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Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants

Nature Cell Biology volume 24, pages 1606–1616 (2022)Cite this article

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An Author Correction to this article was published on 16 January 2023

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Abstract

Asymmetric cell division gives rise to two daughter cells that inherit different determinants, thereby acquiring different fates. Polarized trafficking of endosomes containing fate determinants recently emerged as an evolutionarily conserved feature of asymmetric cell division to enhance the robustness of asymmetric cell fate determination in flies, fish and mammals. In particular, polarized sorting of signalling endosomes by an asymmetric central spindle contributes to asymmetric cell division in Drosophila melanogaster. However, how central spindle asymmetry arises remains elusive. Here we identify a moonlighting function of the Elongator complex—an established protein acetylase and tRNA methylase involved in the fidelity of protein translation—as a key factor for central spindle asymmetry. Elongator controls spindle asymmetry by stabilizing microtubules differentially on the anterior side of the central spindle. Accordingly, lowering the activity of Elongator on the anterior side using nanobodies mistargets endosomes to the wrong cell. Molecularly, Elongator regulates microtubule dynamics independently of its acetylation and methylation enzymatic activities. Instead, Elongator directly binds to microtubules and increases their polymerization speed while decreasing their catastrophe frequency. Our data establish a non-canonical role of Elongator at the core of cytoskeleton polarity and asymmetric signalling.

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Fig. 1: Elongator controls central spindle asymmetry.
Fig. 2: Elongator controls asymmetric Sara endosome segregation and thereby contributes to Notch signalling.
Fig. 3: Elongator is recruited to the central spindle via its direct interaction with microtubules.
Fig. 4: The enzymatic activity of Elp3 is not required for the effects of Elongator on the central spindle.
Fig. 5: Elongator directly controls microtubule dynamics.
Fig. 6: Asymmetric control of microtubule dynamics by Elongator governs the asymmetric trafficking of Sara endosomes.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE72 partner repository with the dataset identifier PXD036556. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Code availability

Custom image processing code specific to this paper have been deposited on our GitHub page in a dedicated repository (https://github.com/deriverylab/2022_Planelles-Herrero). Similarly, our general image registration and wavelet filtering codes can also be found on our GitHub page (https://github.com/deriverylab/GPU_registration and https://github.com/deriverylab/GPU_wavelet_a_trous). Safir denoising code can be found on the Inria website (https://team.inria.fr/serpico/software/nd-safir). All lookup tables applied to images in this paper come from the collection from J. Manton (https://github.com/jdmanton/ImageJ_LUTs).

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Acknowledgements

This work was supported by the Medical Research Council, as part of United Kingdom Research and Innovation (UK Research and Innovation; grant no. MC_UP_1201/13 to E.D.), the Human Frontier Science Program (Career Development Award grant no. CDA00034/2017 to E.D.) as well as by grants from the SNSF, the ERC (Sara and Morphogen), the NCCR Chemical Biology program, the DIP of the Canton of Geneva and the SystemsX EpiPhysX (SNSF) granted to M.G.G. V.J.P.-H. is supported by an EMBO postdoctoral fellowship. We thank the electronics workshop of the LMB, in particular M. Kyte for his help with the Field Programmable Gate Array hardware required for precise synchronization of our microscope. We also thank the LMB Biophysics and Mass Spectrometry facilities, in particular M. Skehel and F. Begum for performing the mass spectrometry and analysing the mass spectrometry data. We thank I. Diaz (Venki Ramakrishnan laboratory) for sharing tRNA. We thank T. Stevens and J. O’Neill for expert advice on statistics, and F. Karch and P. Verstreken for flies. We thank S. Loubéry for their help with microscopy. We thank the members of the Derivery and Gonzalez-Gaitan laboratories, as well as C. Aumeier, B. Baum, S. Bullock and K. Kruse for critical reading of the manuscript.

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  1. These authors contributed equally: Vicente Jose Planelles-Herrero, Emmanuel Derivery.

