significance was determined by a two-tailed, two-sample unequal variance t-test. (B) Microtubule fluorescence intensity distribution in X. laevis (green), X. tropicalis (blue), and X. borealis (purple) spindles. Line scans of rhodamine-tubulin signal along the length of the spindle were measured (n = 257 spindles for X. laevis, n = 242 for X. tropicalis, and n = 470 for X. borealis, from 3 independent egg extracts for each species). Spindle length was normalized to 100% and tubulin intensities were normalized within each dataset. Average tubulin intensities were plotted for each species' spindles. Error bars represent the standard deviation. Representative images of spindles formed from X. laevis, X. tropicalis, and X. borealis sperm nuclei in their respective egg extracts are shown with tubulin signal individually (left) and merged with DNA signal (right). Scale bar is 20 µm.

FIGURE 5 :

 5 FIGURE 5: Sensitivity of X. borealis spindles to perturbation of the RanGTP gradient (A) Phenotype categories quantified in Xenopus egg extract spindle assembly reactions containing Ran mutants. Representative images in X. laevis extracts are shown with tubulin (red) and DNA (blue) signals. Scale bar is 20 µm. Bar graphs of the percentage of spindles in each category are shown for (B) untreated extracts, (C) RanT24N treated extracts, and (D) RanQ69L treated in Xenopus extracts. In B-D, percentages of bipolar (dark gray), multipolar (polka dots), and no spindles found (light gray) are plotted for X. laevis, X. tropicalis, and X. borealis extracts (3 independent extracts were quantified for each species). Error bars represent the standard deviation. Statistical significance was determined by a two-tailed, 3x2 Fisher Exact test.

FIGURE 6 :

 6 FIGURE 6: Titration of X. borealis egg extract with other Xenopus egg extracts (A) X. borealis extract mixed with X. laevis extract. Spindle length (left panel) and width (right panel) are plotted as columns, with colors proportionate to the amount of X. laevis (green) added

FIGURE 7 :

 7 FIGURE 7: Localization of scaling factors katanin and TPX2 on X. borealis spindles (A) Katanin immunofluorescence staining of X. laevis (green), X. tropicalis (blue), and X. borealis (purple) spindles. Line scans of Alexa Fluor 488 signal along the length of the spindle were measured (n = 385 spindles for X. laevis, n = 408 for X. tropicalis, and n = 348 for X.

  Figure 1 269x362mm (300 x 300 DPI)

  Figure 2 256x384mm (300 x 300 DPI)

  Figure 3 231x178mm (300 x 300 DPI)

  Figure 4 266x360mm (300 x 300 DPI)

  Figure 5 267x471mm (300 x 300 DPI)

  Figure 6 256x320mm (300 x 300 DPI)

  Figure 7 259x317mm (300 x 300 DPI)

Cytoskeleton

ACKNOWLEDGEMENTS

We thank members of the Heald lab for support and fruitful discussions, in particular Xiao Zhou for his line scan plugin and Ambika Nadkarni for the purification of Ran mutants. We thank Sofia Medina-Ruiz, Austin Mudd, and Dan Rokhsar for providing early access to X. borealis katanin, TPX2, and XMAP215 sequences. The confocal microscopy performed in this work was done at the UC Berkeley CRL Molecular Imaging Center, supported by NSF DBI-1041078. MK was supported by the UC Berkeley MCB department NIH training grant 4T32GM007232-40. RH was supported by NIH R35 GM118183 and the Flora Lamson Hewlett Chair. RG was supported by an HFSP long term fellowship LT 0004252014-L.

Confocal microscopy was performed on an upright Zeiss LSM 780 NLO AxioExaminer using the Zeiss Zen software. Embryos were illuminated with an LED light source (X-cite 120LED).

For the imaging of histone H3 and tubulin, embryos were imaged using a Plan-Apochromat 20x/1.0 Water objective and laser powers of 12%, on multiple 1024x1024 px plans spaced of 0.683 µm in Z. Images are mean averages of 2 scans with a depth of 16 bits. Channels were acquired simultaneously, and pinhole size was always chosen to correspond to 1 airy unit.

