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. 2021 Sep 27;31(18):3973-3983.e4.
doi: 10.1016/j.cub.2021.06.063. Epub 2021 Jul 22.

Asymmetric nuclear division in neural stem cells generates sibling nuclei that differ in size, envelope composition, and chromatin organization

Chantal Roubinet  1 , Ian J White  2 , Buzz Baum  3
Affiliations

Asymmetric nuclear division in neural stem cells generates sibling nuclei that differ in size, envelope composition, and chromatin organization

Chantal Roubinet et al. Curr Biol. .

Abstract

Although nuclei are the defining features of eukaryotes, we still do not fully understand how the nuclear compartment is duplicated and partitioned during division. This is especially the case for organisms that do not completely disassemble their nuclear envelope upon entry into mitosis. In studying this process in Drosophila neural stem cells, which undergo asymmetric divisions, we find that the nuclear compartment boundary persists during mitosis thanks to the maintenance of a supporting nuclear lamina. This mitotic nuclear envelope is then asymmetrically remodeled and partitioned to give rise to two daughter nuclei that differ in envelope composition and exhibit a >30-fold difference in volume. The striking difference in nuclear size was found to depend on two consecutive processes: asymmetric nuclear envelope resealing at mitotic exit at sites defined by the central spindle, and differential nuclear growth that appears to depend on the available local reservoir of ER/nuclear membranes, which is asymmetrically partitioned between the two daughter cells. Importantly, these asymmetries in size and composition of the daughter nuclei, and the associated asymmetries in chromatin organization, all become apparent long before the cortical release and the nuclear import of cell fates determinants. Thus, asymmetric nuclear remodeling during stem cell divisions may contribute to the generation of cellular diversity by initiating distinct transcriptional programs in sibling nuclei that contribute to later changes in daughter cell identity and fate.

Keywords: asymmetric division; cell fate; chromatin; closed mitosis; cytokinesis; nuclear division; nuclear envelope remodelling; open mitosis; spindle; stem cells.

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Conflict of interest statement

