Feizbakhsh O et al. (2020), A Peak of H3T3 Phosphorylation Occurs in Synchr...

ECB-ART-48709

Cells 2020 Apr 07;94:. doi: 10.3390/cells9040898.

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A Peak of H3T3 Phosphorylation Occurs in Synchrony with Mitosis in Sea Urchin Early Embryos.

Feizbakhsh O , Pontheaux F , Glippa V , Morales J , Ruchaud S , Cormier P , Roch F .

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The sea urchin embryo provides a valuable system to analyse the molecular mechanisms orchestrating cell cycle progression and mitosis in a developmental context. However, although it is known that the regulation of histone activity by post-translational modification plays an important role during cell division, the dynamics and the impact of these modifications have not been characterised in detail in a developing embryo. Using different immuno-detection techniques, we show that the levels of Histone 3 phosphorylation at Threonine 3 oscillate in synchrony with mitosis in Sphaerechinus granularis early embryos. We present, in addition, the results of a pharmacological study aimed at analysing the role of this key histone post-translational modification during sea urchin early development.

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Genes referenced: impact LOC100887844 LOC115919910

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	Figure 1. A peak of T3H3 phosphorylation precedes cell cleavage in S. granularis early embryos. (a) Western blots showing the relative levels of T3H3ph, H3, Actin, T318PP1Cph and PP1C at different times during the first and second embryonic divisions. (b) Quantitative analysis of the above results. For each lane, the T3H3ph (black triangles) and T318PP1Cph (black diamonds) intensity scores were normalised against the amount of Actin signal detected. The values obtained were expressed as a percentage of the maximal signal observed. The graph includes as a visual reference the proportion of embryos that have completed either the first cleavage (grey dots) or the second cleavage (grey squares) at different time points.
	Figure 2. A wave of T3H3 phosphorylation/de-phosphorylation occurs in synchrony with mitotic division in S. granularis early embryos. (a–j) Pictures correspond to confocal equatorial optical sections obtained by imaging embryos immunostained for Tubulin (shown in green, top panels) and T3H3ph (red in the top panels, greyscale in bottom panels). Samples were also labelled with DAPI (blue in top panels, grey-scale in small insets). Images present consecutive stages illustrating progression through the first embryonic division. Scale bar, 25 µm. (a–d) The T3H3ph signal (white arrows) appeared when chromatin condensation was already apparent in the cell nucleus (red arrowheads, insets). (e,f) A strong T3H3ph signal (white arrows) was observed in association with chromatin, both during the metaphase (e) and early anaphase (f). In these stages, a fluorescent signal associated with the cell cortex (red open arrowheads) and the mitotic spindle (white arrowheads) was also visible. (g–h) During the anaphase, the T3H3ph signal (white arrows) rapidly faded away. (i,j) No T3H3ph signal was detected during the telophase.
	Figure 3. A peak of T3H3 phosphorylation accompanies the second mitotic division in S. granularis embryos. (a–f) Pictures correspond to confocal equatorial optical sections obtained by imaging embryos immunostained for Tubulin (shown in green, top panels), T3H3ph (red in the top panels, greyscale in bottom panels) and DAPI (blue in top panels). Images present consecutive stages illustrating progression through the second embryonic division. Scale bar, 30 µm. (a,b) The T3H3ph signal (white arrows) appeared during chromatin condensation. (c,d) A strong T3H3ph signal (white arrows) was observed during the prometaphase (c) and metaphase (d). (e,f) The T3H3ph signal (white arrows) disappears during the anaphase.