Imaging and quantification

Eggs were imaged using the Leica Application Suite (v4.9; Leica Microsystems, Buffalo Grove, IL) with a Wild M7A stereoscope equipped with a Leica MC170HD camera. Sperm cells, sperm nuclei, embryo nuclei, and spindles were imaged using micromanager software [START_REF] Edelstein | Advanced methods of microscope control using µManager software[END_REF] with an Olympus BX51 microscope equipped with an ORCA-ER camera (Hamamatsu Photonics, Hamamatsu city, Japan), and with an Olympus UPlan FL 40x/0.75 air objective. All measurements were made using Fiji. The spindle tubulin, katanin, TPX2, and XMAP215 intensity line scans were measured using an automated Java ImageJ plugin developed by Xiao Zhou (Heald lab, UC Berkeley; https://github.com/XiaoMutt/AiSpindle).

Western blot and analysis

1 µL of egg extract for 3 independent extracts for each species was subjected to SDS-PAGE (12% gel for katanin, 8% gel for TPX2 and XMAP215) and transferred to a nitrocellulose membrane. The anti-TPX2 (1:500 dilution, unpublished), anti-katanin p60 (1:1000 dilution) [START_REF] Loughlin | Katanin contributes to interspecies spindle length scaling in Xenopus[END_REF], or anti-XMAP215 (1:1000, Tony Hyman & Kazu Kinoshita) rabbit antibodies and anti-Ran (1:2000 dilution, BD Biosciences, San Jose, CA) mouse antibody were added for 1 h or overnight at 4°C in PBS + 0.1% Tween-20 + 5% milk. Secondary antibodies (goat anti-rabbit IRDye 800 or goat anti-mouse IRDye 680) were added at 1:10,000 for 1 h in PBS + 0.1% Tween-20. Blots were scanned with an Odyssey Infrared Imaging System (LI-COR Biosciences), and band intensity was quantified with Fiji.

Protein sequence alignments

AUTHOR CONTRIBUTIONS

M.K. performed the experiments and analyzed the data, with help from R.G. M.K. prepared the figures and wrote the manuscript with input from R.H. and R.G.

AUTHOR INFORMATION

The authors declare no competing financial interests. Correspondence should be addressed to R.G. (romain.gibeaux@univ-rennes1.fr) and R.H. (bheald@berkeley.edu).

SUPPLEMENTARY MATERIAL SUPPLEMENTARY TABLES

SUPPLEMENTARY TABLE 1: Ran perturbation phenotypes

Quantification of spindle phenotypes observed in egg extract reactions containing RanT24N or RanQ69L. Data are from 3 independent experiments for each species. Statistical significance was determined by a two-tailed 3x2 Fisher's exact test.

VIDEOS

VIDEO 1: Characterization of X. borealis embryo development; X. laevis vs. X. borealis X. laevis (left) and X. borealis (right) eggs were fertilized with X. laevis or X. borealis sperm, respectively, and simultaneously imaged in separate dishes. Scale bar corresponds to 200 µm.

The video represents 20 h played in 15 s at a rate of 120 frames per second.

SUPPLEMENTARY FIGURE LEGENDS

SUPPLEMENTARY FIGURE S1: X. borealis sperm nuclei length comparison and quantification

Images of sperm nuclei prepared from X. laevis, X. tropicalis, and X. borealis. DNA signal is shown. Scale bar is 10 µm. Length (µm) is plotted as boxplots for X. laevis (green), X. tropicalis (blue), and X. borealis (purple). Each dot represents an individual sperm nucleus (n = 31 for X. laevis, n = 26 for X. tropicalis, n = 30 for X. borealis), the thicker black line indicates the average, and the outlined gray box represents the standard deviation. Statistical significance was determined by a two-tailed, two-sample unequal variance t-test. S3: Effects of cytoplasmic mixing on microtubule distribution (A) Microtubule intensity distribution in spindles formed around X. borealis (purple), X. laevis (green), and X. tropicalis (blue) sperm nuclei in X. borealis egg extract (n = 316 for X. borealis sperm nuclei, n = 307 spindles for X. laevis sperm nuclei, and n = 307 for X. tropicalis sperm nuclei, from 3 independent X. borealis egg extracts). (B) Microtubule distribution in spindles formed in mixed extracts of X. laevis (green) and X. borealis (purple) (n = 301 for 100% X. borealis, 0% X. laevis; n = 338 for 75% X. borealis, 25% X. laevis; n = 322 for 50% X. borealis, 50% X. laevis; n = 295 for 25% X. borealis, 75% X. laevis; and n = 240 for 0% X. borealis, 100% X. laevis over 3 independent experiments). (C) Microtubule distribution in spindles formed in mixed extracts of X. tropicalis (blue) and X. borealis (purple) (n = 283 for 100% X. borealis, 0% X. tropicalis; n = 272 for 75% X. borealis, 25% X. tropicalis; n = 260 for 50% X. borealis, 50% X. tropicalis; n = 272 for 25% X. borealis, 75% X. tropicalis; and n = 305 for 0% X. borealis, 100% X. tropicalis over 3 independent experiments). In A-C, line scans of rhodamine-tubulin signal along the length of the spindle were measured. Spindle length was