Declarations of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
The nuclear envelope of fly neuroblasts is maintained during mitosis and remodeled to generate two sibling nuclei that differ in size and envelope composition (A–D) Mitotic neuroblasts expressing Cherry::Jupiter in combination with either (A) CD8::GFP, (B) Klaroid::GFP, Klarsicht::GFP or BIP-SfGFP::HDEL, (C) NLS::5GFP or Rps13::GFP, or (D) Nup107::GFP or Nup58::GFP. Yellow arrows indicate higher nucleoporin density. (E and F) Electron microscopy performed at various cell cycle stages: (E) magnified regions are marked in yellow in (F); yellow arrowheads show nuclear pores; white arrowheads show holes in the nuclear envelope; N, nuclear compartment; C, cytoplasm. Scale bars are 5 μm. See also Figure S1 and Videos S1, S2, S3, and S4.
Figure 2
Figure 2
A nuclear lamina is required for nuclear envelope maintenance during passage through mitosis (A) Mitotic neuroblast expressing LamDm0::GFP with Cherry::Jupiter. Yellow arrows point the higher LamDm0 density. (B) LamDm0::GFP mean intensity along the nuclear membrane and in the cytoplasm, throughout the cell cycle. (C) LamDm0 mean intensity ratio between the nuclear membrane and the cytoplasm is measured at 5 different stages for 25 cells. Grey area corresponds to a ratio of 1 ± 0.25. Bars indicate mean ± standard deviation. Asterisks denote statistical significance, derived from unpaired t tests: ∗p ≤ 0.05, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001. (D) Immunostaining for LamDm0, dMoesin, and DAPI. (E) Immunostaining for non-phosphorylated LamDm0 on serine 25 and DAPI. (F) Representative time lapse of neuroblast expressing Klaroid::GFP, Cherry::Jupiter, and an RNAi against LamDm0. Yellow stars indicate presence of intact prophase nuclear compartment. (G and H) CLEM showing progressive dispersion of nuclear membranes into the cytoplasm (G) and a loss of nuclear compartment integrity (H) after LamDm0 RNAi expression. (I) Quantification of mitotic neuroblasts expressing an RNAi against LamDm0 and displaying normal or abnormal nuclear division. Number of analyzed cells = 123. (J) Representative time-lapse images of LamDm0 RNAi neuroblast expressing Klaroid::GFP and Cherry::Jupiter, in which nuclear membranes disperse and nuclei fail to reform at mitotic exit. At least 3 independent experiments were performed. Scale bars are 5 μm. See also Figure S2 and Video S5.
Figure 3
Figure 3
Nuclear division is a sealing-dependent process (A) Dividing neuroblasts expressing Cherry::Jupiter and CD8::GFP. The red dashed line represents the site of cortical furrowing. (B) Graph showing the cell and nuclear width from the apical to the basal cortex, for 7 time points. (C) Graph showing the cell and nuclear length along the apico-basal axis for ten cells throughout mitosis. Error bars represent standard deviation and the dashed line indicates anaphase onset. (D) Graph showing the cell and nuclear width at the cleavage furrow region throughout mitosis, averaged from ten cells. Error bars represent standard deviation, dashed line indicates anaphase onset, and blue and green arrowheads indicate start of cortical and nuclear furrowing, respectively. (E) Representative time lapse sequences of mitotic neuroblasts expressing CD8::GFP and Cherry::Jupiter, and failing in cytokinesis after LatrunculinA, CytochalasinD, or ConcanavalinA treatment. (F) Time-lapse images of a Latrunculin-treated neuroblast expressing CD8::GFP and Cherry::Jupiter. The yellow square indicates area used on the kymograph and the yellow arrowheads represent sites of nuclear sealing. (G) Quantification of dividing neuroblasts, neuroblasts failing in cell division but not in nuclear division, or failing in both cell and nuclear division after expression of an RNAi against AuroraB, Pavarotti, or Tumbleweed. The number of cells analyzed for each condition is indicated. (H) Representative time-lapse images of neuroblast expressing an RNAi against AuroraB, CD8::GFP, and Cherry::Jupiter, failing in both cell and nuclear division. Orange arrow shows the absence of central spindle. (I and J) Representative time-lapse images of neuroblasts expressing an RNAi against Tumbleweed, CD8::GFP, and Cherry::Jupiter, failing cell division (I), or failing in both cell and nuclear division (J). Orange arrows show the presence (I) or absence (J) of central spindle. For each experiment, n ≥ 3. Scale bars are 5 μm. See also Figure S3 and Video S6.
Figure 4
Figure 4
Asymmetric nuclear division depends on nuclear sealing at sites dictated by the spindle (A) Dividing neuroblasts expressing Klaroid::GFP and Cherry::Jupiter. Orange and yellow arrows indicate envelope sealing events. (B) Airyscan pictures of neuroblasts expressing CD8::GFP and Cherry::Jupiter. Yellow arrowheads show sealing sites. Kymographs are done along the apico-basal axis. (C) Graph showing the spindle asymmetry ratio and the nuclear diameter ratio (n = 33). Asterisks denote statistical significance, derived from unpaired t tests: n.s., not significant. (D) Plot showing correlation between spindle and nuclear asymmetries (n = 68). (E) Representative time-lapse images of neuroblast expressing CD8::GFP, Cherry::Jupiter, and Galphai. Quantification shows the asymmetric ratio for the cell, nucleus, and spindle at the sealing time. (F) Plot showing correlation between spindle and nuclear asymmetries (on the left) or between cell and nuclear asymmetries (on the right); (n = 8). (G) Image of neuroblast at the nuclear sealing time, showing the asymmetry in the central spindle and nuclear size (green dashed line). (H) Representative time-lapse images of neuroblast expressing Cherry::Jupiter, Patronin::GFP, and GBP-PonLD. Green dashed lines correspond to the nuclei. (I) Graph showing the spindle asymmetry (left) and the nuclear asymmetry (right) for control neuroblasts and after expression of Patronin::GFP and GBP-PonLD (n = 25 and n = 24). Bars indicate mean ± standard deviation. Asterisks denote statistical significance, derived from unpaired t tests: ∗∗∗∗p ≤ 0.0001. For each experiment, n ≥ 3 experiments. Scale bars are 5 μm. See also Figure S4.
