# Length limitation of astral microtubules orients cell divisions in murine intestinal crypts

Jad Saleh, Marc-Antoine Fardin, Amlan Barai, Matis Soleilhac, Olivia Frenoy, Cécile Gaston, Hongyue Cui, Tien Dang, Noémie Gaudin, Audrey Vincent, et al.

► To cite this version:

Jad Saleh, Marc-Antoine Fardin, Amlan Barai, Matis Soleilhac, Olivia Frenoy, et al.. Length limitation of astral microtubules orients cell divisions in murine intestinal crypts. Developmental Cell, 2023, 58 (17), pp.1519-1533.e6. 10.1016/j.devcel.2023.06.004. hal-05408167

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# Length-limitation of astral microtubules orients cell divisions in murine intestinal crypts

## AUTHORS

Jad Saleh <fnref n="1" />, Marc-Antoine Fardin <fnref n="1" />&, Amlan Barai <fnref n="1" />&, Matis Soleilhac <fnref n="1" />, Olivia Frenoy <fnref n="1" />, Cécile Gaston <fnref n="1" />, Hongyue Cui <fnref n="1" />, Tien Dang <fnref n="1" />, Noémie Gaudin <fnref n="1" />, Audrey Vincent <fnref n="2" />,<fnref n="3" />, Nicolas Minc <fnref n="1" />,<fnref n="4" />*# and Delphine Delacour <fnref n="1" />*#

**KEYWORDS:** cell division, spindle positioning, polarity, actomyosin, organoids, astral microtubules

<footnote n="1">¹, Université Paris Cité, CNRS, Institut Jacques Monod, F-75013 Paris, France</footnote>

<footnote n="2">² CANTHER Cancer Heterogeneity Plasticity and Resistance to Therapies, Université de Lille, CNRS, INSERM, CHU Lille, UMR9020-U1277, F-59000 Lille, France</footnote>

<footnote n="3">³ ORGALille Core Facility, CANTHER, Université de Lille, CNRS, INSERM, CHU Lille, UMR9020-U1277, F-59000 Lille, France</footnote>

<footnote n="4">⁴ Equipe Labellisée La Ligue Contre le Cancer</footnote>

<footnote>& Authors participated equivalently</footnote>

<footnote># Lead contacts</footnote>

<footnote>* Corresponding authors:
delphine.delacour@ijm.fr ; nicolas.minc@ijm.fr</footnote>
SUMMARY

Planar spindle orientation is critical for epithelial tissue organization, and generally instructed
from the long cell shape axis or cortical polarity domains. We introduced mouse intestinal
organoids to study spindle orientation in a monolayered mammalian epithelium. Although
spindles were planar, mitotic cells remained elongated along the apico-basal axis and polarity
complexes were segregated to basal poles, so that spindles oriented in an unconventional
manner, orthogonal to both polarity and geometric cues. Using high-resolution 3D imaging,
simulations, cell shape and cytoskeleton manipulations, we show that planar divisions resulted
from a length-limitation in astral microtubules which precludes them from interacting with basal
polarity, and orient spindles from the local geometry of apical domains. Accordingly,
lengthening microtubules affected spindle planarity, cell positioning and crypt arrangement.
We conclude that microtubule length regulation may serve as a key mechanism for spindles
to sense local cell shapes and tissue forces to preserve mammalian epithelial architecture.
INTRODUCTION

Proliferative monolayered epithelia support the morphogenesis, function and renewal of many stem cell niches and organs during development and adult life ¹,². They are characterized by an apico-basal polarity (A-B), and an alignment of mitotic spindles and consequent cell divisions within the tissue planes. These planar divisions position divided daughter cells side by side in the epithelial layer ³,⁵ to promote tissue monolayered architecture, elongation and homeostasis ⁶⁻⁸. Accordingly, a mis-regulation of spindle planarity has been shown to impair cell positioning and tissue integrity, and proposed to drive the emergence of dysplasia, hyperplasia, or cancer stem cell populations in stem cell niches ⁹⁻¹⁴. To date, however, spindle orientation in epithelia has been best studied in model invertebrates and cultured cell lines, which are amenable to high-resolution live imaging and genetic manipulations of spindle associated elements ¹¹,¹⁴. In contrast, studying cell division orientation in complex mammalian tissues and organs, relevant to human physiology and diseases has been in general hampered by the difficulty of performing advanced imaging in live animal models, and by plausible pleiotropic effects of loss of function assays ¹⁵. Therefore, addressing detailed mechanisms regulating spindle orientation in proliferating mammalian epithelial tissues remains an outstanding open endeavor.

In most animal cells and tissues, mitotic spindles are positioned and oriented from forces and torques generated by their astral microtubules (MTs) pulled by dynein motors. Dynein may be evenly distributed over the cytoplasm or cortex, a situation thought to yield length-dependent MT forces, that function to center spindles and align them with the long geometrical axis of the cell ¹⁶⁻¹⁸. Dynein can also be activated at specific sub-cellular cortical polarity domains enriched in dynein activators including NuMA (Nuclear Mitotic Apparatus) and LGN (Leucine-Glycine-Asparagine), such as during asymmetric divisions ¹¹,¹⁹⁻²¹. In tissues, these geometrical and polarity cues often compete to dictate spindle and division positioning ²²⁻²⁵. For instance, the planar orientation of mitotic spindles in columnar monolayered epithelia is thought to rely on the localization of dynein-regulating polarity complexes at the level of lateral junctions, as well as on a complete mitotic rounding that serves to erase the influence of cell geometry and ensures that short mitotic-phase (M-phase) astral MTs bounded by dynamic instabilities reach the cortex to influence spindle orientation ¹¹,¹⁴,²²,²⁶,²⁷. In general, however, how cell shape changes, MT dynamics and polarity effectors intersect to specify spindle planarity in mammalian epithelia remains poorly defined.

Given its high renewal rate throughout adult life, the intestinal tissue provides a prime model to study cell proliferation and division in mammals. The surface of the intestine is lined by a monolayer of tall columnar epithelial cells, with mitotic stem and transit-amplifying cells
exclusively located in curved tissue invaginations called crypts<sup>28,29</sup>. Previous studies of cell
division in the crypt have reported that mitosis is associated with an apical migration of cells
and DNA, which ends up with the assembly of a planar spindle in metaphase that specifies
cytokinesis orthogonal to the tissue layer <sup>30-34</sup>. However, in part because of the lack of
accessibility of the intestine *in vivo*, detailed mechanisms that control planar spindle orientation
and the monolayered architecture of the crypt are still lacking.

Here, we build on intestinal organoids to address mechanisms that control division orientation
in a 3D mammalian epithelium. Organoids are classically generated from isolated crypts
derived from mouse intestinal pieces, and develop regular crypt-like structures with self-
renewing capacity within 4 days of culture in 3D hydrogels, thereby mimicking the organization
and dynamics of the *in vivo* proliferative compartment <sup>35,36</sup>. Using live 3D imaging, physical,
chemical and genetic interventions, we show that dynein-regulating polarity complexes
accumulate at the basal face of mitotic cells and not at lateral junctions, and that mitotic cells
remain largely elongated along the A-B axis, challenging previous generic models for spindle
planarity established in model tissues. We propose a new quantitative model for planar
spindle orientation, based on a local apical cell shape sensing, mediated by a length-limitation
of M-phase astral MTs. This model predicts dose-dependent variations in spindle planarity in
normal organoids and in multiple conditions that affect cell shape and polarity.

RESULTS

Mitotic spindles in intestinal crypts orient orthogonal to the cell long axis

The spherical geometry of intestinal organoid crypts grown in 3D, provides a unique
opportunity to document cell division with an optimal optical resolution along the A-B axis of
the monolayer. We imaged spindle assembly and positioning along this axis, using α-tubulin-
GFP/H2B-mCherry organoids (Figure 1a). We confirmed that as in many columnar epithelia,
centrosomes were initially located close to the apical pole during interphase. Centrosomes
migrated towards the DNA at the onset of mitosis to assemble prometaphase spindles that
initially oscillated to eventually stabilize in metaphase orthogonal to the A-B axis, in the tissue
plane (Figure 1b; Figure S1a,h-i; Videos S1 and S3) <sup>31,33,34</sup>. This planar orientation was
maintained throughout metaphase, anaphase and telophase, yielding cytokinesis that
bisected mother cells along the A-B axis, and placed daughter cells side by side in the
monolayer (Figure 1b; Video S1) <sup>33,34</sup>.
To decipher mechanisms which regulate spindle planarity, we first explored the role of cell geometry, which generally orients spindles along the long cell shape axis³⁷,³⁸. We imaged cell contours in live tissues using td-Tomato organoids. This revealed that interphase columnar cells deform at the onset of prophase from the basal pole, yielding an inverted water drop-like metaphase cell shape, rounder at the apical pole and elongated towards the basal pole (Figure 1c; Figure S1b). These mitotic shape changes were concomitant with a marked basal enrichment of myosin-IIA-GFP as well as P-MLC2, suggesting they are driven by an anisotropic actomyosin-generated cortical tension (Figure 1d-e; Figure S1c-f, Video S2). This singular actomyosin distribution may underpin mitotic cell apical migration and neighboring cell rearrangements on the basal side of the epithelium (Figure S1g). In fact, these cell rearrangements around the dividing cell materialized by the formation of numerous multicellular contacts (3-, 4- and 5-cell contacts), most likely contributed to maintain tissue cohesion and integrity during division (Figure S1g). Therefore, as previously reported in drosophila imaginal disc³⁹, but in contrast to many epithelial tissue and adherent cells in which the actomyosin cortex homogenously remodels to ensure a complete mitotic rounding²²,²⁶,⁴⁰, metaphase cells remain elongated along the A-B axis in intestinal organoids.

Full 3D reconstitution of confocal tissue z-stacks allowed to quantify metaphase cell shapes along with spindle orientations and positions (Figure 1f; Figure S1b). This showed that metaphase cells exhibit a mean aspect ratio of 1.33±0.04 along the A-B axis, and a sphericity of 0.79±0.02 (Figure 1g-i), with a mean angle between mitotic spindles and the cell's long axis of 66.1±3.8° (Figure 1j). In addition, spindles were not centered at the geometrical cell center. Rather, they were shifted asymmetrically at an average position of 40.7±1.5% of the cell long axis length towards the apical pole (Figure 1g,i,k). Importantly, similar elongated mitotic shape, planar and asymmetrically positioned spindles were observed *in vivo* in crypts of mouse jejunum (Figure S2a-d). Therefore, planar cell division in intestinal crypts may not directly follow long-axis geometrical rules, with a spindle oriented nearly orthogonal to the long cell shape axis and positioned asymmetrically towards the apical cell domain.

## Dynein-regulating polarity complexes localize to basal poles of mitotic cells and may not contribute to spindle orientation

These observations prompted us to assay the localization of dynein-associated polarity complexes which are potential candidates to override geometric guiding cues³,⁵,²³,²⁵,⁴¹,⁴². We imaged multiple components of evolutionary conserved polarity complexes, which control spindle orientations in many tissues, including the dynein-regulators NuMA, LGN as well as
the LGN-binding partner afadin <sup>43,44</sup>. During interphase, NuMA was mostly localized within the nucleus, and re-located to spindle poles and to the cortex throughout mitosis, as reported in many systems <sup>45,46</sup> (Figure S2e). However, although we expected the cortical pool of NuMA to localize to lateral poles in face of spindle poles, as described in many epithelia and adherent cells <sup>14</sup>, it was largely enriched to basal poles of metaphase cells, away from and orthogonal to spindles, in both intestinal organoids and *in vivo* jejunum (Figure 2a-b; Figure S2e-f). Similar basal cortical enrichments were found for LGN and afadin (Figure 2c-d; Figure S2g-h). Dynein was in contrast localized throughout the cytoplasm and cortex, and accumulated at spindle poles (Figure S2i-j). These data show that canonical polarity complexes are segregated in an unconventional manner, away from lateral cortices and spindle planes in intestinal crypts. Rather, they appear to follow sites with increased cortical and junctional tension, as evidenced by the basal accumulation of the actomyosin cytoskeleton (Figure S1c-f) and E-cadherin (Figure S2k-n) as previously reported in mammalian cell lines <sup>44,47,48</sup>.