SUPPLEMENTARY FIGURE S2: Analysis of X. borealis CSF spindle properties

SUPPLEMENTARY FIGURE


J Duriez

R Wan

Subtleties in Discrete Element Modelling of wet granular soils

The stress description provided by the discrete element method (DEM) when modelling wet granular materials is investigated for the case of low degrees of saturation within the pendular regime. The stress tensor as computed solely from contact and resultant capillary forces between individual pairs of particles is analysed in depth, being compared with an analytically derived stress expression that accounts for the distributed nature of internal actions such as the liquid pressure acting along wetted surfaces. Both fundamentally different approaches are shown to be equivalent, including for low suction values where wetted surfaces are significant and physical internal forces clearly deviate from the resultant point forces considered by DEM. Under wet conditions, it is demonstrated that the discrete element entity now includes both a particle and half a meniscus as a unit.

INTRODUCTION

A unified mechanical description of granular soils in biphasic (liquid saturated or dry) conditions and triphasic (unsaturated) conditions is still lacking, with the effective stress proposed by [START_REF] Bishop | The principle of effective stress[END_REF] suffering shortcomings that have been amply discussed by [START_REF] Nuth | Effective stress concept in unsaturated soils: Clarification and validation of a unified framework[END_REF]. In fact, the existence of an effective stress under unsaturated conditions had been challenged by Bishop himself [START_REF] Bishop | Some aspects of effective stress in saturated and partly saturated soils[END_REF]. In this connection, refined micro-mechanical modelling investigations have been undertaken to gain insights in the mechanical behaviour of unsaturated granular soils [START_REF] Chateau | Micromechanics of saturated and unsaturated porous media[END_REF][START_REF] Hicher | A microstructural elastoplastic model for unsaturated granular materials[END_REF][START_REF] Wan | Micromechanical analysis of force transport in wet granular soils[END_REF], using in particular the Discrete Element Method (DEM, [START_REF] Cundall | A discrete numerical model for granular assemblies[END_REF] as a pertinent numerical tool for probing into the microstructure [START_REF] Gili | Microstructural deformation mechanisms of unsaturated granular soils[END_REF][START_REF] Richefeu | Shear strength properties of wet granular materials[END_REF][START_REF] Scholtès | On the capillary stress tensor in wet granular materials[END_REF][START_REF] Mani | Liquid migration in sheared unsaturated granular media[END_REF]. A common feature of every DEM model is the consideration of particles interacting through solely point forces. Thus, DEM simulations of wet granular materials simulate directly the solid grains as discrete elements (DE's) and include the fluid phases by introducing extra point forces, compared with simulations in the dry state. These extra forces basically represent capillary forces resulting from the distribution of the liquid and gas phases. At low liquid content in the pendular regime, this spatial distribution takes the form of distinct liquid menisci between separated or contacting particles (Fig. 1).

Despite the description of internal forces with point-wise interaction forces, a total stress tensor can still be defined for DEM models and the objective of this Note is to discuss such a stress description from resultant capillary forces. In particular, we compare DEM with another approach based on analytical homogenization (Chateau et al., 2002). The analytical formulation is first recalled before elaborating the stress tensor expression for DEM models, which is thereafter compared with the homogenization approach, thus providing an extension of a preliminary comparison by [START_REF] Wan | A tensorial description of stresses in triphasic granular materials with interfaces[END_REF].