Authors and Affiliations

  1. Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge, UK

    Vicente Jose Planelles-Herrero, Alice Bittleston & Emmanuel Derivery

  2. Department of Biochemistry, Faculty of Sciences, University of Geneva, Geneva, Switzerland

    Carole Seum, Alicia Daeden, Marcos Gonzalez Gaitan & Emmanuel Derivery

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  1. Vicente Jose Planelles-Herrero
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Contributions

V.J.P.-H. conducted all biochemical experiments as well as all microtubule assays and associated analysis. E.D. and A.B. conducted most fly imaging experiments and associated quantification. A.D. performed additional Elp4 immunostaining and respective imaging. E.D. wrote the codes for automated image processing. C.S. generated the Klp98A–GFP knock-in and conducted genetic interaction experiments. V.J.P.-H., M.G.G. and E.D. designed the project. All authors contributed to writing the paper.

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Correspondence to Marcos Gonzalez Gaitan or Emmanuel Derivery.

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Extended data

Extended Data Fig. 1 Conventions and rationale of the approach that identified Elongator.

(a) Left panel: conventions. We consider a reference frame centred on the central spindle with the y axis parallel to the division plane and the x axis orthogonal to it. The width is defined along the y axis, and the length along the x axis. During division, since the actin ring contracts, the width of the central spindle constricts over time. This constriction is stereotypic between cells and thus can be used to register movies in time a posteriori (methods). Right panel: Average temporal contraction profile of the central spindle length quantified from the Jupiter signal, mean ± SEM (shaded area). Registered time=0 indicates anaphase B onset. n: number of cells. (b) Candidate-based approach that identified Elongator as a key player of central spindle asymmetry. We took the list of all the genes identified by Buffin and Gho13 as being overexpressed in SOP cells, considering only genes with a enrichment of at least a factor two compared to neighbouring cells (green disk, n = 128 genes). This list was cross-referenced with the literature to find proteins within this list that were shown to regulate microtubule dynamics (in flies or other systems, red). We found only one gene matching both criteria, Elp3. Indeed, Elp3 is 2.26-fold overexpressed in SOPs, and Elongator has been suggested to play a role in microtubule dynamics in other systems/species14,15,16, albeit the molecular mechanisms involved was unclear. Note that there are numerous proteins in the Drosophila proteome that affect microtubule dynamics (for example Msps, CLASP, Patronin, Klp10A…), but these were not found specifically overexpressed in SOPs by Buffin and Gho and thus were not further tested here. Conversely, there are numerous proteins that were found specifically overexpressed in SOPs by Buffin and colleagues and that will have an indirect role onto central spindle asymmetry because they control the establishment of the polarity in these cells, which is upstream of central spindle asymmetry (for instance, meru73). But these proteins are not known to directly affect microtubule dynamics and thus were not further tested here. Source numerical data are available in source data.

Source data

Extended Data Fig. 2 Characterization of the antibodies, constructs and RNAi sequenced used in this paper.

(a-b) Characterization of the Elp3 antibody, the Elp3 RNAi sequence and the RNAi-resistant Elp3* construct used in this study. (a) S2 cells stably expressing His-PC-Elp3* (abbreviated HP-Elp3*), or wild-type control (S2) were treated with indicated RNAi before extracts and analysed by Western Blot (WB) against Elp3 (top panels) or acetylated tubulin (bottom panel). His-PC stands for the (His)6 and PC purification tags, respectively. Two different exposures of the western blot are shown to reflect the differences of loadings. Note the band disappearing in the Elp3 WB upon Elp3 RNAi treatment (orange arrowhead). In contrast, the band corresponding to His-PC-Elp3*, shifted upwards because of the PC tag, is not affected by the RNAi treatment. (b) RNAi-treated S2 extracts were subjected to immunoprecipitation using Elp2 antibodies. Note that the band disappearing upon RNAi Elp3 treatment (orange arrowhead) is pulled down by Elp2 antibodies. Altogether, these results demonstrate the specificity of the Elp3 antibody, the Elp3 RNAi sequence and the resistance of the Elp3* construct to the RNAi treatment. n.s.: non-specific band. (c-e) Characterization of the Elp2 (C), Elp4 (D) and Elp5(E) antibodies used in this study. S2 cells were treated with indicated RNAi before extracts were analysed by WB against Elp2 (top panel in c), or Elp4 (top panel in d). Ponceau staining is provided as a loading control (bottom panels in c and d). Note the major band disappearing in the Elp2 (respectively Elp4) WB upon Elp2 (respectively Elp4) RNAi treatment. Similarly, lysates from an identical number of S2 cells stably expressing His-PC-Elp3 and of wild-type control (S2) were processed for immunoprecipitation using anti-PC antibodies followed by Western Blot against PC or endogenous Elp5. Elp5 is specifically enriched upon immunoprecipitation of PC-Elp3, suggesting that it recognizes endogenous Elp5 in enriched fractions. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 Several Elongator subunits are needed for its activity on central spindle asymmetry and Sara endosome segregation.