To identify potential mechanical designs in astral microtubule (MT) force distributions that orient planar spindles in this tissue, we developed 2D mathematical models. Starting from a series of idealized cell shapes elongated along or orthogonal to the tissue plane, we placed spindles asymmetrically towards the apex, as in experiments, and computed the torque exert generated by astral MTs as a function of spindle orientation, to identify rotational equilibrium angles <sup>49-51</sup>. As expected, when MTs were grown to fill the whole cell and exert length-dependent forces (shape-sensing system <sup>49</sup>), spindles oriented along the long cell shape axis, in the A-B axis of the tissue. Similarly, assuming that astral MT forces were scaled to the amount of the dynein-regulator NuMA <sup>51</sup>, also oriented spindles to face NuMA basal domains along the A-B axis (Figure 2e-g). We conclude that previously established generic models for spindle orientation may not simply account for the observed spindle planarity, and that canonical polarity complexes may not influence spindle orientation in this tissue.

## Length-limitations of M-phase astral MTs promote spindle planarity

Because M-phase astral MT growth and lengths are generally bounded by dynamic instabilities, we tested a model based on a length-limitation of astral MTs <sup>52,53</sup>. We reasoned that such limitation could in principle prevent astral MTs from reaching basal poles of elongated mitotic cells, and create an anisotropy of mitotic aster pair shapes now longer along the planar axis. Remarkably, in 2D models, this limitation coupled to length-dependent MT forces robustly predicted planar spindle orientation for a range of cell shapes and model parameters (Figure 2e-h; Figure S3a-d). Interestingly, however, as cells became too
elongated along the A-B axis, spindles eventually turned to a preferred orientation along the long A-B axis, yielding a bi-stable phase diagram, controlled by the distance from MT (+)-tips to the basal poles and cell shape aspect ratio (Figure 2f-h). These modelling results suggest that a cell shape sensing mechanism truncated by a limit in MT length, could in principle function to orient spindles in the plane of intestinal organoids.

To test this hypothesis, we imaged the distribution of astral MTs around spindles. We implemented expansion microscopy of intestinal organoids to visualize and quantify individual astral MTs. This revealed the presence of astral MT (+)-tips in close contacts with both apical and lateral cortices (Figure 3a,c; Figure S3e-g). In sharp contrast, MTs were barely detected in contact with the basal side, with MT (+)-tips located at a distance ~4x higher on average from the basal cortex than the apical one (Figure 3a-c; Figure S3e,h). Importantly, these effects were not caused by a putative anisotropy in MT nucleation, as the number of MTs growing apically vs basally was similar (Figure 3d). In addition, imaging of the (+)-tip associated protein EB3-GFP confirmed this exclusion in live tissues (Figure S3i, Video S3). To further test this differential MT-cortex interaction, we performed laser severing of groups of MTs in α-tubulin-GFP organoids along a line-scan orthogonal to the A-B axis, placed either on the apical or basal side of spindles (Figure 3e-f; Figure S3j-k). Both caused spindle recoils away from the cuts suggesting that astral MTs predominantly exert pulling forces, presumably by engaging with dynein motors at the cortex<sup>54,55</sup>. However, in agreement with the lesser extent of MTs reaching the basal cortex, basal cuts caused significantly smaller recoils than apical ones (Figure 3f; Figure S3k). These data further support an asymmetry in MT cortex contacts along the A-B axis, and a lack of contribution of basal polarity cues to MT forces and spindle orientation.

To directly assay the role of MT length regulation, we next sought to increase astral MTs length and monitor impact on spindle orientation. Kif18B, one important (+)-tip depolymerizing kinesin, was shown to promote MT catastrophe and thus limit the length of M-phase astral MTs in mammalian cells and tissues<sup>53,56</sup>. Interestingly, in intestinal organoids, we found that Kif18B accumulated at the basal domain of metaphase cells, colocalizing with polarity cues, as observed in other cell types and tissues (Figure S3l-n)<sup>57</sup>. This observation suggests that Kif18B could promote MT depolymerization from the basal side to limit MT length. Accordingly, knocking-out Kif18B, using a CRISPR-Cas9 inducible system in organoids, led to significantly longer astral MTs, with basal-facing MTs that now grew long enough to reach the cell cortex (Figure 3g-h; Figure S3o-p).
Strikingly, mitotic spindles in Kif18B-KO organoids were not planar anymore, and rather oriented randomly, with a significant fraction of spindles oriented along the A-B axis (Figure 3i). Importantly, shortening back astral MTs using a low-dose nocodazole treatment in Kif18B-KO organoids restored planar spindle orientation (Figure S3q-r), suggesting that spindle orientation defects in this mutant may be primarily associated to a mis-regulation of MT length. Finally, in Kif18B-KO organoids, NuMA was no more accumulated at the basal cortex but dispersed in multiple patches around the cell surface, yet still away from spindle poles, reinforcing the lack of direct contribution of polarity effectors to spindle orientation in this tissue (Figure S3s-t). Together, these results suggest that a length-limitation of basal mitotic astral MTs mediated in part by depolymerizing kinesins, may prevent MTs from interacting with basal polarity factors and allow spindles to orient in the tissue plane by probing the geometry of the apical fraction of dividing cells.

### A 3D model for spindle position and orientation in intestinal crypts

In order to test further the length-limitation hypothesis, we explored the parameter space in the model phase diagram (Figure 2f-h) by experimentally altering cell shapes. We first elongated cells in the tissue plane, to validate a general influence of apical cell shape. We grew organoids in L-WNR medium, an exogenous global Wnt3a treatment, which results in the formation of hyper-proliferative and undifferentiated cystic structures<sup>58,59</sup>, in which the epithelial monolayer displays a flat squamous morphology with cells now elongated in the tissue plane (Figure S4a). As predicted by models, and not surprisingly, spindles oriented parallel to the long cell shape axis in the tissue plane in this condition (Figure 4a-d; Figure S4a). To increase mitotic cell elongation along the A-B axis, we next affected contractility by inhibiting myosin-IIA activity. Both blebbistatin treatment or myosin-IIA-KO drastically impaired mitotic shape changes and rounding, with cells remaining significantly more elongated along the A-B axis than controls (Figure 4a-c; Figure S4b-e). This resulted from a loss of cellular rearrangements and the formation of fewer multicellular contacts in the basal environment of metaphase cells (Figure S4d-h). These results confirmed the primary function of basal actomyosin contractility in reshaping mitotic cells and its contacts with neighbors in this tissue. Importantly, as predicted in models (Figure 2f,h), in these more A-B elongated metaphase cells, spindle orientation was not planar anymore, with many cells aligning their spindles closer to the A-B axis (Figure 4a-b,d). Importantly, in these conditions, polarity complexes detached from the cell cortex (Figure S4i-j) and spindles were still positioned off-center towards the apical cell poles (Figure 4b), ruling out here again any putative contributions of basal polarity to spindle orientation.
To test our hypothesis in these conditions affecting cell shapes using realistic 3D cell geometries, we next turned to simulations. We adapted a gradient descent strategy that can predict both spindle orientation and position in 3D from the 3D geometry of mitotic cells, the localization of polarity cues and length limitation in astral MTs <sup>25</sup>. Using 3D segmentations of individual cell contours, spindle poles and basal domains from fixed tissues, we reconstituted experimental cell shapes, polarity and spindle orientation and position in mitotic cells (Figure 4e). By inputting the experimental geometry and a limit for MT growth at the basal pole in the model, to account for Kif18B enrichment there, we ran simulations starting from random positions and orientations, and searched for equilibrium. The simulations were robust to initial conditions and predicted with accuracy in 3D, the planarity of spindles and their apical shifts in control and L-WNR, as well as their reorientation along the A-B axis in more elongated blebbistatin-treated cells (Figure 4e-g; Figure S5a-c, and Videos S4-5). In contrast, models in which astral MTs filled whole cell volumes, or models based on a predominant influence of NuMA domains, yielded poor agreements with experiments (Figure S5d-g). Importantly, the model predicted deviations to planarity in a dose-dependent manner, among more or less elongated wt or blebbistatin treated cells, and also the correct orientation of spindles within the tissue plane, when shape anisotropies were present in this plane (Figure 4e-f). These direct comparisons between 3D model predictions and experiments strongly support that length-limitations of astral MTs can account for spindle position and planar orientation in intestinal crypts.

## Lengthening astral MTs affect epithelial tissue layering

This truncated shape sensing may have many advantages for the regulation of tissue layering and cell density. For instance, it may allow cells to adapt division orientation to planar tissue forces, tilting spindles along the A-B axis if cells become over-compressed by their neighbors, providing a potential homeostatic mechanism to regulate cell density and monolayered architecture <sup>17</sup>. Accordingly, in Kif18B-KO organoids, as a consequence of spindle mis-orientation, planar polarity of cytokinesis and placement of daughter cells were largely impaired (Figure 5a-b). Hence, the regular basal nuclear arrangement, which is a hallmark of polarized columnar epithelia, was lost three days after KO induction (Figure 5a,c-d). Finally, nuclei densities were significantly reduced in the Kif18B-KO (Figure 5e), showing how spindle mis-orientation may impact crypt architecture and cell density. These results directly demonstrate how a modulation in astral MT length may impact spindle planarity and architecture of a mammalian tissue.
# DISCUSSION

## A new model for planar spindle orientation in proliferative epithelia

How spindle orientation is regulated in proliferative tissues and stem cell niches is of fundamental importance for organ morphogenesis and homeostasis, and highly relevant to human disorders¹⁰,³⁹,⁶⁰,⁶¹. Here, by documenting with high temporal and spatial resolution spindle orientation together with MT dynamics, cell shape and polarity in a 3D mammalian proliferative organoid, we propose a new model for the control of planar spindle orientation and monolayered tissue architecture. This model is based on a partial cell shape sensing resulting from a length-limitation of M-phase astral MTs, mediated by an enrichment of depolymerizing promoting factors at the basal pole. This limitation is thus coupled to the A-B polarity of the columnar tissue, and allows initially apically positioned centrosomes to stop their basal migration at a position shifted towards the apex, and astral MTs to probe the local apical fraction of mitotic cells to orient spindles in the tissue plane (Figure 5f; Figure S5h,k). This mechanism has similarities with previous models proposed for asymmetric aster pair positioning and orientation in some large zygotes featuring anisotropic MT asters²⁵,⁶² (Figure 5f). Importantly, our model contrasts with established ones in many epithelia, in which the role of polarity effectors including NuMA, LGN or afadin recruited to lateral cortices, is thought to be predominant to orient spindles in the tissue plane¹⁴. In intestinal crypts, their unconventional localization to the basal domain of mitotic cells suggest they may not contribute to orient spindles, and raise the question of how they may be segregated there.

We found that both myosin-IIA and Kif18B inhibition affected their localization at the basal cortex (Figure S4i-j; Figure S3s-t). Therefore, we suggest that basal polarity recruitment could be in part driven by enhanced local cortical tension associated to basal actomyosin activity and cellular rearrangements (Figure 1d-e; Figures S1a-g), as proposed in drosophila⁵¹ or epithelial cell lines⁴⁴,⁴⁷,⁴⁸, or from yet unknown mechanisms mediated by Kif18B or MT-(+) tip contacts⁵⁷. Other plausible mechanisms contributing to this basal localization might include a local clearance of polarity effectors from the apex from chromosome-derived signals⁶³,⁶⁴, or a recruitment associated to the numerous basal multicellular contacts formed in mitosis⁵¹.

Interestingly, asymmetric actomyosin activity in other epithelial cells and tissues has been proposed to directly influence spindle orientation. In drosophila, one study notably reported
that local actomyosin enrichment may override geometrical cues to orient spindles in face of
actomyosin rich zones⁶⁵. In cultured cells, a recent local optogenetic assay showed an
opposite behavior with spindles orienting orthogonal to the most contractile cortical zone⁶⁶.
Our findings in the crypt align more with this latter finding, at least in terms of geometrical
outputs. However, our results favor a more indirect role for basal actomyosin asymmetric
enrichment, primarily impacting spindle orientation through its role in controlling mitotic cell
shape changes. We currently do not understand which mechanism may enrich actomyosin at
the basal poles of mitotic crypt cells. We suggest it may reflect the need for apical migrations
of dividing cells in columnar tissues, as proposed in the mouse nervous system or zebrafish
retina⁶⁷,⁶⁸. Furthermore, actomyosin is not only enriched at the base of the metaphase cell
but also in its direct neighbors, together with E-cadherin and afadin (Figure S1c-f; Figure S2g-
l). This basal actomyosin pool may promote multi-junctional remodeling and cell cohesion, as
proposed in other contexts⁶⁹,⁷⁰, given the lack of multicellular contacts and basal neighbor cell
remodeling when myosin is inhibited in organoids (Figure S4e-h).