	Figure 4. The echinoidea Haspin homologs display a conserved Ser/Thr kinase domain. Clustal-W protein alignment showing the C-terminal portion of Haspin in different mammalia and echinoidea species. Invariant amino acid positions are shown in red and labelled with asterisks. The Haspin Ser/Thr kinase domain is shaded in green and its HRD catalytic loop is shown in red. Residues implicated in H3 recognition and/or interacting with ATP/Mg2+ in the human Haspin are respectively marked with red and blue dots [34,39,40]. The vertebrate HBIS motif and its homolog region in sea urchins are shaded in blue. The positively charged residues of the vertebrate motif are marked with orange dots [42], but do not appear to be conserved. The RAS motif, where the Ser is one of the residues phosphorylated by the Polo-kinase during Haspin activation is indicated by an orange rectangle [42]. We aligned the following sequences: Hsap, Homo sapiens (NP_114171.2); Mmus, Mus musculus (NP_038578.2); Spur, Strongylocentrotus purpuratus (JT120475.1); Sgra, Sphaerechinus granularis (GAVR01006044.1) and (GAVR01015122.1); Echl, Evechinus chloroticus (GAPB01047772.1); Pliv, Paracentrotus lividus (GEDS01040703.1) and Hpul, Hemicentrotus pulcherrimus (IACU01096736.1).
	Figure 5. Exposure to the CHR-6494 inhibitor delays cell division in S. granularis early embryos. (a) Graph representing the proportion of embryos that have completed the first cleavage at different time points (min after fertilisation). The different curves describe culture behaviour in the presence of increasing concentrations of CHR-6494 or its vehicle DMSO, all added at 15 min after fertilisation. (b) Relative levels of T318PP1Cph observed at different time points in embryos treated with DMSO, 0.5 µM CHR-6494 or 10 µM Roscovitine. The T318PP1Cph form was detected by Western blot using a specific antibody. The levels of CDK1 were visualised in the same membrane with the PSTAIR antibody and used as a loading control. (c) Quantitative analysis of the above results. Plotted values were normalised against the CDK1 levels and expressed as a percentage of the maximal signal observed.
	Figure 6. CHR-6494 and Roscovitine administration delays cell division and impacts cleavage rates in S. granularis embryos. (a–c) Plots represent the proportion of embryos that have completed their first cleavage at different time points (minutes PF). Final concentrations for CHR-6494 and Roscovitine were respectively 0.5 µM and 10 µM. For each experimental condition, a culture sample was collected 20 min after drug addition and processed for T3H3ph immunostaining (see Figure 7). (a) Drug addition at 70 min PF (red arrowhead) caused a general delay in the cleavage onset. (b) Drug addition at 90 min PF delayed cleavage onset and perturbed culture synchronicity. (c) No delay in cleavage onset was observed, but culture synchronicity was still perturbed in embryos treated at 110 min PF.
	Figure 7. Neither CHR-6494 nor Roscovitine prevent T3H3 phosphorylation during the first embryonic division in S. granularis. (a–c) Embryos were assigned to five different phenotypic categories, according to their morphology. For each sample, representative images illustrating the different phenotypes found are shown in the same row. Pictures correspond to confocal equatorial optical sections obtained imaging embryos immunostained for Tubulin (shown in green), T3H3ph (red and inverted grey-scale in the small insets) and DAPI (blue). Scale bar, 25 µm. The relative proportions of the different categories are reported on each image and have been plotted as a series of histograms shown on the right panel of each row. The number of embryos considered for the quantitative analyses is also shown on the corresponding graphs. Abbreviations used: P, Prophase; Pm, Prometaphase; M, Metaphase; A, Anaphase; T, Telophase. (a) All embryos were in prophase in a sample collected before drug addition, at 70 min PF. In the control batch, samples collected at 90 min PF included 26% of embryos in prometaphase, all displaying high levels of T3H3ph (white arrow). This phenotypic category was absent in the drug-exposed batches. (b) Samples collected at 110 min PF contained embryos in prometaphase and metaphase, all showing a bright T3H3ph staining (white arrows). In the batch exposed to Roscovitine, all the embryos in metaphase presented ectopic spindle poles (red arrowhead). (c) In the 130 min PF samples, embryos in prometaphase and metaphase displayed normal levels of T3H3ph (white arrows) and the staining disappeared during the anaphase.

References [+] :

Ashtiyani, AtHaspin phosphorylates histone H3 at threonine 3 during mitosis and contributes to embryonic patterning in Arabidopsis. 2011, Pubmed