Switching of INCENP paralogs controls transitions in mitotic chromosomal passenger complex functions

Abstract

A single inner centromere protein (INCENP) found throughout eukaryotes modulates Aurora B kinase activity and chromosomal passenger complex (CPC) localization, which is essential for timely mitotic progression. It has been proposed that INCENP might act as a rheostat to regulate Aurora B activity through mitosis, with successively higher activity threshold levels for chromosome alignment, the spindle checkpoint, anaphase spindle transfer and finally spindle elongation and cytokinesis. It remains mechanistically unclear how this would be achieved. Here, we reveal that the urochordate, Oikopleura dioica, possesses two INCENP paralogs, which display distinct localizations and subfunctionalization in order to complete M-phase. INCENPa was localized on chromosome arms and centromeres by prometaphase, and modulated Aurora B activity to mediate H3S10/S28 phosphorylation, chromosome condensation, spindle assembly and transfer of the CPC to the central spindle. Polo-like kinase (Plk1) recruitment to CDK1 phosphorylated INCENPa was crucial for INCENPa-Aurora B enrichment on centromeres. The second paralog, INCENPb was enriched on centromeres from prometaphase, and relocated to the central spindle at anaphase onset. In the absence of INCENPa, meiotic spindles failed to form, and homologous chromosomes did not segregate. INCENPb was not required for early to mid M-phase events but became essential for the activity and localization of Aurora B on the central spindle and midbody during cytokinesis in order to allow abscission to occur. Together, our results demonstrate that INCENP paralog switching on centromeres modulates Aurora B kinase localization, thus chronologically regulating CPC functions during fast embryonic divisions in the urochordate O. dioica. Abbreviations: CCAN: constitutive centromere-associated network; CENPs: centromere proteins; cmRNA: capped messenger RNA; CPC: chromosomal passenger complex; INCENP: inner centromere protein; Plk1: polo-like kinase 1; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; SAC: spindle assembly checkpoint; SAH: single α-helix domain.

Keywords: Aurora B kinase; Inner Centromere Protein (INCENP); abscission; cytokinesis; kinetochore; tunicate.

Figure 1.

O. dioica kinetochore and inner centromere complements and epigenetic marks. (a) Orthologs that were detected (green) in the O. dioica genome are projected onto the human complement (modified from [1]). Centromere proteins (CENPs) of the CCAN are represented by single letters. (b) Identification of O. dioica centromeres using CenH3. Top left: Six monocentric chromosomes in mitotic phase were indicated by numbers. Middle: CenH3-GFP (arrow) flanked H3pS28 signal on the centromeres. Bottom: CenH3-GFP localized at the centrosome proximal side of separating sister chromatids at anaphase. (c) The distribution of major histone H3 phosphorylation and methylation modifications on O. dioica chromosomes. H3pT3 and H3pS28 localized on the inner centromeres, and H3pT11 localized on the outer centromeres. In striking contrast, H3pS10, H3pS31, H3K4me2 and H3K9me3 localized on the chromosome arms. Bars, 3 µm.

Figure 2.

Temporal expression pattern of the CPC components and Plk1a during O. dioica development. (a) Developmental expression analysis of core CPC components and Plk1a. Aurora B and Plk1a (left), and two coexisting INCENP and Survivin paralogs (right), were highly expressed during mitotic proliferation and gonad development. Paired histogram bars correspond to the following developmental stages from left to right: oocytes, 2–8 cells, 1 hpf, hatching, metamorphosis, Day 2, Day 3, Day 4, Day 5 and Day 6. Mean RT-qPCR values normalized to EF1β transcripts (n = 3) are shown with SE. (b) Plk1a protein was enriched during mitotic development as determined by western blot using odPlk1a antibody. Equal amounts of whole animal lysates from various stages were loaded. (c) Domain comparison of Homo sapiens and O. dioica INCENP homologs. N terminal CEN, single α-helix (SAH) and C terminal IN box domains are indicated. Identities (blue) and similarities (red) between O. dioica INCENP paralog IN boxes and the single human ortholog IN box are shown. The CDK1 consensus phosphorylation site (SS₂₀₉P) is positioned on INCENPa and is absent in INCENPb. (d) Sequence alignment of the INCENP IN box with predicted secondary structure in several model species. W801 (Xenopus numbering), involved in Aurora B interaction, is marked in red and F837, involved in Aurora B activation, is marked in green. Aurora B phosphorylation sites “TSS” are marked in blue. Hs, Homo sapiens; Gg, Gallus gallus; Xl, Xenopus laevis; Od, Oikopleura dioica; Ci, Ciona intestinalis; Bf, Branchiostoma floridae; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans.