Length-regulation of M-phase MTs, mitotic rounding and spindle orientation

Astral MTs control spindle positioning in organisms ranging from yeast to mammals <sup>16</sup>. While most models have thus far largely assumed that astral MTs grow to reach the cortex, to probe cell shapes and polarities, recent studies have suggested that M-phase astral MTs which are bounded by dynamic instabilities, may be just long enough in mitosis to reach the cortex of fully rounded cells <sup>71</sup>. Accordingly, impairment of mitotic rounding can prevent MTs from properly interacting with the cortex, yielding to chromosome segregation and spindle orientation defects <sup>26,72</sup>. In intestinal organoids, using live imaging and expansion microscopy, we found that astral MTs rarely reach the basal domain of the metaphase cell. Based on these observations, we propose that this cell-cycle regulated length-limitation may be exploited in the organoid tissue to allow the spindle to only probe apical cell shapes, position asymmetrically and orient in the tissue plane. In support to this, we found that Kif18B, a MT-depolymerizing kinesin which limits astral MT length in numerous mammalian cell lines and primary cultures <sup>53,56,57</sup>, localizes to the basal pole of metaphase cells in intestinal organoids (Figure S3m-n). Loss of Kif18B does not disrupt initial apical centrosome migration (Figure S5h,j-k) but leads to significantly longer MTs and random spindle orientation (Figure 3g-i). Therefore, although we cannot fully discard a more indirect mechanism by which Kif18B could regulate spindle orientation, we propose it may primarily function to negatively regulate MT growth to allow spindles to probe local apical shapes.

In addition to Kif18B, local modulation of MT dynamics or dynein activity could also emerge
directly from enhanced basal actomyosin activity, or from putative higher basal organelle
crowding, as proposed in other systems<sup>25,73,74</sup>. While the impact of cell shape on division orientation was first reported more than 150 years ago<sup>75</sup>, mechanisms by which geometries are being sensed in multicellular tissues are still at their infancy<sup>18</sup>. Our study provides an important new generic mechanism to orient spindles along the short axis in monolayered mammalian epithelia, and calls for a better exploration of mechanisms regulating astral MT length and mitotic shape changes in multicellular tissues.

Functions of planar divisions for crypt dynamics and architecture

Planar cell divisions are key for maintaining the monolayered architecture of certain epithelial tissues. Conversely, programmed alterations of planar divisions along the A-B axis of the tissue are required for epithelial stratification such as during skin development in the mouse embryo<sup>76,77</sup>. In the curved 3D geometry of intestinal crypts, we found that cell divisions orient parallel to the local plane of curvature of the tissue. We propose that such organization of cell divisions may contribute to promote a near-isotropic expansion of the crypt tip while maintaining a regular monolayered architecture. Accordingly, altering spindle planarity in Kif18B-KO resulted in mis-placed daughter cells, often protruding into the apical lumen (Figure 5a-e). However, beyond its primary conserved function in negatively regulating M-phase MT lengths, Kif18B has also been recently proposed to play a role in the DNA damage response<sup>78</sup>. Therefore, in addition to its spindle orientation regulatory role, we cannot fully exclude other Kif18Bfunctions in maintaining tissue architecture.

Whether such spindle orientation defects could lead to the long-term formation of tumor masses, epithelial dysplasia or hyperplasia, or if mechanisms such as apoptosis or daughter cell reintegration may safeguard the intestinal tissue, remain to be tested with additional genetic manipulations <sup>79</sup>. In addition, many of the dividing cells in the crypt are Lgr5+-stem cells, which may undergo symmetric fate divisions for self-renewal or asymmetric divisions to generate a daughter cell that becomes fated. As such, whether alteration of planar spindle positioning affects the auto-renewal properties of organoids and their homeostasis, as previously described for instance in Apc-mutated intestinal tissues, is another important open question <sup>80,81</sup>. More work on the regulation and function of oriented division in mammalian stem cell niches, tissues and organs may help to better appreciate the emergence of human epithelial disorders.

Limitations of the study

Our study establishes a new mechanism for planar spindle orientation based on a limitation of
MT length, presumably independent of canonical polarity cues, including LGN and NuMA. This
model derives from the unconventional localization of these factors on the basal side of
intestinal dividing cells, away from lateral cortices and spindle poles. However, in the present study, we do neither address the detailed mechanisms that promote this atypical localization, nor the general function of polarity cues. This would require the generation of fluorescent knock-ins, optogenetic tools or proper loss of function alleles, that we failed to generate due to technical difficulties in transfecting this primary culture. Another limitation is that we do not currently understand if the spindle orientation mechanism that we propose is specific to intestinal crypts, mammalian epithelia, or it emerges from the particular topology, dynamics and mechanics of the tissue.
## Acknowledgments

We thank René-Marc Mège and Benoit Ladoux (Institut Jacques Monod, IJM) for helpful discussions, Paula Martin Gil, Valeria Rocchi and Jeremy Sallé (IJM) for technical help, as well as Yohanns Bellaiche (Institut Curie) for reading the manuscript. We thank Renata Basto, Ana-Maria Lennon-Dumenil, Robert S. Adelstein and Danijela Vignjevic for providing mice. Confocal microscopy and spinning-disc analyses were performed in the ImagoSeine microscopy facility (IJM). This work was supported by grants from the Groupama Foundation – Research Prize for Rare Diseases 2017 (to D.D), the Fondation pour la Recherche Médicale (FRM) (to C.G.), the Fondation ARC (to J.S.), the Human Frontier Science Program (RGP0038/2018) (to D.D), the LabEx "Who Am I?" #ANR-11-LABX-0071 and the Université de Paris IdEx #ANR-18-IDEX-0001 funded by the French Government through its "Investments for the Future" program (to N.M. and D.D.), the INCA PLBIO20-150, Cancéropole Ile-de-France (to N.M. and D.D.), La Ligue Contre le Cancer (EL2021.LNCC/ NiM), the ANR-20-CE13-0015 (to D.D.) and the Fondation Bettencourt Schueller ("Coup d'élan prize") (to N.M.).

**Author contributions:** J.S, M.A.F, A. B, M.S., O.F, C.G., T.D., HC, N.M. and D.D. performed experiments. N.M. designed and performed models and simulations. J.S, M.A.F., A.B., C.G., N.M. and D.D. designed the experiments. J.S, M.A.F, A.B., C.G., M.S., H.C., N.M. and D.D. performed analyses. J.S, N.M. and D.D. coordinated the overall research and experiments, and wrote the manuscript.

**Conflict of interest:** The authors declare no conflict of interest.
FIGURE LEGENDS

**Figure 1:** An atypical metaphase cell rounding and spindle orientation in intestinal organoids. **a** Spinning-disk analysis of α-tubulin-GFP (green) and H2B-mCherry (blue) distribution in intestinal crypt organoids. Yellow arrows point on organoid crypt-like structures. L, lumen. Scale bar, 50µm. **b** Time lapse images of α-tubulin-GFP (green) and H2B-mCherry (blue) during mitosis. Yellow arrowheads point on migrating centrosomes. Yellow dotted lines highlight spindle axis. Scale bar, 10µm. **c** Time lapse images of tdTomato organoids showing mitosis progression. Yellow star points on a dividing cell. Yellow arrowheads point on daughter cell separation and re-integration in the epithelial monolayer. Scale bar, 5µm. **d** Statistical analysis of the percentage of total signal intensity for myosin-IIA-KI-GFP at the apical, lateral or basal cortex in interphase (grey) or during mitosis (red). Interphase: myosin-IIA signal intensity at the apical cortex = 41.88±1.38 % (mean±S.E.M), lateral cortex = 31.6±0.78 %, basal cortex = 26.47±0.89 %. Mitosis: myosin-IIA signal intensity at the apical cortex = 30.64±0.84 % (mean±S.E.M), lateral cortex = 31.16±0.28 %, basal cortex = 38.26±1.02 %. N = 4 experiments, n = 30 cells. Two-way ANOVA test and Sidak's multiple comparison test, ****p < 0.0001, ns non-significant. **e** Time-lapse of a live-imaged cell division in myosin-IIA-KI-GFP organoid crypt. Yellow star points on a dividing cell. Yellow arrowheads point on daughter cell separation and re-integration in the epithelial monolayer. Scale bar, 10µm. **f** Representative 3D rendering of metaphase cell (magenta) and neighboring cells (grey) after segmentation of cell membranes based on confocal z-stacks in crypt organoids. Spatial coordinates are shown. **g** Confocal analysis of α-tubulin (green) and E-cadherin (magenta) distribution in organoid metaphase cells. Scale bar, 10µm. **h** Quantitative analyses of the aspect ratio (distance between the spindle axis and the basal membrane over the distance between the spindle axis and the apical membrane) (n = 20 cells) and sphericity (n = 9 cells) of the organoid metaphase cells. Aspect ratio = 1.33±0.04 (mean±S.E.M), sphericity = 0.79±0.02. **i** Representative 3D rendering after segmentation of cell membranes and spindle poles from confocal z-stacks of organoid metaphase cells. Cell shape is depicted in magenta and in transparency, spindle poles in green. Distances between the spindle axis (black dotted line) and the apical (grey arrow) or basal membrane (red arrow) are shown. Spatial coordinates are shown. **j** Statistical analysis of the spindle orientation relative to planar axis (grey) or long cell axis (light grey). Spindle orientation relative to planar axis = 8.61±1.52° (mean±S.E.M), to long cell axis = 66.13±3.8°. N = 3 experiments, n(planar axis) = 25 cells, n(long cell axis) = 12 cells. **k** Statistical analysis of the percentage of the distance between the spindle axis and the apical (grey) or basal (light grey) membrane. Distance from apical
membrane = 40.728±1.485 % (mean±S.E.M), from basal membrane = 59.272±1.485 %. N =
3 experiments, n = 13 cells. Paired t-test, ****p <0.0001.

**Figure 2:** A 2D model for spindle orientation predicts that length limitation in M-phase astral microtubules may account for spindle planarity. **a** Confocal analysis of the distribution of E-cadherin and NuMA in mouse organoid metaphase cells. NuMA signal intensity is color-coded with Fire LUT table from ImageJ on the right panel. Color scale bar indicates the gray value intensity. Yellow arrow points to basal accumulation of NuMA. Scale bar, 5 µm. **b** Statistical analysis of NuMA signal intensity at the apical, lateral or basal cortex. NuMA signal intensity at the apical cortex = 21.65±1.01 % (mean±S.E.M), lateral cortex = 29.88±1.01 %, basal cortex = 40.65±1.24 %. n = 30 cells. One-way ANOVA test and Tukey's multiple comparison test, ****p <0.0001. For each experiment, three independent experiments were carried out. **c** Confocal analysis of the distribution of E-cadherin and LGN in human organoid metaphase cells. LGN signal intensity is color-coded with Fire LUT table from ImageJ on the right panel. Color scale bar indicates the gray value intensity. Yellow arrow points to basal accumulation of LGN. Scale bar, 5 µm. **d** Statistical analysis of LGN signal intensity at the apical, lateral or basal cortex. LGN signal intensity at the apical cortex = 27.86±0.57 % (mean±S.E.M), lateral cortex = 30.41±0.45 %, basal cortex = 41.77±0.79 %. n = 30 cells. One-way ANOVA test and Tukey's multiple comparison test, *p* = 0.012, ****p <0.0001. For each assay, three independent experiments were carried out. **e** Schemes representing the core hypothesis of the distribution of microtubule (MT) length and polarity domains in the 3 tested models for spindle orientation. **f** Rotational potential energy profiles plotted as a function of spindle orientation angles with respect to the planar axis predicted by the 3 different models. The black dot marks the preferred spindle orientation at the minimum of the potential profiles. **g** Predicted preferred spindle orientation for a range of cell shapes with increasing aspect ratios in the tissue plane or along the A-B axis. Note how the model based on cell shape coupled to length-limitation in MTs transits from predicting planar spindles to A-B oriented spindles when cells become over-elongated, along the A-B axis. **h** Phase diagram of the predicted preferred spindle orientations for the model based on cell shape and a limit in MT lengths, drawn as a function of the 2 control parameter, the distance of MT- (+) tips to the basal side and the elongation of mitotic cell shapes.