(a) Dividing SOP showing Jupiter–GFP (top row) or Jupiter–GFP with Rainbow RGB LUT (bottom row). Image corresponds to maximum intensity z-projection of the central spindle (SDCM). (b) Enrichment in pIIb. Thick line: median; thin line: quartile; n: number of central spindles analysed. Statistics: Kruskal–Wallis non-parametric ANOVA1 test. P-values of the respective post-hoc tests are indicated. Control and Elp3RNAi datasets are the same as those presented in Fig. 1d, shown here for convenience. (c-d) Elp3 Δ4/Δ4 MARCM clones. (c) Dividing SOP showing Sara endosomes (iDelta20 and mRFP-Pon in the indicated genotype (pIIa cortex; dashed blue). Time in seconds. Note that Sara endosomes are symmetrical in Elp3 mutant (arrowheads). The Elp3Δ4 mutant is a null allele of Elp3 that lacks most of the protein, including the SAM and KAT domains. (d) Quantification of the effects in (c): iDelta20 percentage in pIIa after abscission in SOPs of indicated genotypes (mean ± SEM; Statistics in were performed using a two-sided Welch test (p value indicated); n: SOPs analysed. (e) Dividing SOP showing Sara endosomes (iDelta20) and mRFP-Pon (pIIa cortex; dashed blue). Note the endosomes in the pIIb daughter cell upon Elp1, Elp2 and Elp3 depletion (arrows). (f) Percentage of iDelta20 in pIIa after abscission in SOPs (mean ± SEM; Statistics: one-way ANOVA (P < 0.0001), followed by a Tukey post-hoc test (p value indicated); n: SOPs analysed. The control and Elp3RNAi datasets are the same as those presented in Fig. 2b, shown here for convenience. As expected for stable multiprotein complexes, depletion of the Elongator subunits Elp1, 2 and 3 lead to the same phenotype. However, depletion of Hsc70-4 does not, suggesting that Elongator effect on microtubule dynamics does not require Hsc70-4. Scale bars: 2 µm (a); 5 µm (c,e). Images in this figure are Maximum intensity z-projection of entire cells (SDCM). Source numerical data are available in source data.

Source data

Extended Data Fig. 4 Elp3 depletion induces a short bristle phenotype.

(a) Scanning Electron Microscopy of the notum of representative control, Elp3 RNAi and Elp3 RNAi + Elp3* flies. Dashed lines indicate panier expression region. (b) Microchaete length in the panier region (Mean ± SEM). Statistics: Kruskal–Wallis test (p-value as indicated; n: microchaete number). Note the smaller bristles in the panier region in the Elp3 RNAi genotype. This is rescued by expression of a RNAi-resistant wild-type version of Elp3 (Elp3*). (c) Dividing SOP showing mRFP-Pon after immunolabelling with antibodies against endogenous Elp2 and α-tubulin. Images corresponds to single confocal plane (top panels) or maximum intensity z-projection of two planes (point scanning confocal microscopy). For mRFP-Pon: safir denoising filter (see methods). Top panel displays cell in metaphase and bottom panel a cell in early anaphase. Note that the cell on the bottom panel is at a too early stage of anaphase to display central spindle asymmetry, which gradually builds up during anaphase B (see Fig. 1a). (d,e) Fly nota were stained for tubulin and endogenous Elp2 (d) or Elp4 (e) to image epithelial cells (that is, non-SOP cells). Images corresponds to a maximum intensity z-projection (point scanning confocal microscopy). Both Elp2 and Elp4 antibodies label the mitotic spindle (orange arrow) and central spindle (blue arrow) in epithelial cells. Scale bar: 100 µm (a) and 5 µm (c-e). Source numerical data are available in source data.

