Centralspindlin and chromosomal passenger complex behavior during normal and Rappaport furrow specification in echinoderm embryos

Abstract

The chromosomal passenger (CPC) and Centralspindlin complexes are essential for organizing the anaphase central spindle and providing cues that position the cytokinetic furrow between daughter nuclei. However, echinoderm zygotes are also capable of forming "Rappaport furrows" between asters positioned back-to-back without intervening chromosomes. To understand how these complexes contribute to normal and Rappaport furrow formation, we studied the localization patterns of Survivin and mitotic-kinesin-like-protein1 (MKLP1), members respectively of the CPC and the Centralspindlin complex, and the effect of CPC inhibition on cleavage in mono- and binucleate echinoderm zygotes. In zygotes, Survivin initially localized to metaphase chromosomes, upon anaphase onset relocalized to the central spindle and then, together with MKLP1 spread towards the equatorial cortex in an Aurora-dependent manner. Inhibition of Aurora kinase activity resulted in disruption of central spindle organization and furrow regression, although astral microtubule elongation and furrow initiation were normal. In binucleate cells containing two parallel spindles MKLP1 and Survivin localized to the plane of the former metaphase plate, but were not observed in the secondary cleavage plane formed between unrelated spindle poles, except when chromosomes were abnormally present there. However, the secondary furrow was sensitive to Aurora inhibition, indicating that Aurora kinase may still contribute to furrow ingression without chromosomes nearby. Our results provide insights that reconcile classic micromanipulation studies with current molecular understanding of furrow specification in animal cells.

Fig. 1. Cytokinesis in sea urchin embryos requires Aurora B kinase activity

A. Sensitivity of recombinant LvAurora kinase to the Aurora kinase inhibitor VX-680 was assayed using Histone H3 as a substrate, and phosphorylation was detected by Western blotting with mouse anti-phospho-(Ser10) histone H3. B. Fertilized S. purpuratus eggs were cultured up until nuclear envelope breakdown (NEB) and then transferred into either 0.1% dimethyl sulfoxide (DMSO) or 50 μM VX-680. Extracts were then probed by Western blotting for phospho-(Ser10) histone H3. C. Quantification of furrowing phenotypes for L. pictus eggs cultured during mitosis in either 0.1% DMSO (control, n = 143) or VX-680 (n = 343). Bars denote standard error for 13 separate experiments. D. Timelapse imaging of L. pictus eggs treated beginning at NEB with either 0.1% DMSO (a–e) or 50 μM VX-680 (f–j). Bar: 50 μm.

Fig. 2. Aurora kinase inhibition blocks Histone H3 phosphorylation and alters anaphase microtubule organization in echinoderm zygotes

Panels A–I. S. purpuratus zygotes were treated with either 0.1% DMSO or 100 μM VX-680 from NEB to anaphase onset, then fixed and probed for phospho-(Ser10) histone H3 and DNA, and imaged by confocal microscopy. Panels J–O. D. excentricus zygotes at NEB were transferred into either 0.1% DMSO (panels J, L, N) or 50 μM VX-680 (panels K, M, O), cultured and fixed during anaphase (panels J–M) and telophase (panels N,O). Embryos were stained for tubulin and imaged by confocal microscopy. Bar: 20 μm.

Fig. 3. Survivin localization during mitosis in sea urchin zygotes

S. purpuratus zygotes were fixed and probed for tubulin (green) and Survivin (red) at various stages of first mitosis, and imaged by confocal microscopy. Panels A–C show a metaphase cell: Survivin localized to chromosomes. Panel D–I depict embryos during early and late anaphase: Survivin localized to the central spindle midzone and weakly to equatorial astral microtubules. As cytokinesis progressed, Survivin concentrated at the ingressing furrow and midbody (panels J–L). Bar: 45 μm.