**Figure 3:** Existence of a length-limit for M-phase astral MTs that impact spindle orientation. **a** Projected confocal z-stacks of α-tubulin distribution in isolated organoid metaphase cells. En-face and lateral views are presented. Apical domain areas boxed in blue and basal domain areas boxed in yellow are presented in the low panels. Green arrowheads
point to astral MTs contacting the apical cortex. Corrected scale bar, 10µm. b Representative 3D rendering of a metaphase cell (magenta), neighboring cells (grey), spindle poles and microtubules (green) after segmentation of a confocal z-stack of E-cadherin and α-tubulin localization. Are depicted A-B views tilted to show basal cortex. c Statistical analysis of the distance between the astral MT tip and the apical, lateral or basal cortex. Distance between astral MT tip and the apical cortex = 0.466±0.045 µm (mean±S.E.M), the lateral cortex = 0.349±0.025 µm, the basal cortex = 1.685±0.086 µm. N = 3 experiments, n = 11 cells. N = 104 apical astral MTs, n = 102 lateral astral MTs, n = 107 basal astral MTs. Two-way ANOVA with Tukey's multiple comparisons test, ****p <0.0001; ns, non-significant. d Statistical analysis of the number of astral MTs at the apical, lateral and basal sides. Number of astral MTs at the apical side = 16.3±1.09 (mean±S.E.M), at the lateral side = 7.6±0.5, at the basal side = 14.2±1.06. N = 3 experiments, n = 11 cells. Unpaired t-test, ****p <0.0001; ns, non-significant. e Scheme showing the position of apical or basal laser cut (yellow dotted lines) of astral MTs. f Statistical analyses of the spindle pole displacement in the y-axis after apical or basal laser cut in organoid metaphase cells. n = 6 cells for each condition. Unpaired t-test, ****p <0.0001. g Confocal analysis of α-tubulin distribution in metaphase cells in control-KO or Kif18B-KO organoids. Yellow dotted line delimits the metaphase cell. Green arrowheads point to astral MTs contacting the basal cortex. L, lumen. Scale bar, 10µm. h Statistical analysis of the length of basal astral MTs in metaphase cells of control-KO or Kif18-KO organoids. n = 99 astral MTs in 13 control-KO cells, n = 103 astral MTs in 17 Kif18-KO cells. Unpaired t-test, ****p <0.0001. i Statistical analysis of the spindle axis orientation relative to the planar axis of the epithelium in metaphase cells of control-KO or Kif18-KO organoids. n = 32 control-KO cells, n = 50 Kif18-KO cells. Unpaired t-test, ****p <0.0001. For each experiment, three independent experiments were carried out.

**Figure 4: Metaphase cell shape impacts planar spindle orientation in organoid cells.** a Representative 3D rendering of cell shape and spindle pole positioning after segmentation of cell membranes and NuMA spindle-associated signal from confocal z-stacks of control (DMSO-treated), L-WNR-cultured, blebbistatin-treated or induced myosin-IIA-KO organoid metaphase cells. Cell shape is depicted in magenta, spindle poles in green. Spatial coordinates are shown. b In control (DMSO-treated), L-WNR-cultured, blebbistatin-treated or induced myosin-IIA-KO organoid metaphase cells, is shown the confocal image of α-tubulin or NuMA (green) and nuclei (blue). Scale bar, 5µm. c Statistical analyses of organoid metaphase cell aspect ratio in control (DMSO-treated), L-WNR-cultured, blebbistatin-treated or induced myosin-IIA-KO organoid metaphase cells. Aspect ratio of metaphase cells in control organoids = 1.33±0.04 (mean±S.E.M), in L-WNR-cultured organoids = 0.53±0.04, in
blebbistatin-treated organoids = 2.01±0.12, in myosin-IIA-KO organoids = 1.78±0.13. n (control organoids) = 20 cells, n (L-WNR organoids) = 10 cells, n (blebbistatin-treated organoids) = 10 cells, n (myosin-IIA-KO organoids) = 10 cells. One-way ANOVA, with Tukey's multiple comparisons tests, **p = 0.0011, ****p < 0.0001. d Statistical analyses of the spindle axis orientation relative to the plane of the epithelium. Angle deviation in control organoids = 8.61±1.52° (mean±S.E.M), in L-WNR-cultured organoids = 11.28±3.74°, in blebbistatin-treated organoids = 37.11±5.04°, in myosin-IIA-KO organoids = 34.88±5.22°. n (control organoids) = 25 cells, n (L-WNR organoids) = 9 cells, n (blebbistatin-treated organoids) = 25 cells, n (myosin-IIA-KO organoids) = 25 cells. One way-ANOVA, ***p = 0.0001, ****p < 0.0001. ns, non-significant. For each experiment, three independent experiments were carried out. e (Top) 3D reconstitution of experimental cells obtained from 3D z-stacks of a representative dividing cell labeled for E-cadherin and NuMA to extract cell shape (magenta), spindle orientation (green line) and NuMA accumulation at the basal cortex (grey domain). (Bottom) Corresponding simulation output, based on MT length-dependent forces (shape sensing) and an exclusion of MTs length in the basal domain region (orange zone). The red traces represent the history of spindle axis displacement in the simulation. Both experimental and simulated cells are viewed in different planes with respect to A-B axis. f. Predicted spindle orientation angle with respect to the A-B axis, plotted as a function of the experimental axis for 10 individual control, L-WNR treated or blebbistatin-treated crypt cells. Insets provide representative examples of experimental and simulated spindle orientation in the different treatments. The dotted line is a linear fit, with a slope of 0.96 (1 being a perfect agreement between models and experiments). R² is the correlation coefficient between the fit and the data. Expt, experimental. g. Experimental and theoretical prediction of spindle asymmetric position towards the apical cell poles in the indicated conditions.

**Figure 5: Impact of spindle mis-orientation in epithelial organization.** **a** Confocal microscopy analysis of the distribution of the E-cadherin (magenta) and nuclei (blue) in control-KO and Kif18B-KO organoids. Representative cytokinesis events are boxed in yellow and presented in bottom left. Yellow dotted lines highlight cell division axis. L, lumen. Scale bar, 50µm. **b** Statistical analysis of cytokinesis angle relative to the planar axis in control-KO or Kif18B-KO organoids. n = 13 control-KO cells, n = 26 Kif18B-KO cells. Cytokinesis angle (control-KO) = 10.3±1.68°, cytokinesis angle (Kif18B-KO) = 50.8±4.9°. Unpaired t-test, ****p < 0.0001. **c** Statistical analysis of interphase nuclear positioning (distance from the centroid of the interphase nucleus to the basal membrane) (n = 100 cells). Nuclear positioning (control-KO) = 0.271±0.003 µm (mean±S.E.M), (Kif18B-KO) = 0.399±0.01 µm. Unpaired t-test, ****p < 0.0001. **d** Statistical analysis of distance between adjacent interphase nuclei (n = 100 cells).
Distance between nuclei (control-KO) = 0.559±0.015 µm (mean±S.E.M), (Kif18B-KO) = 0.939±0.032 µm. Unpaired t-test, ****p < 0.0001. e Statistical analysis of nucleus density (n = 10 fields). Nucleus density (control-KO) = 0.031±0.001 µm (mean±S.E.M), (Kif18B-KO) = 0.022±0.001 µm. Unpaired t-test, ***p = 0.0002. f Scheme depicting the proposed model of spindle polarity modulation in organoid metaphase cells.
## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed and will be fulfilled by the lead contacts, Delphine Delacour (delphine.delacour@ijm.fr) and Nicolas Minc (nicolas.minc@ijm.fr).

### Materials availability

Materials generated in the current study are available from the lead contacts upon request. There are restrictions to the availability of materials due to collaborations or MTAs.

### Data and code availability

Source data files have been deposited at Zenodo (10.5281/zenodo.7804776). Microscopy data reported in this paper will be shared by the lead contacts on request. Original codes for segmentation prior to MeshLab 3D viewing and cell contact mapping have been deposited at Zenodo (10.5281/zenodo.7802531). DOI is listed in the key resources table. Any additional information required to reanalyze the data reported in this paper is available from the lead contacts upon request.

## EXPERIMENTAL MODEL AND SUBJECT DETAILS

### Organoid cultures and transfection

Wild-type C57/BI6 adult male mice were provided by the animal house facility of the Institut Jacques Monod. Mice were housed in EOPS (Environment without Specific Pathogenic Organisms) environment, and handled in accordance with French regulation for animal care. Experiments were performed with ethical approval of the Comité d'Ethique Buffon (Institut Jacques Monod). Mice used for intestinal crypt isolation were between 6 and 12 weeks old. After euthanization by cervical dislocation, the small intestine was harvested, flushed with PBS to discard luminal content and cut longitudinally open. The tissue was then cut into small pieces of 3-5 mm and further washed in PBS. The pieces of intestinal tissue were then incubated on ice for 10 min in a tube containing 5 mM EDTA. The tube was then vortexed for 2 min to release villi from the tissue. After EDTA removal, the intestinal pieces were placed in cold PBS and vortexed vigorously for 3 min to ensure crypt release. This process was
repeated 3 times, with each fraction recovered. The third and fourth fractions are usually concentrated in crypts, so these were combined and passed through a 70-µm cell strainer to remove remaining villi and centrifuged at 1000 RPM for 5 min. The pellet (crypts) was then washed in advanced DMEM/F12 (#12634010 Thermo Fisher Scientific, Waltham, Massachusetts, USA) and centrifuged. The final pellet is resuspended in 50 µl of 1:1 ratio of advanced DMEM/F12 and ice-cold Matrigel (#734-1100 VWR, Radnor, PA, USA) and plated as domes. Incubation at 37°C for 20-30 min allowed Matrigel polymerization. Organoid culture was performed in IntestiCult™ Organoid Growth medium (#06005 STEMCELL Technologies, Vancouver, Canada), from here on termed ENR medium. Organoids were routinely grown in Matrigel with IntestiCult™ Organoid Growth medium and passaged every 7 to 10 days. Medium was changed every 2 days. Live-imaging or immunofluorescence experiments were performed on 3-4 days organoids. For cystic growth, intestinal organoids were cultured with L-WNR medium supplemented with 10µM CHIR99021 for 10 days. The L-WNR cell line was purchased from ATCC (ATCC CRL-3276TM). The L-WNR medium was produced according to the ATCC recommendations.

α-tubulin-GFP and EB3-RFP expression was carried out by lentiviral transduction using the commercial lentiviral biosensors LentiBrite (#17-10206; #17-10222, Merck, Darmstadt, Germany) and following an already established protocol for lentiviral transduction in intestinal organoids<sup>82</sup>. This process involves dissociating intestinal organoids into single cells using TrypLE Express (#12605010 Thermo Fisher Scientific, Waltham, Massachusetts, USA) and seeding on a layer of Matrigel along the lentiviral vectors overnight, before covering with another layer of Matrigel the next day to allow growth into 3D organoids. The medium was enriched with 10 µM of CHIR99021 and 10 µM of Y-27632 (#72054 and #72304 STEMCELL Technologies, Vancouver, Canada) to prioritize stem cell proliferation and improve single cell survival respectively. After 48 to 72h, fluorescent organoids start appearing and can be isolated and expanded.

H2B-mCherry organoids were generated from H2B-mCherry-knock-in mice provided by Renata Basto (Institut Curie, Paris). VillinCreERT2-tdTomato organoids were generated from mice provided by Danijela Vignjevic (Institut Curite, Paris) <sup>83</sup>. Myosin-IIA-GFP-knock-in mice <sup>84</sup> were provided by Robert S. Adelstein (NHLBI, Bethesda) and Ana-Maria Lennon-Dumesnil (Institut Curie, Paris). Myosin-IIA-KO/mTmG mice <sup>83</sup> were kindly provided by Danijela Vignjevic (Institut Curite, Paris) and generated by crossing Myosin-IIA-KO <sup>85</sup> and mTmG mice <sup>86</sup>. Cre recombinase for Myosin-IIA-KO was induced with 100nM of 4-hydroxytamoxifen for 24h (#SML1666, Sigma-Aldrich).