Source data

Extended Data Fig. 5 Purification and characterization of the Drosophila Elongator complex.

(a) Stable S2 cell lines expressing His-PC-SNAP tagged version of each of the six Elongator subunits (or a His-PC-SNAP empty vector) were established. The endogenous complex assembling around tagged subunits was then analysed by quantitative Mass Spectrometry. Purification via the Elp3 or Elp4 subunit are the most efficient. (b) Left panel: western blot evaluation of an analytical sucrose gradient (10-30%) after a Protein C affinity step. The full complex migrates as a single unit in the 20% sucrose fractions. Right panel: coomassie-stained gradient SDS–PAGE gel of Elongator complex in the indicated fraction (fraction kept for in vitro analyses). (c) Left panel: chromatogram of the size exclusion (superose 6 increase) step of the purification of His-PC-tagged Elp3 Elongator complex. Right panel: coomassie-stained gradient SDS–PAGE analysis of the main peak of the size exclusion column (fraction used in in vitro analyses). The right panel is identical to Fig. 3d, shown here for convenience. (d) Identification of the subunits of the Elongator complex purified from His-PC-Elp3 by MS. This experiment also demonstrates that the Drosophila homologues of Elp5 and Elp6 are indeed anon-i1 and poly, respectively, previously predicted based on sequence analysis57,58. (e) Coomassie-stained gradient SDS–PAGE gel of Elongator complex purified using a His-PC-SNAP-tagged Elp3 subunit WT or Y531A starting from the same amounts of cells. The Y531A mutation does not affect the assembly of the complex. (f,g) Biochemical characterization of the Drosophila Elongator complex purified in b. (f) Affinity of the direct interaction between Elongator and tRNALys measured by MST (see methods). (g) tRNA modification activity of Elongator (mean ± SD, see methods). Drosophila Elongator behaves similarly to yeast Elongator. n = 3 independent experiments. Statistics: one-way ANOVA followed by a Tukey multiple comparison test (p value indicated). Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 6 Elongator subcomplexes do not bind stabilized microtubules.

(a) Coomassie-stained SDS–PAGE gradient gel of a partially purified Elp23 subcomplex (note the absence of Elp1), which copurifies with Hsc70-4. This is a by-product of the sucrose-gradient purification of the Full Elongator complex in the His-PC-SNAP–Elp3 stable cell line (Extended Data Fig. 5b). PC: protein C tag. (b-c) The affinity of the Elp23 + Hsc70-4 subcomplex for microtubules is less than that than of the full complex. (b) The subcomplex shown in (a) was fluorescently labelled with Alexa 488-BG, and the resulting fluorescent complex was incubated with Rhodamine-labelled, GMPCPP-stabilized microtubules (12.5 µM tubulin, 100 nM complex) and imaged by TIRFM. Alternatively, binding to microtubules was assessed by microtubule pelleting assays as in Fig. 3e and compared to the full complex (c). (d) Coomassie-stained SDS–PAGE gradient gel of a purified Elp456 subcomplex tagged with mScarlet on Elp4 and produced in E. coli. (e-f) The Elp456 subcomplex does not bind to microtubules. (e) Microtubule binding of the subcomplex shown in (d) was assessed as in (c) using 1 µM mScarlet–Elp456 subcomplex. The Elp456 subcomplex does not bind quantitatively to microtubules. Note that our purification of the mScarlet–Elp456 subcomplex contains some residual uncleaved GST-mScarlet-Elp4, but that since no mScarlet signal is observed on microtubules, this implies that a potentially dimeric GST-tagged mScarlet–Elp456 also does not bind to microtubules. Alternatively, binding to microtubules was assessed by microtubule pelleting assays and analysed using coomassie staining (f). Altogether these results presented in this figure suggest that the quantitative binding to Microtubules shown in Figs. 3,5 requires the full Elongator complex. Note that this is confirmed by analysis of microtubule dynamics (Fig. 5h,i). Scale bars: 5 μm. Unprocessed blots are available in source data.