Fig. 4. Mklp1 localization in S. purpuratus zygotes during furrow specification and ingression

Zygotes were fixed at various stages of mitosis and processed for tubulin (green) and MKLP1 (red) localization, and imaged using confocal microscopy. During anaphase MKLP1 localized to the central spindle (panels A–C), but as anaphase progressed was increasingly found on the tips of astral microtubules in the equatorial cleavage plane (panels D–I). During furrow ingression, MKLP1 concentrated at the spindle midzone and on microtubule bundles associated with the ingressing furrow (panels J–L); see also Fig. 5. Bar: 45 μm.

Fig. 5. MKLP1 concentrates on furrow-associated microtubule bundles

Dividing S. purpuratus zygotes fixed and probed for tubulin (green) and MKLP1 (red), and imaged by confocal microscopy. Panels A–F illustrate MKLP1 localization at microtubule tips during an early stage of furrowing. Panels D–F are high magnification views of panels A–C. Panels G–L illustrate a long axis view of MKLP1 localization in a dividing sea urchin zygote, where individual microtubule bundles are visibly associated with the ingressing furrow. Panels J–L are high magnification views of panels G–I. Bars: 20 μm.

Fig. 6. Centralspindlin localization is dependent upon CPC function

Beginning at NEB S. purpuratus zygotes were cultured in either 0.1% DMSO or 50 μM VX-680, then fixed and processed to reveal tubulin (green), MKLP1 (red) and DNA (blue) localization and imaged by confocal microscopy. Panels D–F, G–I, and J, L show progressive stages from anaphase through early telophase, when normal cells would be cleaving (compare with Fig. 4 panels G–L). Note not only the loss of normal MKLP1 localization, but also the altered microtubule organization (see also Fig. 2 panels J–O). Bar: 45 μm.

Fig. 7. Cytokinesis in binucleate eggs

Binucleate L. pictus and S. purpuratus cells were generated by transferring late anaphase/early cleavage zygotes into ice cold seawater for twenty minutes to disrupt astral microtubule elongation and furrow specification. Eggs were then warmed to normal culture temperature whereupon they resumed development as binucleate cells. Panel A. Binucleate eggs undergoing second division imaged by polarization microscopy (also see Movie 2, Supporting Information). Panel B. Quantification of furrowing phenotypes of binucleate eggs where the primary furrow corresponds to cleavage in the plane of the former metaphase plate, and the secondary furrow is cleavage between the two parallel spindles. Furrowing was scored as either successfully completed (cleaved), furrows regressed, or no initiation; cold-shocked cells whose chromosomes failed to fully segregate to produce two discrete and separate nuclei were discarded. Panel C. Microtubule staining of cells containing two parallel spindles reveals the presence of a true central spindle in the primary cleavage plane. In contrast, microtubule tips in the secondary cleavage plane clearly overlap but, unless chromosomes are abnormally present there (see Fig. 9), they do not form a well defined midzone. Bars: 10 μm. Panel D. En face views of microtubule and Ser19-phosphorylated myosin regulatory light chain (P-MLC) localization in cleaving binucleate cells. The primary cleavage furrow is clearly characterized by more robust P-MLC localization than the secondary cleavage furrow. Bar: 10 μm.

Fig. 8. CPC preferentially localizes to the primary cleavage plane in binucleate cells

Survivin staining of binucleate cells at three successive stages of cleavage. Even when the primary furrow had ingressed to near completion and Survivin was at its most concentrated (panels I–L) Survivin was still not detectable in the ingressing secondary furrows (arrowhead, panel J). Color code as in Fig. 3. Bar: 25 μm.

Fig. 9. Chromosome position determines CPC recruitment pattern

Survivin accumulation in both primary and secondary cleavage planes was seen in binucleate cells where the spindles were in sufficiently close proximity that chromosomes were captured by microtubules from both of the parallel spindles. Panels A–D, E–H, and I–L show successive stages. Staining and color code as in Fig. 3. Bar: 25 μm.