ANALYTICAL HOMOGENIZATION OF WET GRANULAR SOILS

The stress state of an unsaturated soil depends on the solid, liquid and gas phases involved, including the interfaces between them. In fact, liquid-gas interfaces and surface tension contribute necessarily to the total stress as they enter into the expression of the total energy of the unsaturated mixture [START_REF] Nikooee | Effective stress in unsaturated soils: A thermodynamic approach based on the interfacial energy and hydromechanical coupling[END_REF][START_REF] Madeo | A continuum model for deformable, second gradient porous media partially saturated with compressible fluids[END_REF]. [START_REF] Chateau | Micromechanics of saturated and unsaturated porous media[END_REF], Chateau et al. (2002) derived the stress homogenization of a wet granular soil, expressing the total stress as a function of the 1 various internal forces including an interfacial term:

Σ= 1 V ( ∫ V s σ dV +∫ V l σ dV +∫ V g σ dV +∫ S lg π dS ) (1)
Eq. ( 1) accounts for surface tension internal forces along the interface S lg through the stress-like tensor π that is expressed in terms of surface tension γ and δ lg , the identity tensor restricted to S lg (Chateau et al., 2002). The magnitude of this stress contribution from interfaces has not yet been quantified, leading [START_REF] Wan | Micromechanical analysis of force transport in wet granular soils[END_REF] to neglect it in independent derivations. Also, [START_REF] Wan | A tensorial description of stresses in triphasic granular materials with interfaces[END_REF] did not include such surface term in a previous comparison with the stress expression for DEM models, that will be presented in the next section.

Accouting herein for all volume phases and this interface, we note that the stresses within the fluid phases are uniform pressure fields. As for the solid phase (the granular skeleton), the corresponding stress is defined from the stresses within grains [START_REF] Bagi | Stress and strain in granular assemblies[END_REF]. This assumption may lead to stress components that are difficult to interpret when rotations of individual grains are taken into account [START_REF] Nicot | On the definition of the stress tensor in granular media[END_REF][START_REF] Smith | The effects of particle dynamics on the calculation of bulk stress in granular media[END_REF]; however such discussion is outside the scope of this Note. Eq. ( 1) is then rewritten, based on individual grain particles p and converting the volume integrals into surface integrals, considering particle equilibrium without body forces (see e.g. [START_REF] Nicot | On the definition of the stress tensor in granular media[END_REF]:

Σ= 1 V ∑ p ∫ S p (σ ⃗ n)⊗⃗ x dS+ V l V u l δ + V g V u g δ - 1 V ∫ S lg γ δ lg dS (2)
Eq. ( 2) involves tractions through σ ⃗ n and we note matric suction and surface tension respectively act along the wetted surface and the menisci contours where the three phases intersect, while contact forces are considered to act on vanishing surfaces (Fig. 2).

Finally, the total stress tensor is obtained as (see Chateau et al., 2002 and the Appendix): DEM STRESS MODELLING OF WET GRANULAR SOILS DEM model formulation DEM models for wet granular soils usually differ through the expression of the capillary forces used to simulate the liquid menisci. For fully wettable particles where the contact angle is zero, the exact expression from matric suction and surface tension is: [START_REF] Richefeu | Shear strength properties of wet granular materials[END_REF]; [START_REF] Mani | Liquid migration in sheared unsaturated granular media[END_REF] used empirical relations to express directly the capillary force as functions of the particle distance and the meniscus volume. The present work adopts a model we developed within the Yade code (Šmilauer et al., 2010) from the one by [START_REF] Scholtès | On the capillary stress tensor in wet granular materials[END_REF] that is based on systematic numerical solutions of Laplace (1806) equation as per [START_REF] Lian | A theoretical study of the liquid bridge forces between two rigid spherical bodies[END_REF]; [START_REF] Soulié | Influence of liquid bridges on the mechanical behaviour of polydisperse granular materials[END_REF].

Σ= 1 V ∑ c 1,2 ⃗ f 2 c ⊗ ⃗ l 12 -s χ-B+u g δ χ = 1 V ( V l δ + ∑ p R p∫ S p l ⃗ n⊗⃗ n dS ) B= 1 V ( ∑ p R p ∫ Γ p ⃗ γ⊗⃗ n dS+ ∫ S lg γ δ lg dS ) (3)
⃗ f cap =π R sin (α) 2 (2 γ+ R s)⃗ e (4)
Accordingly, the model includes capillary forces at each (separated or contacting) particle pair for which Laplace's equation can be solved, assuming a constant suction and a zero contact angle. The resulting liquid bridge configuration corresponds to uniform suction conditions, i.e. thermodynamic equilibrium.

In addition to these capillary forces, contact forces are applied at touching particles according to particle relative displacements and a standard elastic-plastic contact law. More details about the DEM model have been presented by [START_REF] Wan | A tensorial description of stresses in triphasic granular materials with interfaces[END_REF].