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Extended Data Fig. 7 Perturbation of tubulin acetylation by AtatRNAi does not affect central spindle asymmetry and symmetric endosome segregation.

(a-b) Validation of the AtatRNAi construct. (a) Notum of a fly expressing AtatRNAi under the control of the Pnr promoter after immunolabeling using Oregon Green-514 anti-β-tubulin and Atto 647N anti-K40 acetylated-α-tubulin antibodies (SDCM). White line: Pnr expression domain limit. Image corresponds to maximum intensity z-projection of epithelium by SDCM. (b) Quantification of the normalized K40 acetyl-α-tubulin/β-tubulin ratio at the central spindle in conditions shown in a (see methods). Mean±SEM. Statistics: two-sided Mann–Whitney test (p-value<0.0001; n: central spindle number). AtatRNAi leads to a marked decrease of K40-α-tubulin acetylation. (c) Dividing SOP of indicated genotype showing Jupiter–GFP (top row) or Jupiter–GFP with Rainbow RGB LUT applied (bottom row). Anterior/pIIb orientation as determined by the mRFP-Pon signal. Image corresponds to maximum intensity z-projection of the central spindle (SDCM). (d) Quantification of the effects seen in c: pIIb enrichment (see methods) in indicated genotypes. Median±95% confidence interval; n: number of central spindles analysed. Statistics: two-sided Student t-test. The control dataset is the same as the one presented in Fig. 1d, shown here for convenience. Central spindle asymmetry is not affected upon Atat depletion. (e) Dividing SOP showing Sara endosomes (iDelta20) and mRFP-Pon in the indicated genotype (pIIa cortex; dashed blue). Image corresponds to Maximum intensity z-projection of entire cells (SDCM). (f) Quantification of the effects seen in (e): iDelta20 percentage in pIIa after abscission in SOPs of indicated genotypes (mean ± SEM; Statistics: two-sided Student t-test; n: SOPs analysed. The control dataset is the same as the one presented in Fig. 2B, shown here for convenience. Sara endosome asymmetric segregation is not affected upon Atat depletion. (g) Summary of the AtatRNAi and Elp3RNAi phenotypes: Elp3RNAi phenotype cannot be due to a defect of α-tubulin acetylation. Scale bars: 10 µm (a); 2 µm (c); 5 µm (e). Source numerical data are available in source data.

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Extended Data Fig. 8 The enzymatic activity of Elp3 are not required for Elongator effects on the central spindle.

(a) Average central spindle in SOPs after immunolabelling using anti-K40-Acetyl-α-Tubulin antibodies. n: number of spindles analysed. (b) K40-Acetyl-α-Tubulin intensity profile across the spindle (dashed orange in a). While in control SOPs the pIIb peak is ≈25% higher than that of the pIIa, this is abolished in Elp3RNAi mutants. (c) Enrichment in pIIb. Thick line: median; thin line: quartile. n: number of spindles analysed. Statistics: two-sided Mann–Whitney test (P-values indicated). The β-Tubulin datasets are reproduced from Fig. 1g. (d) Acetyl-tubulin/tubulin signal ratio at the central spindle in indicated genotypes (see methods). Mean±SEM. n: number of spindles. Statistics: two-sided, paired Student’s t-test between pIIa/pIIb values for each spindle. Elp3 depletion does not affect central spindle acetylation. (e) Left panel: S2 cells depleted for indicated Elongator complex subunits (or GFP control) were analysed by quantitative Western blot against β-tubulin and K40-Acetyl-α-tubulin. A 2-fold dilution series of the GFP-RNAi control was loaded to demonstrate linearity of the antibodies. Right panel: acetyl-tubulin/tubulin ratio in RNAi-depleted extracts. Depletion of Elongator subunits does not affect tubulin acetylation. (f) Dividing SOP showing Jupiter–GFP with (top row) or without the Rainbow RGB LUT (bottom row). Maximum intensity z-projection of the central spindle (SDCM). Control panel is reproduced from Fig. 4d. (g) Enrichment in pIIb. Thick line: median; thin line: quartile; n: number of central spindles. Statistics: Kruskal–Wallis test. P-values of the respective post-hoc tests indicated. Control, Elp3 RNAi and Elp3 RNAi + Elp3* datasets are reproduced from Fig. 1d. (h) Dividing SOP showing Sara endosomes (iDelta20) and mRFP-Pon (pIIa cortex; dashed blue). Maximum intensity z-projection of entire cells (SDCM). Note the endosomes in the pIIb cell upon Elongator depletion (arrows). Control panel reproduced from Fig. 4f. (i) Percentage of iDelta20 in pIIa after abscission in SOPs (mean ± SEM; Statistics: one-way ANOVA (P < 0.0001), followed by a Tukey post-hoc test (p value indicated); n: number of SOPs. Control, Elp3 RNAi and Elp3 RNAi + Elp3* datasets reproduced from Fig. 2b. Scale bars: 1 µm (a); 2 µm (f); 5 µm (h). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 9 Characterization of the Elongator-induced increase of microtubule growth speed.