Kif18B-KO organoids were generated by a CRISPR-Cas9 strategy using the Edit-R All-in-one lentiviral system from Horizon Discovery (Cambridge, UK). A set of 3 lentiviral sgRNAs were
used to target Kif18B (#GSGM11839-246783580: GGTCAGAACACCCAGTTAAT; #GSGM11839-246783577: GTGTTTGCCCTATGGCGCCAC; #GSGM11839-246783579: GTGGTGTTGAGGTCCCGAGT). Control-KO organoids were generated using a non-targeting sgRNA (#GSGM11811, Horizon Discovery). Organoids were transduced with lentiviral particles containing an inducible Cas9 along the sgRNAs for the targeted gene. Selection was then performed using 2ug/ml of puromycin (#A1113803, Thermo Fisher Scientific) for at least 2 weeks before inducing Cas9 with 600 ng/ml of doxycycline (#D9891 Merck).

For human organoid preparation, the project was approved by the Scientific Committee of the tumor bank of Lille and the Department of Pathology of the Lille University Hospital. The patient had signed an informed consent. Left colectomy was carried out for a colon adenocarcinoma in the Department of General and Digestive Surgery of the Lille University Hospital. The patient had not been treated with neoadjuvant chemotherapy. The normal mucosa sample (1 cm2 harvested >10cm distant from the tumor) was cut into small pieces (<3mm3) and washed thoroughly with 1X PBS buffer supplemented with combined antibiotics (normocin, gentamicin and amphotericin B). Mucosa fragments were then incubated in 25 ml of 1X PBS with 2.5mM EDTA at 4°C for 30 min under slow rotation. Mechanical release of colonic crypts was then performed three times in 10 mL 1X PBS. Fractions of colonic crypt suspension were pooled, centrifuged, resuspended in advanced DMEM/F12 medium and filtered through a 70-µm cell strainer. After counting, cells were resuspended in Matrigel and seeded in 40 µl domes in the wells of a 24-well plate. After Matrigel solidification, domes were covered with complete colon organoid medium (advanced DMEM/F12 medium supplemented with 1X Glutamax (Invitrogen), 1 X HEPES (Sigma-Aldrich), B-27® Supplement Minus Vitamin A (Thermo Fisher Scientific), 1 X N2 (Thermo Fisher Scientific), 1 mM N-acetyl-L-cysteine (Sigma-Aldrich), 50% v/v Wnt3a/RSPO1/Noggin-conditioned medium, 50 ng/ml EGF (Peprotech), 0.5 µM A83-01 (Tocris), 10 mM Nicotinamide (Sigma-Aldrich), 10 nM Gastrin (Sigma-Aldrich) and 10 µM Y-27632 dihydrochloride (Tocris), as recommended in Sato et al.⁸⁷. Resulting colonic organoids were named COL-2920xi. Complete medium without Y-27632 was then renewed every two days and organoids were passaged through mechanical disruption every week.

## METHOD DETAILS

### Antibodies and reagents

Mouse monoclonal antibody directed against α-tubulin (clone DM1A, IF dilution, 1:100) was purchased from Sigma-Aldrich. Rabbit polyclonal antibody directed against EpCAM (#ab71916, IF dilution, 1:100), rabbit polyclonal against afadin (#ab11337, IF dilution 1:100),
rabbit polyclonal antibody directed against α-tubulin (#ab18251, IF 1:100) were from Abcam. Mouse monoclonal antibody directed against E-cadherin (clone 36, #610181, IF dilution, 1:50) was from BD Biosciences. Rabbit polyclonal antibody directed against Phospho-Myosin Light Chain 2 (Ser19, #3671, IF dilution, 1:100) and rabbit monoclonal antibody directed against E-cadherin (clone 24E10, #3195S, IF dilution 1:100) were from Cell Signaling Technology. Mouse monoclonal antibody against dynein (#MAB1218, IF dilution 1:100) was from Millipore. Rabbit monoclonal antibody directed against NuMA (EP3976, #ab109262, IF dilution, 1:100) was from Abcam. Rabbit polyclonal antibody directed against non-muscle myosin heavy chain II-A antibody (clone poly19098, #909801, WB dilution 1:500) was from Biolegend. Mouse monoclonal antibody directed against GAPDH (#60004-1-Ig, WB dilution 1:5000) was from Proteintech. Rabbit polyclonal antibody against Kif18B (#A303-982A-T, WB dilution 1:500; #orb612187, IF dilution 1:100) were from ThermoFisher Scientific and Biorbyt, respectively. Goat anti-mouse-Alexa-488, 568, anti-rabbit-Alexa488, 568 or 647 were from Life Technologies (Paisley, UK). Nuclei were stained with Hoechst 33342 solution incubation (Life Technologies) at a 1:1000 dilution. Blebbistatin, Y-27632 and nocodazole were from Sigma Aldrich (Saint-Louis, MO, USA). Spy650-Tubulin was obtained from Cytoskeleton (Denver, USA).

## Drug treatments

Organoids were incubated in 25µM blebbistatin or 150nM nocodazole for 1.5h, then washed out with PBS and prepared for immunostaining or live-imaging. Cells were incubated for 1.5h in DMSO as controls.

## Biochemical analysis

For western blots, organoid lysates were prepared 3 days after plating using per condition 6 wells of a 24-well plate. Matrigel was depolymerized by incubating with 1 ml of Gentle Cell Dissociation Reagent (#07174 STEMCELL Technologies, Vancouver, Canada) for 30 min at 4°C and centrifugation for 5 min at 500 x g at 4°C. The pellet was resuspended in lysis buffer containing 25mM Tris / 5 mM NaCl / 1mM EDTA / 1mM EGTA / 0.5% NP40 / 1% Triton TX100, and incubated on ice for 30 min. The solution was then passed 10 times through a syringe equipped with a 23G needle and centrifuged at 10 000 RPM at 4°C for 10 min. Supernatant total protein content was measured by Bradford assay (Biorad). For each condition, 50µg of proteins was loaded per well in Novex Tris-Glycine pre-cast gels (ThermoFischer Scientific). Proteins were detected with either HRP-linked goat anti-mouse IgG antibody (dilution 1:10,000; Sigma-Aldrich) or HRP-linked donkey anti-rabbit IgG antibody (dilution 1:10,000,
Invitrogen), and visualized on ImageQuant LAS4000 (GE-Healthcare, Buckinghamshire, UK).
Signal quantification was performed using Fiji software.

## Immunostaining

Routinely, organoids were fixed using 4% paraformaldehyde for 30 min, then permeabilized using 0.025% saponin solution in PBS for 30 min. Blocking step was performed in 0.025% saponin/1% BSA solution for 45 min, before proceeding to incubation with primary antibody at 4°C overnight. The next day, the primary antibody was removed and the organoids washed 3 times in PBS for 10 min each, before adding the secondary antibody and left to incubate for 2h at room temperature. Finally, organoids were washed 3 times again for 10 minutes before incubating in Hoechst 33342 for 15 min to stain nuclei. Immunostained samples were mounted in home-made Mowiol solution.

For the MT immunostaining, we used an established protocol that maintain the MT integrity in intestinal organoids<sup>88</sup>. Briefly, organoids were isolated from the Matrigel and fixed in a methanol/formaldehyde solution (92% methanol, 8% formaldehyde). The blocking step was performed by incubating organoids in 10% goat serum solution in PBS with 0.1% Triton X-100. Immunostained organoids were mounted in home-made Mowiol solution on a slide.

For E-cadherin immunostaining *in vivo*, mouse jejunum was processed as previously described <sup>89</sup>. Briefly, samples were fixed for 2 hours in 4% PFA and paraffin embedded. 5 µm tissue sections were de-waxed in a xylene bath, rehydrated in isopropanol and in solutions with decreasing ethanol concentrations. Tissue sections were then blocked in 10% goat serum (Sigma-Aldrich) for 1 h. Primary antibody incubation was performed at 4°C overnight and secondary antibody incubation at room temperature for 2 h, both in 1% goat serum solution. Hoechst33342 staining was used to detect nuclei. Tissue sections were mounted in home-made Mowiol 488 solution.

For NuMA immunostaining *in vivo*, 1-mm pieces of mouse jejunum were fixed in 4% PFA overnight under shaking. After PBS wash, tissue permeabilization was performed in 1% Triton X-100 / PBS solution for 1 h, before saturation in 1% BSA / 3% goat serum / 0.2% Triton X-100 / PBS solution for 1 h. Incubation with primary or secondary antibodies were done in 0.1% BSA / 0.3 % goat serum / 0.2 % Triton X-100 / PBS overnight at 4°C. Hoechst33342 staining was used to detect nuclei. Immunostained samples were mounted in Vectashield (Vector Laboratories, Burlingame, CA).

## Live imaging

Dynamics experiments on α-tubulin-GFP/H2B-mCherry, myosin-IIA-KI-GFP, EB3-RFP or Spy650-Tubulin organoids were performed using an inverted Zeiss microscope equipped with a CSU-X1 spinning disk head (Yokogawa – Andor), using Zeiss 40X and 63X water objectives.
Spindle pole oscillations during metaphase have been analyzed using the ImageJ plugin MTrack J⁹⁰.

## Laser ablation experiments

Laser ablation experiments were performed using a spinning disk microscope equipped with a CSU-XI spinning disk head, using a 63X oil objective. The ablation was done using a pulsed 355 nm ultraviolet laser at a power of 30% and a thickness of 3, interfaced with an iLas system (Roper Scientific) piloted in Metamorph.

## Two-dimensional models for spindle orientation

2D models to predict spindle orientation were adapted from Minc et al. ⁴⁹ and Bosveld et al. ⁵¹. These models were developed and executed through Matlab (Mathworks) scripts which can be made available upon demand. Starting from the shape of a cell, the model positioned spindle poles and traced MT asters radiating from spindle poles to the contour. For length limitations, we added a fixed maximal length to MTs, normalized to cell length along the A-B axis, which was varied in figure 2e. Each MT is associated to a force, $f_{MT}$, which varies depending on hypothesis. For models based on shape sensing, we posited that $f_{MT} = aL_{MT}^2$ with $L_{MT}$ the length of the MT and a, an arbitrary constant. The scaling to the square was chosen as it best represents a length-dependent system in which MTs pull at the surface ⁴⁹,⁹¹. We note however that other models previously proposed to center and orient spindles with the long axis, including those based on pulling in bulk cytoplasm or MT pushing at the surface and limited by buckling, yielded to the same outputs. For NuMA based models, the force per MT was $f_{MT} = b[NuMA]*L_{MT}^2$, with b an arbitrary constant, and [NuMA] is the local concentration of NuMA around cells, inputted as a normalized Gaussian distribution with a peak located at the basal pole of cells. The script then looped to compute the torque exert generated by all MTs as a function of all possible spindle angles, and computed a rotational energy potential as a primitive of the torque ⁵⁰ (Figure 2f). Free parameters in the model were the number of MTs, the size of the spindle and the spatial extension of asters, which had little influence on model prediction (Figure S3a-d). For the NuMA model, we added one other parameter which is the width of the Gaussian, which was estimated from experimental images, and which did not influence prediction for domain size relatively small as compared to cell contour. Key parameters that altered spindle orientation outputs were thus cell shape, and the asymmetry of spindle positioning, as well as the maximal length of MTs related to cell length (Figure 2g-h and Figure S3a-d).

## Three-dimensional simulations for spindle position and orientation
The simulation package used for 3D models was developed in Matlab and was adapted from Pierre et al. 25 and Ershov et al. 92. This package includes a module to extract and reconstitute 3D shapes, spindle pole positions and polarity domains from segmented experimental image, and to add predefined hypothesis similar to those used above in 2D. In 3D, the exclusion of MTs from the basal pole was introduced as a gradient from the basal pole of length limitation, which was scaled to cell length along the A-B axis. The size of spindles and polar basal domains were defined directly from each experimental stack. Once parameters were defined as inputs, the model placed the spindle in a random position and orientation, traced MTs from spindle poles to measure their length and associated a force to each, and computed both global forces and torques exerted by MT asters. To search for minima, the model used a random walk, which follows the minimization of torques and forces. This was achieved by randomly modulating one of the 5 spatial parameters (3 for the position and 2 for the orientation of spindles), and recalculating the force torque at the subsequent step. The simulations followed the direction of force/torque minimization and stopped at a position/orientation once the iteration returns to this equilibrium after a given number of iterations (e.g. 300 runs) (Figure 4e-g, Figure S5a-c, and Videos S4-5). The length of the simulations, the duration needed to identify a stable equilibrium, the noise added to explore other parameters, and other intrinsic parameters of the loop, can be modulated, but were fixed for all the simulations performed in this work. Finally, to ensure that the spindle position/orientation identified did not correspond to a local minimum which could have been biased by the initial conditions, the simulations were run typically 3-4 times from another random starting position/orientation. Finally, once a position was found, the model recalculated the torque landscapes as a function of the 2 possible angles (Figure S5c).