Stress homogenization in DEM

In addition to interaction forces, a stress tensor representation of the DEM model is still desirable for a meaningful analysis at the macroscopic level. Stress homogenization of the DEM assembly is again obtained from surface integral of external tractions acting on the discrete elements. Since the only external tractions are point forces in the DEM model, it follows immediately that:

Σ=- 1 V ∑ p ∑ ⃗ f p c ⃗ f p c ⊗⃗ x (5)
It is herein noted that DEM simulations encompass only summations of forces and moments without the need for any information on the points of force application ⃗ x , if torques are disregarded.

For dry conditions, considering application points of contact forces to lie on the particle surfaces seems natural. In doing so, Eq. ( 5) leads to the homogenized contact stress:

Σ=σ cont = 1 V ( ∑ c 1,2∈V ⃗ f 2 c ⊗ ⃗ l 12 -∑ p∈∂V ∑ ⃗ f ext ⃗ f p ext ⊗⃗ x ) (6)
The first term of Eq. ( 6) gathers contributions of every contact within the volume V. The second term involving external forces sustained by the particles at the boundaries is classically neglected [START_REF] Nicot | On the definition of the stress tensor in granular media[END_REF]. For triaxial simulations with 20,000 particles, this induces a 2% discrepancy between the homogenized stresses and the ones deduced from the rigid boundaries used in the simulations (Fig. 3). Note that the perfect match between stresses deduced from Eq. ( 6) and at the boundaries demonstrates the quasi-static nature of the simulations.

In order to account for unsaturated conditions, [START_REF] Scholtès | On the capillary stress tensor in wet granular materials[END_REF]; [START_REF] Richefeu | Shear strength properties of wet granular materials[END_REF] supplemented the contact stress tensor with a so-called capillary stress tensor that includes the capillary forces such that Σ =σ cont +σ

cap

. By analogy to the leading term of Eq. ( 6), the capillary stress tensor was assumed to take a similar form without any formal proof. Thus, summing over all menisci-bonded particle pairs:

σ cap = 1 V ∑ m∈V ⃗ f 2 cap ⊗ ⃗ l 12 (7)
The derivation of Eq. ( 7) from Eq. ( 5) would in fact require considering a common application point for ⃗ f 1 cap and ⃗ f 2 cap such that ⃗ x 1 -⃗ x 2 = ⃗ l 12 , but this might be considered as physically unrealistic for stretched menisci as seen in Fig. 4(a). A more physical choice as illustrated in Fig. 4(b) would lead to: As for the contact forces, the DEM model does not define any application points for capillary forces. It turns out that Eq. ( 7), Fig. 4(a), is finally more suitable to analyze the DEM model through an homogenized stress tensor. Indeed, σ cap as calculated from Eq. ( 7) is in closer agreement with (Σσ cont ) than is σ cap as computed from Eq. ( 8); see Fig. 5. In this comparison, the total stress is deduced from the rigid boundaries, whereas the contact stress is calculated from Eq. ( 6).

σ cap =- 1 V ∑ p∈V ∑ m on p ⃗ f p cap ⊗ ⃗ x m (8)

STRESS DESCRIPTIONS: HOMOGENIZATION VS DEM APPROACHES Preliminaries

Following both the homogenization and DEM approaches, a capillary stress tensor appears as the difference between the total stress and the contact stress, neglecting the gas pressure. The capillary stresses as derived in the analytical approach is:

σ hom cap =Σ-σ cont =-s χ -B (9)
whereas, within the DEM framework,

σ DEM cap =Σ-σ cont = 1 V ∑ m∈V ⃗ f 2 cap ⊗ ⃗ l 12 (10)
A preliminary investigation on whether both modelling procedures provide equivalent stress desciptions of unsaturated granular materials was first undertaken by [START_REF] Wan | A tensorial description of stresses in triphasic granular materials with interfaces[END_REF], neglecting the interface stress contributions in σ hom cap . Numerical results along hydraulic loading paths showed discrepancies in the respective computed mean capillary stresses, which might be due to the following reasons: (i) The DEM model does not account for the fluid volumes as such. Instead, it includes them indirectly through the resultant forces they induce on the solid phase.