(a-e) SNAP-tag-free Elongator behaves the same as SNAP-tagged Elongator (Fig. 5). The effects on microtubule dynamics of a purified full Elongator complex devoid of SNAP tag (Fig. 3d and Extended Data Fig. 5c) were analysed by TIRFM as in Fig. 5. Conditions: 16.5 µM GTP-tubulin, 10% HiLyte 647-tubulin, in the absence (blue data points) or in the presence of 100 nM His-PC-Elongator (green data points). (a,b) Microtubule growth rate at both the minus- (a) and plus-ends (b) under the conditions described above. Data points represent individual growth events quantified from at least two independent experiments. Thick line: median; thin line: quartile. Statistics: two-sided Mann–Whitney test (p-values<0.0001 in both cases; n: microtubule number). (c,d) Cumulative lifetime distribution of microtubules grown from each end under the conditions described above. Elongator increases the microtubule lifetime at both the minus- (c) and the plus-ends (d). Lines: gamma distribution fits. Mean lifetime estimate ±error (that is lifetime at half cumulative distribution, dashed lines): 217 ± 14 s for control (n = 209; blue triangles) and 324 ± 24 s for Elongator (n = 132; green triangles); at the plus end: 163 ± 7 s for control (n = 391; blue circles) and 212 ± 12 s for 25 nM Elongator (n = 237; green circles). Estimate of microtubule catastrophe rate by fitting lifetime histograms by a mono-exponential decay model: Minus end: Control 0.0040 ± 0.0015 sec−1/ Elongator 0.0026 ± 0.0010 sec−1; Plus end: Control 0.0050 ± 0.0022 sec−1, Elongator 0.0037 ± 0.0014 sec−1. (e) Increase of the polymerization speed versus increase of the mean microtubule lifetime (± error) at both ends under the conditions described above. (f) Western blot analysis of in vitro co-sedimentation assays with dynamic microtubules and purified YY531AA Elongator complex (P pellet; S, supernatant). In the presence of microtubules, all the subunits of Elongator identified by immunoblotting are found in the pellet. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 10 Elongator asymmetry controls central spindle asymmetry and thereby polarized trafficking of Sara endosomes.