Figure 5
Figure 5
Final nuclear sizes are achieved via differential growth of the two daughter nuclei (A) Scatterplots showing nuclear diameter of neuroblast and GMC right after nuclear sealing or during G1. Bars indicate mean ± standard deviation. Asterisks denote statistical significance, derived from unpaired t tests: ∗∗∗∗p ≤ 0.0001. (B) Graph showing the diameter of a neuroblast and GMC nucleus, from nuclear sealing until G1. (C) Neuroblasts expressing Klaroid::GFP and Cherry::Jupiter; yellow arrows show cytoplasmic reservoir of nuclear membrane. (D) Representative time-lapse images of neuroblast expressing the ER marker Bip-SfGFP::HDEL and Cherry::Jupiter, taken with Airyscan. Yellow arrows show cytoplasmic reservoir of nuclear membrane. (E) Graph showing the asymmetric ER segregation at sealing time, using two different probes. The sum intensity is measured in the entire neuroblast and GMC volume, and the ratio Nb/GMC is plotted. Bars indicate mean ± standard deviation. Asterisks denote statistical significance, derived from unpaired t tests: n.s., not significant. (F) Electron micrographs. ER/nuclear membranes are highlighted in pink. (G) Representative time-lapse images of a neuroblast cultured on ConcanavalinA expressing CD8::GFP and Cherry::Jupiter. (H) Graph corresponding to the neuroblast presented in (G), showing the size of both nuclei throughout the cell cycle. (I) Representative time-lapse images of a Latrunculin-treated neuroblast expressing CD8::GFP. Yellow arrowheads indicate reservoir of available nuclear membrane. (J) Graph from a representative Latrunculin-treated neuroblast showing the size of both nuclei throughout the cell cycle. (K) Representative pictures showing wild-type and binucleated neuroblasts expressing CD8::GFP and Cherry::Jupiter, containing two nuclei with different or similar size. (L) Scatterplot showing the nuclear area ratio between sibling nuclei for wild-type, DMSO-, Latrunculin-, Cytochalasin-, or Concanavalin-treated neuroblasts. Grey area on the graph represents a nuclear area ratio equal to 1 ± 0.25. Number of analyzed cells = 148. Asterisks denote statistical significance, derived from unpaired t tests: n.s., not significant, ∗∗∗∗p ≤ 0.0001. For each experiment, n ≥ 3. Scale bars are 5 μm. See also Figure S5 and Video S7.
Figure 6
Figure 6
Asymmetric nuclear division affects chromatin organization (A) Representative time-lapse images of a neuroblast expressing the DNA marker His2A::RFP and the cortical marker Sqh::GFP. (B) Graph showing His2A::RFP mean intensity over time for GMC and neuroblast nuclei, n = 5. Error bars represent standard deviation. (C) Wild-type neuroblasts fixed and stained for DAPI, H3K4me2, Tubulin, and Phalloidin. Yellow arrows indicate similar H3K4me2 intensity on the two pools of sister chromatids. (D) Wild-type neuroblasts fixed and stained for DAPI, H3K4me2, and Prospero. White dashed lines show the nuclear area during and after nuclear growth. (E) For cells from early anaphase to late telophase, graph (left) shows nuclear size ratio on the x axis versus ratio (neuroblast/GMC) of sum intensity for DAPI across the entire nuclear volume on the y axis, and (right) the equivalent for H3K4me2 on the y axis. (F) Binucleated neuroblast stained for DAPI and H3K4me2. The graph shows the nuclear area ratio (left) and the DAPI or H3K4me2 sum intensity ratio (right) for binucleated neuroblasts displaying two nuclei with different size (n = 10). Bars indicate mean ± standard deviation. Asterisks denote statistical significance, derived from unpaired t tests: ∗∗p ≤ 0.01. For each experiment, n ≥ 3. Scale bars are 5 μm. See also Figure S6.
Figure 7
Figure 7
Asymmetric nuclear division generates two daughter nuclei that differ in size, envelope composition, and chromatin organization before the nuclear import of cell fate determinants (A) Graph showing the timing of the successive steps related to nuclear division, cytokinesis, and nuclear import of Prospero. (B) Diagram representing different steps of the process of asymmetric nuclear division and cell fate determinant release. NPCs disassemble 330 s before anaphase onset. The nuclear envelope is then asymmetrically sealed in a spindle-dependent manner 200 s after anaphase onset to generate two daughter nuclei with moderate differences in size. This asymmetry is increased by differential nuclear growth during telophase, until nuclear size stabilization 480 s after anaphase onset. This is associated with a differential packing of the chromatin and an asymmetric distribution of histone marks between the new neuroblast and GMC nuclei, before the nuclear import of cell fate determinants (980 s after anaphase onset).
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