(ii) Also, the DEM reduces the physical internal force distributions into resultant forces

⃗ f = ∫ S σ ⃗ n dS .
Obviously, this has no impact on (rigid) particles displacements, and strains within granular soils. However, computing stresses involves the integral ∫ S σ ⃗ n⊗⃗ x dS . Because the physical internal forces are reduced to a resultant point force, the stress computation in the DEM model leads to calculating

⃗ f ⊗⃗ x = (∫ S σ ⃗ n dS ) ⊗⃗ x≠ ∫ S σ ⃗ n⊗⃗ x dS .
The consistency between the two approaches is herein further investigated, starting from the exact expressions of the mean capillary stresses, i.e.

p hom cap = 1 3 tr(σ hom cap ) (11) p DEM cap = 1 3 tr (σ DEM cap ) (12)
For sake of clarity, indices 2 and 12 are omitted in the DEM formulation, i.e.

p DEM cap = 1 3 V ∑ m tr ( ⃗ f cap ⊗ ⃗ l )= 1 3 V ∑ m ⃗ f cap . ⃗ l =- 1 3 V ∑ m R 1 sin (α 1 ) 2 (2 γ +R 1 s)(R 1 +R 2 -u n ) =- 1 3 V ∑ m R 2 sin (α 2 ) 2 (2 γ +R 2 s)(R 1 +R 2 -u n )
(13) As for the analytical model, we get from Eq.

(3) since ⃗ γ .⃗ n=0 for a zero contact angle:

p hom cap =- 1 3 tr(s χ )- 1 3 tr ( γ V ∫ S lg δ lg dS) =- s 3 V ( V l tr (δ)+ ∑ p R p∫ S p l tr (⃗ n⊗⃗ n) dS ) - γ 3 V S lg tr(δ lg ) =- 1 3 V [ s ( 3 V l + ∑ p R p S p l ) +2 γ S lg ] =- 1 3 V ∑ m [ s ( 3 V m + ∑ p=1 2 R p 3 (1-cos α p ) ) +2 γ S m ] (14)
It is easy to show that the two approaches secure agreement in the case of high suction, i.e. a low degree of saturation, as observed by [START_REF] Wan | A tensorial description of stresses in triphasic granular materials with interfaces[END_REF]. In such a case, filling angles are small, suction terms outweight surface tension terms in Eq. ( 13) and menisci volume and surface are negligible in Eq. ( 14). Finally, interparticle penetration can be considered as small, which is classical in DEM for contacting quasi-rigid spheres, and also occurs for noncontacting ones as menisci break after a small gap under high suction. Thus, a common expression is finally obtained:

p DEM cap ≈p hom cap ≈ -2 3 V s∑ m π R m 3 α m 2 (15)
Indeed, the two discrepancies reasons alluded to previously disappear for very low saturation. In particular, negligible wetted surfaces S lead to (∫ S σ ⃗ n dS ) ⊗⃗ x ≈ ∫ S σ ⃗ n⊗⃗ x dS .

Due to intricacies through Laplace's equation between the menisci geometrical variables and the surface tension and suction, an equivalence between Eq. ( 13) and ( 14) cannot be ruled out for a higher saturation where physical internal forces are distributed along significant surfaces. Such case is thus numerically investiguated in the following.

Examples

The capillary stress tensors expressed in Eqs. ( 9) and ( 10) are now compared numerically considering the DEM simulation of axisymmetric stress loading at constant suction, imposed on a numerical specimen of dense silt-like packing with 58 μm as mean diameter, see [START_REF] Wan | A tensorial description of stresses in triphasic granular materials with interfaces[END_REF] In spite of the fundamental differences in paradigm between the two approaches, Fig. 6 shows that the capillary stresses as computed from DEM and homogenization approaches do coincide for both low and high suctions values. The suction values here considered, 10 and 300 kPa, respectively correspond to saturation between 8% and 12%, or 0.03% and 0.055%, and to filling angle distributions around 30⁰ or 8⁰.

Fig. 1 .

 1 Fig. 1. Wet granular material with idealized spherical grains (top) and discrete elements sustaining both contact and capillary forces (bottom).

Fig. 2 .

 2 Fig. 2. Mechanical actions on the soil particles in wet conditions (point contact forces between particles are excluded).

Fig. 3 .

 3 Fig. 3. Stress homogenization of the DEM model during triaxial loading (50 kPa confining pressure) in dry conditions. The second term of Eq. (6) is included for exact stress computations, and dropped for approx. ones.

Fig. 5 .