## Segmentation and 3D rendering

Cell shape or spindle pole 3D segmentations are performed based on confocal z-stacks of cell membranes (i.e. E-cadherin, EpCAM) or MTs (i.e. α-tubulin, NuMA), respectively. Segmentations are performed manually to guarantee the best match with the initial data. Masks corresponding to cell shapes, spindle poles, organoid lumen and basal environment ("exterior") were generated on each slice of confocal z-stacks using Fiji 93.

For generating the 3D rendering of organoid cells, mask outlines were translated into meshes, which are 3D surfaces connecting the perimeters on each slice, by using a Python program adapted from an initial program provided by Emmanuel Faure (LIRMM, Montpellier). Briefly, for a given cell, the mask was first translated into a list of points lying on the perimeters for every position along z where the cell is present. The list of points was then translated into a list of polygonal faces linking the points together. Visualization of meshes was done with the Meshlab software (www.meshlab.net).
Measurements of geometric properties of the segmented cells, in particular their volume V and surface area A, were provided by Meshlab and used to compute the cell sphericity: $s=\pi(1/3(6V)/2/3/A)^{94}$. From each pixel of the segmented cells, we computed the distance to the lumen. For each cell, the mean cell height H in the apico-basal direction was computed as the difference between the furthest and closest pixels from the lumen, averaged for every position along z where the cell is present. The mean cell width was computed from the cell volume and height assuming a cylindrical geometry and using $W=2(V/\pi H)^{1/2}$. The mean spindle positioning with respect to the apico-basal axis was computed by averaging the distance from each point of the spindle to the lumen.

Multicellular contact distribution was computed from the ImageJ organoid cell shape masks and by using a custom Python program. The multicellular contacts were identified as any pixel in the vicinity of the borders of three or more cell masks (within 20 pixels). The “degree” of the multicellular contact (3-cells, 4-cells etc.) was given by the number of different cell masks in the vicinity of each multicellular contact. Positioning of computed multicellular contacts was then visualized together with cell meshes in Meshlab.

## Expansion microscopy of organoids

Expansion microscopy protocol was adapted from Gambarotto et al. ⁹⁵. Matrigel domes containing mouse organoids were dissociated by using Gentle Cell Dissociation Reagent. After centrifugation, Matrigel debris were eliminated with the supernatant and organoid pellet kept. Organoids were incubated overnight at 37°C in PBS containing 2% acrylamide / 1.5% formaldehyde.

For gelation, organoids were washed in PBS and pelleted by centrifugation. After resuspension PBS, organoids were layed on a parafilm piece in a humid chamber. After PBS removal with a tip, gelation solution (19.3% sodium acrylate / 10% acrylamide / 0.1% N,N'-methylenbisacrylamide / 0.5% TEMED / 0.1% Ammonium persulfate, in PBS) was added. Gelation solution containing organoids was covered with a 5mm coverslip, incubated 5 min on ice and then 1 hour at 37°C. The coverslip and the gel were placed in a 24-well plate filled with 500µl of denaturation buffer (200mM SDS / 200mM NaCl / 50mM Tris water, PH9). After 15 min agitation at room temperature, the gel detached from the coverslip, was placed in a 1.5ml microtube filled with denaturation buffer and incubated at 95°C for 1h30min.

For immunofluorescence, organoids were transferred in a 24-well plate, blocked and permeabilized during incubation in 1% BSA / 0.025% saponin, in PBS solution for 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C in the 1% BSA / 0.025% saponin, in PBS solution, under gentle shacking. The day after, after 3 PBS washes, secondary antibodies were incubated 4 hours at 4°C and then 1 hour at room temperature upon gentle shacking. After three PBS washes, nuclei were labelled with Hoechst 33342.
For the organoid expansion, the gel was transferred in a 6-well plate filled with ddH2O and wash for 1 hour without agitation. After ddH2O change, the gel was incubated overnight at room temperature. The day after, the expansion index was evaluated based on gel length before and after expansion. For image acquisition, the expanded gel was placed in a poly L-lysine-coated glass chamber after exceeding water removal.

## QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analyses were performed using Prism (GraphPad Software, San Diego, CA, USA, version 9.0). Statistical details of experiments can be found in the figure legends. Unless otherwise stated, experiments were replicated 3 times independently. No sample size estimations and no blinding were performed.
## SUPPLEMENTAL INFORMATION

Supplemental Figures S1-S5 and Videos S1-S5 are available in the online version of the paper.

## SUPPLEMENTAL VIDEOS

**Video S1: Planar orientation of cell division in intestinal organoids, related to Figure 1b and Figure S1a.**

**Description:** Time lapse imaging of α-tubulin-GFP (green) dynamics during mitosis in H2BmCherry (blue) organoids. Images were acquired every 30 sec. Frame rate is 15 fps.

**Video S2: Basal actomyosin distribution during cell division in intestinal organoids, related to Figure 1e and Figure S1c.**

**Description:** Time lapse imaging of myosin-IIA-GFP in intestinal organoids. Scale bar, 10µm. Images were acquired every 15 sec. Frame rate is 15 fps.

**Video S3: Dynamics of the (+)-tip associated EB3-GFP during mitosis onset and metaphase, related to Figure 3a-d.**

**Description:** Time lapse imaging of EB3-RFP in prophase (left panel) and metaphase (right panel) cells in intestinal organoids. Images were acquired every 4 or 1 sec, respectively. Frame rate is 10 fps.

**Video S4: Simulations of spindle orientation during metaphase in intestinal organoids, related to Figure 4e.**

**Description:** Time-lapses of 3D gradient descent simulations to predict planar spindle orientation, started with spindles close to the apical pole (left) or the basal pole (right). Note the similar final predicted equilibrium.

**Video S5: Simulations of spindle orientation according to NuMA polarity, cell shape or MT length limitation model during metaphase in intestinal organoids, related to Figure 4e.**

**Description:** 3D rotating views of spindle orientations in a representative experimental cell, and as predicted by 3D simulations based on the different indicated hypothesis (NuMA polarity, full cell shape, or cell shape coupled to length-limitation in MTs).
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**Key resources table**

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Mouse monoclonal anti-α-tubulin (clone DM1A)</td><td>Sigma-Aldrich</td><td>Cat#T9026</td></tr><tr><td>Rabbit polyclonal anti-EpCAM</td><td>Abcam</td><td>Cat#ab71916</td></tr><tr><td>Rabbit polyclonal anti-afadin</td><td>Abcam</td><td>Cat#ab11337</td></tr><tr><td>Rabbit polyclonal anti-alpha-tubulin</td><td>Abcam</td><td>Cat#ab18251</td></tr><tr><td>Mouse monoclonal anti-E-cadherin (clone 36)</td><td>BD Biosciences</td><td>Cat#610181</td></tr><tr><td>Rabbit polyclonal anti-Phospho-MyosinLightChain2</td><td>Cell Signaling</td><td>Cat#3671</td></tr><tr><td>Rabbit monoclonal anti-E-cadherin (clone 24E10)</td><td>Cell Signaling</td><td>Cat#3195S</td></tr><tr><td>Mouse monoclonal anti-dynein</td><td>Millipore</td><td>Cat#MAB1218</td></tr><tr><td>Rabbit monoclonal anti-NuMA</td><td>Abcam</td><td>Cat#ab109262</td></tr><tr><td>Rabbit polyclonal anti-non-muscle Myosin Heavy chain II-A (clone poly19098)</td><td>Biolegend</td><td>Cat#909801</td></tr><tr><td>Mouse monoclonal anti-GAPDH</td><td>Proteintech</td><td>Cat#60004-1-Ig</td></tr><tr><td>Rabbit polyclonal anti-Kif18B</td><td>ThermoFisher Scientific</td><td>Cat#A303-982A-T</td></tr><tr><td>Rabbit polyclonal anti-Kif18B</td><td>Biorbyt</td><td>Cat#orb612187</td></tr><tr><td>Goat anti-mouse-Alexa488</td><td>Life Technologies</td><td>Cat#A11001</td></tr><tr><td>Goat anti-mouse-Alexa568</td><td>Life Technologies</td><td>Cat#A11004</td></tr><tr><td>Goat anti-rabbit-Alexa488</td><td>Life Technologies</td><td>Cat#A11008</td></tr><tr><td>Goat anti-rabbit-Alexa568</td><td>Life Technologies</td><td>Cat#A11011</td></tr><tr><td>Goat anti-rabbit-Alexa647</td><td>Life Technologies</td><td>Cat#A21071</td></tr><tr><td>HRP-linked goat anti-mouse IgG antibody</td><td>Sigma-Aldrich</td><td>Cat#A9044</td></tr><tr><td>HRP-linked donkey anti-rabbit IgG antibody</td><td>Invitrogen</td><td>Cat#31460</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>Lentiviral biosensors LentiBrite α-tubulin-GFP</td><td>Merck</td><td>Cat#17-10206</td></tr><tr><td>Lentiviral biosensors LentiBrite EB3-RFP</td><td>Merck</td><td>Cat#17-10222</td></tr></tbody></table>
<table><tr><td>Edit-R All-in-one lentiviral system, set of 3 lentiviral sgRNAs to target Kif18B</td><td>Horizon Discovery</td><td>Cat#GSGM11839-246783580, #GSGM11839-246783577, #GSGM11839-246783579</td></tr><tr><td>Edit-R All-in-one lentiviral system, non-targeting sgRNA</td><td>Horizon Discovery</td><td>Cat#GSG11811</td></tr></table>

## Biological samples

<table><tr><td>Human colon organoids COL-2920xi</td><td>Audrey Vincent (CANTHER, Lille)</td><td>ORGALille Core Facility</td></tr></table>

## Chemicals, peptides, and recombinant proteins

<table><tr><td>DMEM/F12</td><td>Thermo Fisher Scientific</td><td>Cat#12634010</td></tr><tr><td>Matrigel</td><td>VWR</td><td>Cat#734-1100</td></tr><tr><td>IntestiCult™ Organoid Growth medium</td><td>STEMCELL Technologies</td><td>Cat#06005</td></tr><tr><td>TrypLE Express</td><td>Thermo Fisher Scientific</td><td>Cat#12605010</td></tr><tr><td>CHIR99021</td><td>STEMCELL Technologies</td><td>Cat#72054</td></tr><tr><td>Y-27632</td><td>STEMCELL Technologies</td><td>Cat#72304</td></tr><tr><td>4-hydroxytamoxifen</td><td>Sigma-Aldrich</td><td>Cat#SML1666</td></tr><tr><td>Puromycin</td><td>Thermo Fisher Scientific</td><td>Cat#A1113803</td></tr><tr><td>Doxycycline</td><td>Merck</td><td>Cat#D9891</td></tr><tr><td>EGF</td><td>Peprotech</td><td>Cat#236-EG</td></tr><tr><td>B-27® Supplement Minus Vitamin A</td><td>Thermo Fisher Scientific</td><td>Cat#12587010</td></tr><tr><td>N-2 Supplement</td><td>Thermo Fisher Scientific</td><td>Cat#17502048</td></tr><tr><td>A83-01</td><td>Tocris</td><td>Cat#2939</td></tr><tr><td>Gastrin</td><td>Sigma-Aldrich</td><td>Cat#G9020</td></tr></table>
<table><tr><td>Y-27632</td><td>Tocris</td><td>Cat#1254</td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>Cat#H3570</td></tr><tr><td>Blebbistatin</td><td>Sigma-Aldrich</td><td>Cat#B0560</td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>Cat#M1404</td></tr><tr><td>Spy650-Tubulin</td><td>Spirochrome, Cytoskeleton</td><td>Cat#CY-SC503</td></tr><tr><td>Gentle Cell Dissociation Reagent</td><td>STEMCELL Technologies</td><td>Cat#07174</td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>Cat#G9023</td></tr><tr><td>Vectashield</td><td>Vectashield, Eurobio</td><td>Cat#H-1000</td></tr></table>