(a) Percentage of Sara endosomes (iDelta20) in pIIa after abscission in indicated genotypes (mean ± SEM; Statistics: ANOVA1 (P < 0.0001), followed by a Tukey post-hoc test (p value indicated); n: number of SOPs analysed). Control, Elp3 RNAi and Elp3 RNAi + Elp3* datasets are reproduced from Fig. 2b. (b-c) Central spindle asymmetry reversal upon polar targeting of Elongator. (b) SOP expressing Elp3 RNAi + GFP-Elp3*+Elp3RNAi (or control) were processed for K40-Acetyl-α-Tubulin immunolabelling (maximum intensity z-projection, SDCM). (c) Distribution of central spindle asymmetry values in the population of cells described in (b) showing a statistically significant trend for central spindle asymmetry to be reversed. Statistics: Fisher exact test on the proportion of cells with a negative enrichment in pIIb daughter cell (1/26 for Control versus 7/22 for Elp3 RNAi + GFP-Elp3*+GBP-Pon). (d) Percentage of Sara endosome in pIIa cell as a function of the extent of relocalization of Elongator to the anterior cortex in SOP cells expressing Elp3 RNAi + GFP-Elp3*+GBP-Pon (or GFP-Elp3*+GBP-Pon using a different allele of GFP-Elp3*). In this experiment a phenotypic series is generated. The extent of relocalization of Elongator to the anterior cortex positively correlates with the extent of Sara endosomes being targeted to the wrong, pIIb cell. This panel corresponds to unbinned data from panel 6c. Statistics: two-sided Spearman’s correlation coefficient on unbinned data (p value<0.0001). (e) Elongator does not affect the recruitment/activity of Klp98A in SOPs. Sara endosomes (iDelta20) and endogenous Klp98A (GFP knock-in) where imaged in indicated genotypes. Cargo recruitment is a proxy of kinesin activity, thus, the Klp98A–GFP intensity on Sara endosomes can be used as a proxy for kinesin recruitment/activity in Elongator-depleted cells. We specifically looked for a particular situation where, in an Elp3-depleted SOP, two endosomes would be in different cells, but in the same confocal plane, so depth cannot affect fluorescence measurement. Even in this best-case scenario, we could not detect any difference in terms of Klp98A intensity between the two endosomes. (f) Paradigm for the polarized motility of Sara endosomes during SOP division. Scale bars: 5 µm (b, c-left and e), 2 µm (c-right). Source numerical data are available in source data.

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Supplementary Tables 1 and 2.

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Supplementary Video 1

Dynamics of central spindle symmetry breaking in control cells. Time-lapse video of a dividing control SOP showing Jupiter–GFP/mRFP-PonLD (left) and Jupiter–GFP with the Rainbow RGB LUT for signal density (right). Spinning disk confocal imaging (images are maximum intensity z-projections of the entire SOP cell). Registered time is indicated in seconds. Registered time = 0 indicates anaphase B onset. Note that the central spindle becomes asymmetric during anaphase, with an enrichment of microtubule density on the anterior (Pon-positive) side of the spindle. Scale bar, 5 µm.

Supplementary Video 2

Central spindle asymmetry is abolished in Elp3-depleted SOPs. Time-lapse video of a dividing Elp3RNAi SOP showing Jupiter–GFP/mRFP-PonLD (left) and Jupiter–GFP with the Rainbow RGB LUT for signal density (right). Spinning disk confocal imaging (images are maximum intensity z-projections of the entire SOP cell). Registered time is indicated in seconds. Registered time = 0 indicates anaphase B onset. Note that the central spindle never becomes asymmetric. Scale bar, 5 µm.

Supplementary Video 3

Asymmetric Sara endosome segregation is abolished in Elp3 mutants. Dividing SOP showing Sara endosomes (iDelta20) and mRFP-Pon in the indicated genotype. Images are maximum intensity z-projections of entire cells (SDCM). Elapsed time is indicated in seconds. Note that Sara endosomes are asymmetrical in controls but symmetrical in Elp3 mutants. Scale bar: 5 µm.

Supplementary Video 4

Elongator directly stabilizes microtubules. Biotinylated, rhodamine-labelled, GMPCPP-stabilized seeds (red) were anchored on a glass surface via fibrinogen–NeutrAvidin, 12.5 µM GTP-tubulin (10% labelled with HiLyte 647-fluorescent GTP–tubulin, cyan) was then added in the presence or absence of 25 nM (His)6-PC-SNAP–Elongator and microtubule dynamics were observed by TIRFM (top). Time is indicated in seconds. Note that the SNAP-tagged Elongator is not labelled with any BG-conjugated dye here. Control in the absence of Elongator was performed with the same buffer used for the experiment in the presence of Elongator. Kymographs of the microtubules displayed in the top panels (orange lines; bottom). Note that microtubules grow faster and undergo less catastrophe in the presence of Elongator. Scale bar, 10 µm.

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Planelles-Herrero, V.J., Bittleston, A., Seum, C. et al. Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants. Nat Cell Biol 24, 1606–1616 (2022). https://doi.org/10.1038/s41556-022-01020-9

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