 5 Fig. 5. Stress homogenization of the DEM model during triaxial loading (20 kPa confining pressure) in unsaturated conditions (5 kPa suction ; S r ≈ 21+/-5%).

  for details. While obtaining σ DEM cap from the DEM model is straightforward, the computed fluid phase distribution at the same time enables the determination of σ hom cap . Interpreting the DEM model through the analytical approach i.e. σ hom cap aims at verifying whether the stress state of the DEM model i.e. σ DEM cap coincides with the stress state of the simulated wet granular system.

Fig. 6 .

 6 Fig. 6. Capillary stresses for triaxial loading with 10 kPa of confining pressure and different suctions.Capillary stresses are computed from the surface and lineal distributions through analytical homogenization, Eq. (9), or from resultant forces, Eq. (10), through DEM.

  ,V s ,V g total, liquid, solid, or gaseous volumes of a wet granular soil

		contact or negative for separated spheres
	u l ,u g	liquid or gas pressures
	V ,V V m	meniscus liquid volume
	⃗ x	application points of tractions
	α	filling angle defining the wetted surface
	α m	mean filling angle of a liquid bridge bonded pair
	γ	liquid-gas coefficient	surface	tension
	⃗ γ	lineic surface tension forces as sustained by solid particles
	Γ p	contact lines of a solid particle p where the three phases intersect
	δ	identity tensor
	δ lg	identity tensor restricted to S lg (projection tensor)
	χ Σ , σ σ cont σ cap	capillary stress contribution proportional to s macroscopic i.e. total or microscopic stress contact stress: stress contribution from contact forces capillary stress: stress contribution from the liquid-gas mixture	LIST OF NOTATIONS B capillary stress contribution proportional to γ ⃗ e meniscus orientation ⃗ f p c , ⃗ f p cap contact or capillary force sustained by particle p ⃗ n solid outward normal
	σ DEM cap ,σ hom cap	expressions for σ (resultant capillary forces) or cap from DEM analytical (distributed internal forces) approaches	p DEM cap , p hom cap R 1 , R 2 ,R p R m	mean pressure of σ DEM cap , σ hom cap radii of particles 1,2,p mean radius of a liquid bridge bonded pair
					S lg , S m	liquid-gas interface surface at the sample or menisci scale
					S p , S p l	total or wetted surface of solid particle p
					Such equivalence between σ hom cap saturation ratio s=u g -u l S r σ DEM cap matric suction tensors for any suction implies that the and ⃗ t tractions on solid particles DE's do not correspond only to solid particles, but also encompass halves of the menisci volume and surface, so that a complete stress description can be u n overlap between discrete elements: positive for geometrical

l

ACKNOWLEDGMENTS

This work is supported by the Natural Science and Engineering Research Council of Canada and Foundation Computer Modelling Group within the framework of a Government-Industry Partnership (NSERC-CRD) Grant. The authors also acknowledge gratefully various discussions of ideas with Félix Darve and Vincent Richefeu from Laboratoire 3SR and François Nicot from IRSTEA, all in Grenoble, France.

obtained from the DE's only. This surprising property of DE's in models of wet granular materials is related to the relevancy of considering a common application point for the capillary forces, as shown in the above.

Note that obtaining such above equivalence, which was previously suggested by Chateau et al. (2002) without much formal justication, is not possible if interfaces are neglected [START_REF] Wan | A tensorial description of stresses in triphasic granular materials with interfaces[END_REF].

CONCLUSIONS

Wet granular materials include internal forces that are distributed in nature, such as liquid pressures or surface tension acting along wetted surfaces or interfaces. However, the Discrete Element Method still shows a consistent stress description from resultant point forces for any saturation in the pendular regime. Indeed, the DE's of corresponding DEM models encompass fluid volumes and interface surfaces in addition to the solid particles volumes.

The present work is currently being extended to consider non-zero contact angles and surface tension along every interface.

APPENDIX: TOTAL STRESS DERIVATION FOR A WET GRANULAR SOIL

The explicit derivation of Eq. ( 3) from ( 2) is obtained by taking into account all external tractions acting on particles, with σ ⃗ n=-⃗ t due to the considerations of external normals in the divergence theorem.

Furthermore, all vectors ⃗ x ∈S p are expressed from the particles centers: ⃗ x=R p ⃗ n ; this choice being immaterial by virtue of the particles' equilibrium. Then,

1) The last contact force term is classically rewritten summing over all binary contacts (1,2), which demonstrates Eq. ( 3).