Experimental models: Cell lines and organoid lines

<table><tr><td>L-WNR cell line</td><td>ATCC</td><td>Cat# CRL-3276</td></tr><tr><td>α-tubulin-GFP/H2B-mCherry organoids</td><td>This manuscript</td><td>N.A.</td></tr><tr><td>VillinCreERT2-tdTomato organoids</td><td>This manuscript</td><td>N.A.</td></tr><tr><td>Myosin-IIA-GFP-knock-in organoids</td><td>This manuscript</td><td>N.A.</td></tr><tr><td>Myosin-IIA-KO/mTmG organoids</td><td>This manuscript</td><td>N.A.</td></tr><tr><td>Control-KO organoids</td><td>This manuscript</td><td>N.A.</td></tr><tr><td>Kif18B-KO organoids</td><td>This manuscript</td><td>N.A.</td></tr><tr><td>EB3-RFP organoids</td><td>This manuscript</td><td>N.A.</td></tr></table>

Experimental models: Organisms/strains

<table><tr><td>Wild-type C57/BI6 mice</td><td>Institut Jacques Monod animal house facility, Paris</td><td>N.A.</td></tr><tr><td>H2B-mCherry-knock-in C57/BI6 mice</td><td>Renata Basto (Institut Curie, Paris)</td><td>N.A.</td></tr><tr><td>VillinCreERT2-tdTomato C57/BI6 mice</td><td>Danijela Vignjevic (Institut Curie, Paris)</td><td>N.A.</td></tr></table>
<table><tr><td>Myosin-IIA-GFP-knock-in C57/BI6 mice</td><td>Robert Adelstein (National Heart, Lung and Blood Institute, Bethesda), Ana-Maria Lennon-Dumesnil (Institut Curie, Paris)</td><td>N.A.</td></tr><tr><td>Myosin-IIA-KO/mTmG C57/BI6 mice</td><td>Danijela Vignjevic (Institut Curie, Paris)</td><td>N.A.</td></tr></table>

## Software and algorithms

<table><tr><td>FiJi</td><td>Schindelin et al. <sup>93</sup></td><td>https://fiji.sc/</td></tr><tr><td>Metamorph</td><td>Molecular Devices</td><td>https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy</td></tr><tr><td>ImageJ plugin MTrack J</td><td>Meijering et al. <sup>90</sup></td><td>https://imagescience.org/meijering/software/mtrackj/</td></tr><tr><td>Matlab</td><td>MathWorks</td><td>mathworks.com/products/matlab.html</td></tr><tr><td>Meshlab</td><td>Visual Computing Lab</td><td>www.meshlab.net</td></tr><tr><td>Prism 9</td><td>GraphPad</td><td>www.graphpad.com/features</td></tr><tr><td>2D models for spindle orientation</td><td>Bosveld et al. <sup>51</sup>, Minc et al. <sup>49</sup></td><td>N.A.</td></tr><tr><td>3D models for spindle orientation</td><td>Ershov and Minc <sup>92</sup>, Pierre et al. <sup>25</sup></td><td>N.A.</td></tr><tr><td>GenerateMesh.py code</td><td>This paper</td><td>https://zenodo.org/badge/latestdoi/623985418</td></tr><tr><td>GetJunctions.py code</td><td>This paper</td><td>https://zenodo.org/badge/latestdoi/623985418</td></tr></table>
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Saleh et al., Figure 5
# SUPPLEMENTAL INFORMATION

## Length-limitation of astral microtubules orients cell divisions in intestinal crypts

Saleh Jad <fnref n="1" />, Fardin Marc-Antoine <fnref n="1" />&, Barai Amlan <fnref n="1" />&, Soleilhac Matis <fnref n="1" />, Frenoy Olivia <fnref n="1" />, Gaston Cécile <fnref n="1" />, Cui Hongyue <fnref n="1" />, Dang Tien <fnref n="1" />, Noémie Gaudin <fnref n="1" />, Vincent Audrey <fnref n="2" />,<fnref n="3" />, Minc Nicolas <fnref n="1" />,<fnref n="4" />* and Delacour Delphine <fnref n="1" />*

## Supplementary Figures

<footnote n="1">¹, Université Paris Cité, CNRS, Institut Jacques Monod, F-75013 Paris, France</footnote>

<footnote n="2">² CANTHER Cancer Heterogeneity Plasticity and Resistance to Therapies, Université de Lille, CNRS, INSERM, CHU Lille, UMR9020-U1277, F-59000 Lille, France</footnote>

<footnote n="3">³ ORGALille Core Facility, CANTHER, Université de Lille, CNRS, INSERM, CHU Lille, UMR9020-U1277, F-59000 Lille, France</footnote>

<footnote n="4">⁴ Equipe Labellisée La Ligue Contre le Cancer</footnote>
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**Figure S1.** Cell rearrangements and mitotic cell shape changes during mitosis in organoid crypt-like structures, Related to Figure 1. a Confocal analysis of α-tubulin (green) and EpCAM (magenta) in interphase, prophase, metaphase or anaphase cells in intestinal organoids. Nuclei are stained in blue. Orange asterisk pinpoints the cells of interest. L, lumen. Scale bars, 5 µm. b Representative 3D rendering of dividing cells (magenta) and neighboring cells (grey) after segmentation of cell membranes based on confocal z-stacks in crypt organoids. Are shown apico-basal, apical or basal views. Spatial coordinates are shown. c Confocal analysis of myosin-IIA-KI-GFP (green) and E-cadherin (magenta) in a metaphase cell. L, lumen. D, dividing cell. N, neighboring cell. Scale bar, 5µm. d Analysis of the percentage of total signal intensity for myosin-IIA-KI-GFP at the apical, lateral or basal cortex during mitotic steps. n = 10 cells for each mitotic step. e Analysis of the percentage of total signal intensity of P-MLC2 in the apical, lateral or basal cortex of prophase, metaphase, anaphase and telophase cells. n = 16 prophase cells, 14 metaphase cells, 11 anaphase cells, and 11 telophase cells. f Confocal analysis of P-MLC2 (magenta) and E-cadherin (green) distribution in prophase and prometaphase / metaphase cells in intestinal organoids. Scale bars, 10µm; scale bars in the inserts, 5µm. g Representative 3D rendering of the metaphase cell of interest (magenta) and neighboring cells (grey) after segmentation of cell membranes based on a representative confocal z-stack in intestinal organoids. 3-cell (cyan), 4-cell (red) and 5-cell (blue) contacts are depicted on the tilted, lateral and basal views. h Color-coded t-projection of time-lapse series of α-tubulin-GFP signal at a spindle pole in prometaphase or metaphase in control organoids. The first image (t0) correspond to onset of the specific mitotic phase and is colored magenta. The last image (t) corresponds to the end of this mitotic phase and is colored in green. Spindle pole track on the whole duration of the mitotic phase is shown in white. Scale bar, 2µm. i Statistical analysis of the amplitude of spindle pole oscillations in prometaphase or metaphase in control organoids. The mean amplitude in prometaphase (n = 20 cells) is 2.551±0.124 µm along the x axis (mean±S.E.M), and 2.449±0.199 µm along the y axis. In metaphase (n = 30 cells), the amplitude along the x axis is 1.021±0.040 µm, and 1.189±0.048 µm along the y axis. One-way ANOVA test and Tukey's multiple comparison test, ****p<0.0001.
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**Figure S2.** Spindle positioning and localization of polarity cues in metaphase cells in mouse jejunum and intestinal organoids, Related to Figures 1 and 2. **a-b** Confocal analysis of E-cadherin (magenta) localization in mouse intestinal crypts. Nuclei are stained in blue. Yellow dotted lines in **a** delimit crypt compartments. Yellow arrow shows a metaphase cell. Yellow line in **b** surrounds a metaphase cell. Scale bar, 10µm. **c** Statistical analysis of the aspect ratio (distance between the spindle axis and the basal membrane over the distance between the spindle axis and the apical membrane) (n = 10 cells). Aspect ratio (in vitro) = 1.378±0.05 (mean±S.E.M), aspect ratio (in vivo) = 1.553±0.057. Unpaired t-test, \*p = 0.03. **d** Statistical analysis of the metaphase plate positioning (distance between the middle of the metaphase plate and the apical membrane) (n = 10 cells). Metaphase plate positioning (in vitro) = 0.411±0.019 (mean±S.E.M), (in vivo) = 0.451±0.015. Unpaired t-test, ns, non-significant. **e** Confocal analysis of the distribution of endogenous NuMA (green) during a mitotic cycle in intestinal organoids. Nuclei are stained in blue. Yellow arrow points to NuMA cortical distribution. L, lumen. Scale bar, 15µm. **f** Confocal analysis of the distribution of endogenous NuMA *in vivo* in mouse jejunum crypts. Crypt tissue is delimited by a yellow dotted line. Nuclei are stained in blue. Yellow arrow points to NuMA cortical distribution. L, lumen. Scale bar, 20µm. **g** Confocal analysis of afadin together with E-cadherin in organoid metaphase cells. Afadin signal intensity is color-coded with Fire LUT table from ImageJ on the right panel. Color scale bar indicates the gray value intensity. Yellow arrow points to basal accumulation of afadin. Scale bar, 5 µm. **h** Statistical analysis of afadin signal intensity at the apical, lateral or basal cortex. Afadin signal intensity at the apical cortex = 16.61±1.43 % (mean±S.E.M), lateral cortex = 22.39±1.31 %, basal cortex = 51.06±1.39 %. n = 30 cells. One-way ANOVA test and Tukey's multiple comparison test, \*p = 0.02, ****p <0.0001. **i** Confocal analysis of dynein together with E-cadherin in organoid metaphase cells. Dynein signal intensity is color-coded with Fire LUT table from ImageJ on the right panel. Color scale bar indicates the gray value intensity. Scale bar, 5 µm. **j** Statistical analysis of dynein signal intensity at the apical, lateral or basal cortex. Dynein signal intensity at the apical cortex = 36.56±1.1 % (mean±S.E.M), lateral cortex = 33.19±0.63 %, basal cortex = 30.26±1.02 %. n = 30 cells. Two-way ANOVA test and Tukey's multiple comparison test, \*p = 0.03, ****p <0.0001, ns non-significant. **k** Confocal analysis of E-cadherin in organoid metaphase cells. E-cadherin signal intensity is color-coded with Fire LUT table from ImageJ on the right panel. Color scale bar indicates the gray value intensity. Yellow arrow points to basal accumulation of E-cadherin. Scale bar, 5 µm. **l** Statistical analysis of E-cadherin signal intensity at the apical, lateral or basal cortex. E-cadherin signal intensity at the apical cortex = 23.97±0.7 % (mean±S.E.M), lateral cortex = 32.87±0.43 %, basal cortex = 43.16±0.94 %. n = 30 cells. Two-way ANOVA test and Tukey's multiple comparison test, ****p <0.0001. **m** Confocal analysis of Membranes-tdTomato in organoid metaphase cells. tdTomato signal intensity is color-coded with Fire LUT table from ImageJ on the right panel. Color scale bar indicates the gray value intensity. Scale bar, 5 µm. **n** Statistical analysis of tdTomato signal intensity at the apical, lateral or basal cortex. TdTomato signal intensity at the apical cortex = 33.84±0.85 % (mean±S.E.M), lateral cortex = 32.76±0.54 %, basal cortex = 32.61±0.83 %. n = 30 cells. Two-way ANOVA test and Tukey's multiple comparison test, ns non-significant. For each experiment, three independent experiments were carried out.
a

Aster extension

<figure><img src="image_67.png" /></figure>

φ = 45°

φ = 90°

φ = 135°

<figure><img src="image_68.png" /></figure>

b

MT number

<figure><img src="image_69.png" /></figure>

n=11

n=51

n=91

<figure><img src="image_70.png" /></figure>

c

Spindle size, s (relative to cell radius)

<figure><img src="image_71.png" /></figure>

s=0.4

s=0.8

s=1.2

<figure><img src="image_72.png" /></figure>

d

Spindle offset along A-B axis (% of cell radius)

<figure><img src="image_73.png" /></figure>

Offset=0%

Offset=30%

Offset=60%

<figure><img src="image_74.png" /></figure>

e

Metaphase

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>

f

Interphase

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /></figure>

Prophase

<figure><img src="image_81.png" /></figure>

<figure><img src="image_82.png" /></figure>

<figure><img src="image_83.png" /></figure>

Metaphase

<figure><img src="image_84.png" /></figure>

<figure><img src="image_85.png" /></figure>

<figure><img src="image_86.png" /></figure>

Anaphase

<figure><img src="image_87.png" /></figure>

<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>

Cytokinesis

<figure><img src="image_90.png" /></figure>

<figure><img src="image_91.png" /></figure>

<figure><img src="image_92.png" /></figure>

g

Lateral side

<figure><img src="image_93.png" /></figure>

h

Basal side

<figure><img src="image_94.png" /></figure>

i

j

<figure><img src="image_95.png" /></figure>

k

<figure><img src="image_96.png" /></figure>

Control : no ablation

<figure><img src="image_97.png" /></figure>

<figure><img src="image_98.png" /></figure>

Apical laser ablation

<figure><img src="image_99.png" /></figure>

<figure><img src="image_100.png" /></figure>

Basal laser ablation

<figure><img src="image_101.png" /></figure>

n

<figure><img src="image_102.png" /></figure>

o

<figure><img src="image_103.png" /></figure>

p

<figure><img src="image_104.png" /></figure>

q

<figure><img src="image_105.png" /></figure>

r

<figure><img src="image_106.png" /></figure>

t

<figure><img src="image_107.png" /></figure>

Saleh et al., Figure S3

<figure><img src="image_108.png" /></figure>

<figure><img src="image_109.png" /></figure>

<figure><img src="image_110.png" /></figure>
**Figure S3.** Astral MT participation in spindle positioning in intestinal organoids, Related to Figures 2 and 3. **a** Evolution of the predicted preferred spindle orientation with respect to the tissue plane as a function of aster extension parameter Φ, in the 2D model. **b** Predicted preferred spindle orientation with respect to the tissue plane as a function of MT numbers per aster, *n*, in the 2D model. **c** Predicted preferred spindle orientation with respect to the tissue plane as a function of spindle size related to cell radii, *s*, in the 2D model. **d** Predicted preferred spindle orientation with respect to the tissue plane as a function of spindle positional offset along the A-B axis in the 2D model. **e-h** Expansion microscopy and confocal analysis of α-tubulin (green) and E-cadherin (magenta) in organoid metaphase cells. Nuclei (blue) are labeled with Hoechst 33342. Apical (f), lateral (g) and basal (h) cortical domains are presented. Nuclei (blue) are labeled with Hoechst 33342. Corrected scale bar, 5µm. **i** Time-lapse imaging of astral MTs during metaphase in EB3-RFP organoids. Green arrowheads point on astral MTs contacting the apical and lateral cortex, black arrowheads on astral MTs contacting the basal cortex. Metaphase cell shape is delimited by a yellow dotted line. Scale bar, 10µm. **j** Representative spinning disk image of EB3-RFP signal in organoid metaphase cells before and after MT laser ablation. The laser cut line on basal astral MTs are shown in yellow dotted lines. Scale bar, 5µm. **k** Left, representative spinning disk image of α-tubulin-GFP signal in organoid metaphase cells. Positions of apical or basal laser cut are shown in yellow dotted lines. Scale bar, 5µm. **Right**, kymograph analysis of the α-tubulin-GFP signal at the spindle poles in control (no laser ablation), or after apical or basal laser cut in organoid metaphase cells. Yellow vertical line indicates the time of the laser cut. Scale bar, 5µm. **l** Confocal analysis of Kif18B signal (green) and DNA (blue) labeled with Hoechst33342 in metaphase Kif18B-KO organoids. Scale bar, 10µm. **m** Confocal analysis of Kif18B signal (green), membranes-tdTomato (magenta) and DNA (blue) during interphase and cell division in control organoids. Scale bar, 10µm. **n** Statistical analysis of Kif18B signal intensity at the apical, lateral or basal cortex during metaphase. Mean Kif18B signal intensity at the apical cortex = 24.77±0.91 % (mean±S.E.M), lateral cortex = 31.41±0.46 %, basal cortex = 44.08±1.14 %. *n* = 28 cells. One-way ANOVA test and Tukey's multiple comparison test, ****p <0.0001. **o** Western blot analysis of Kif18B expression in control-KO or Kif18B-KO organoids 3-days after induction. GAPDH was used as a loading control. **p** Statistical analysis of Kif18B expression in control-KO or Kif18B-KO organoids 3-days after induction. Relative expression of Kif18B in Kif18B-KO organoids = 0.479±0.021 (mean±S.E.M). Unpaired t-test, ****p <0.0001. **q** Confocal analysis of α-tubulin signal in metaphase Kif18B-KO organoids following a treatment with DMSO or a low dose of nocodazole. Scale bar, 5µm. **r** Statistical analysis of the spindle orientation angle relative to the planar axis of the epithelium in metaphase Kif18B-KO organoids cells treated with DMSO (*n* = 93 cells) or a low dose of nocodazole (*n* = 71 cells). Unpaired t-test, ****p <0.0001. **s** Confocal analysis of NuMA signal (green) in metaphase Kif18B-KO organoids. DNA (blue) is labeled with Hoechst 33342. Scale bar, 10µm. **t** Statistical analysis of NuMA signal intensity at the apical, lateral or basal cortex in metaphase Kif18B-KO organoids. Mean NuMA signal intensity at the apical cortex = 31.9±0.54 % (mean±S.E.M), lateral cortex = 34.28±0.35 %, basal cortex = 33.57±0.62 % (*n* = 30 cells). One-way ANOVA test and Tukey's multiple comparison test, **p**=0.005, *ns* non-significant. For each experiment, three independent experiments were carried out.
<figure><img src="image_111.png" /></figure>
**Figure S4.** Impact of the metaphase cell shape in spindle orientation in intestinal organoids, Related to Figure 4. a Confocal analysis of EpCAM (magenta) and NuMA (green) distribution in metaphase cells in cystic organoids after L-WNR culture and CHIR treatment. Nuclei are shown in blue. Yellow arrows point to a metaphase cell. Scale bar, 10µm. b Western blot analysis of myosin-IIA expression in control-KO or myosin-IIA-KO organoids 3-days after induction. GAPDH was used as a loading control. c Statistical analysis of myosin-IIA expression in control-KO or myosin-IIA-KO organoids 3-days after induction. Relative expression of myosin-IIA in myosin-IIA-KO organoids = 0.317±0.077 (mean±S.E.M). Five independent experiments were carried out. Unpaired t-test, ****p <0.0001. d Representative confocal image of E-cadherin (red) and nuclei (blue) localization in a crypt organoid after blebbistatin treatment. White lines delimit the cells to be segmented. Yellow arrow shows the metaphase cell of interest. L, lumen. Scale bar, 20 µm. e Representative 3D rendering of the metaphase cell of interest shown in d (magenta) and neighboring cells (grey) after segmentation of cell membranes based on the representative confocal z-stack in blebbistatin-treated organoids. 3-cell (cyan), 4-cell (red) and 5-cell (blue) contacts are depicted on the lateral and basal views. Spatial coordinates are shown. f Quantitative analysis of the proportion of 3-, 4- and 5-cell contacts in control (DMSO-treated) or blebbistatin-treated organoids. g Confocal analysis of E-cadherin distribution in metaphase cells in DMSO- or blebbistatin-treated organoids. E-cadherin intensity map was generated with Physics LUT table from ImageJ. Color scale bar indicates the gray value intensity. Yellow arrow points to metaphase cell. Scale bar, 5µm. h Statistical analysis of E-cadherin distribution in metaphase cells of DMSO- or blebbistatin-treated organoids. n = 10 cells for each experimental condition. Two-way ANOVA and multiple comparisons. ****p <0.0001. i Confocal analysis of NuMA distribution in metaphase cells in DMSO- or blebbistatin-treated organoids. NuMA intensity map was generated with Physics LUT table from ImageJ. Color scale bar indicates the gray value intensity. Metaphase cell is delimited with a yellow line. Yellow arrowheads point to an increase of NuMA signal at the basal cortex. Scale bar, 5µm. j Statistical analysis of NuMA distribution in metaphase cells of DMSO- or blebbistatin-treated organoids. n = 10 cells for each experimental condition. Two-way ANOVA and multiple comparisons. ****p <0.0001. ns, non-significant.
a

<figure><img src="image_112.png" /></figure>

Initial spindle at
basal pole

<figure><img src="image_113.png" /></figure>

Initial spindle at
apical pole

<figure><img src="image_114.png" /></figure>

b

Simulation history

<figure><img src="image_115.png" /></figure>

c

<figure><img src="image_116.png" /></figure>

d

<figure><img src="image_117.png" /></figure>

Planar View 1

<figure><img src="image_118.png" /></figure>

Planar View 2

<figure><img src="image_119.png" /></figure>

Apical-Basal

<figure><img src="image_120.png" /></figure>

<figure><img src="image_121.png" /></figure>

3D Shape
Model
prediction

<figure><img src="image_122.png" /></figure>

<figure><img src="image_123.png" /></figure>

<figure><img src="image_124.png" /></figure>

3D NuMa
Model
prediction

<figure><img src="image_125.png" /></figure>

<figure><img src="image_126.png" /></figure>

<figure><img src="image_127.png" /></figure>

e

<figure><img src="image_128.png" /></figure>

f

<figure><img src="image_129.png" /></figure>

g

<figure><img src="image_130.png" /></figure>

h

Control organoids

<figure><img src="image_131.png" /></figure>

i

Blebbistatin-treated organoids

<figure><img src="image_132.png" /></figure>

j

Kif18B-KO organoids

<figure><img src="image_133.png" /></figure>

k

<figure><img src="image_134.png" /></figure>
**Figure S5. 3D simulation of spindle positioning in organoids, Related to Figure 4 and Discussion.**

**a.** 3D simulation for the same cell as presented in Figure 4e, but with initial positions of spindles close to the basal (left) or apical pole (right). The red trace shows the history of the spindle center during the simulations. **b.** Corresponding traces of spindle position (x,y,z) and orientations (θ and Φ) during the gradient descent simulation. Note how the traces become flat at the end of simulations indicating that an equilibrium has been found. **c.** Spindle torque color maps plotted as a function of all possible orthoradial and azimuthal angles, highlighting the identified global minimum and the experimental spindle orientation in 3D. **d.** Experimental and model predicted spindle orientation for the same cell as in Figure 4e, using models based on the full cell shape or based on NuMA polarity at the basal pole. **e.** Predicted spindle orientation angle with respect to the A-B axis, plotted as a function of the experimental axis for 10 individual control, blebbistatin-treated or L-WNR treated crypt cells, for a 3D model based on cell shape. Note the poor agreement for control cells. **f.** Predicted spindle orientation angle with respect to the A-B axis, plotted as a function of the experimental axis for 10 individual control, and L-WNR treated crypt cells, for a 3D model based on cell polarity. Blebbistatin treated cells are absent from this analysis as they fail to properly assemble a basal NuMA domain. Note the poor agreement for both control and L-WNR cells. **g.** Experimental and theoretical prediction of spindle asymmetric position towards the apical cell poles in experiments vs models based on cell shape or NuMA polarity domains. Expt, experimental. **h** Time-lapse sequence of α-tubulin-GFP in control organoids at the onset of mitosis. Green arrowheads point to centrosomes migrating from the apical domain. Scale bar, 5µm. **i** Time-lapse sequence of α-tubulin-GFP at the onset of mitosis in blebbistatin-treated organoids. Green arrowheads point to centrosomes migrating from the apical domain. Scale bar, 5µm. **j** Time-lapse sequence of Spy650-Tubulin at the onset of mitosis in Kif18B-KO organoids. Green arrowheads point to centrosomes migrating from the apical domain. Scale bar, 5µm. **k** Statistical analysis of the initial centrosome relative position to the apical surface at the onset of mitosis. n (control) = 14 cells, n (blebbistatin-treated organoids) = 11 cells, n (Kif18B-KO organoids) = 14 cells. One-way ANOVA test and Tukey's multiple comparison test, ns non-significant.
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