Figure 3.

The counteracting effects of protein phosphatases on Aurora B kinase activity in somatic cells. (a) Temporal distribution of Aurora B in mitosis. Aurora B localized on chromosome arms at early prophase, enriched on centromeres from late prophase until metaphase coinciding with the onset of H3pS28 on centromeres, relocated to the central spindle at anaphase and midbody at telophase. Bar, 3 µm. (b) Mitotic H3pS28 on centromeres was governed by Aurora B to antagonize protein phosphatases. Left: Inhibition of Aurora B (AZD1152) after metaphase arrest abolished H3pS10 and H3pS28 in mitotic cells. Middle: Inhibition of protein phosphatases by Calyculin induced Aurora B and H3pS28 redistribution along chromosome arms. Right: Inhibition of protein phosphatases also induced H3pT3 redistribution along chromosome arms. Bars, 10 µm.

Figure 4.

INCENP paralog deployments correlate with temporal and spatial activity of Aurora B in mitosis. (a) INCENPa localized throughout the nucleus during interphase, focused on centromeres during early prophase, coinciding with the onset of centromeric H3pS28, and disappeared afterwards. Bars, 2 µm. (b) INCENPb was undetectable during interphase and prophase, was first detected on centromeres during prometaphase and peaked during metaphase, coinciding with maximum levels of H3pS28. INCENPb relocated to the central spindle during anaphase, and concentrated in the midbody during telophase. Bars, 2 µm. The diagram in the middle illustrated the localization of CPCa and CPCb, representing INCENPa and INCENPb respectively, during each mitotic phase.

Figure 5.

Plk1 docking on INCENPa is a prerequisite for centromeric enrichment of CPCa during late prophase. (a) Plk1 localized on centrosomes and briefly enriched on centromeres during late prophase concomitant with the onset of centromeric H3pS28. (b) Plk1 colocalized with Aurora B on centromeres during late prophase and on the central spindle during anaphase. Inhibition of Plk1 (BI2536) abolished Plk1 staining on centromeres and centrosomes, and Aurora B spread along chromosome arms. (c) Plk1 inhibition resulted in centromeric H3pS28 and INCENPa spreading throughout chromosomes, without affecting CenH3 on centromeres. The INCENPa^S209A mutant failed to localize to centromeres. The graph on the right shows the percentage of pro-metaphase cells that showed nuclear or centromeric INCENP localization for INCENPa WT or the S₂₀₉A mutant. Mean values (n = 3) are shown with SE. Bars, 3 µm.

Figure 6.

Effects of Plk1 or Aurora B inhibition on CPCb localization and phenotypes arising from INCENP paralog knockdowns. (a) INCENPb was enriched on centromeres and colocalized with H3pS28 in metaphase arrested cells (MG132 treatment). Inhibition of Plk1 (BI2536) resulted in a significant delocalization of INCENPb and H3pS28 from centromeres to chromosome arms. Inhibition of Aurora B (AZD1152) abolished INCENPb and H3pS28 staining on chromosomes. (b) Inhibition of Plk1 (BI2536) abolished H3pT3 on centromeres, whereas inhibition of Aurora B (AZD1152) resulted in some H3pT3 delocalization from centromeres to chromosome arms. Bars, 3 µm. (c) Line graphs show fluorescence intensity (a.u.: arbitrary units) of INCENPb, H3pS28 and H3pT3 under respective treatment conditions in (a) and (b). The x axis represents distance along chromosomes. (d) Aurora B was absent on chromosomes and the meiotic spindle was not formed at meiotic metaphase I in INCENPa RNAi oocytes. In INCENPb RNAi oocytes, Aurora B localized to chromosomes and meiotic spindles assembled. (e) Neither H3pS10 nor H3pS28 were present on chromosomes and chromosomes were not condensed at meiotic metaphase I in INCENPa RNAi oocytes. In INCENPb RNAi oocytes H3pS10 localized on bivalents and H3pS28 localized to centromeres. Bars, 2 µm. (f) Line graphs show fluorescence intensity (a.u.: arbitrary units) of pAurora, H3pS10 and H3pS28 under respective treatment conditions in (d) and (e). The x axis represents distance along chromosomes.

Figure 7.

INCENPb is required for the localization and activity of Aurora B on the central spindle and midbody during late mitosis. (a) In wild type early embryonic division, Aurora B localized on chromosome arms at prophase, on centromeres at metaphase, relocated to the central spindle at early anaphase, and was enriched on the midbody during telophase and cytokinesis. (b) In INCENPb RNAi embryos, Aurora B localized on chromosome arms at prophase, and on centromeres at metaphase. Aurora B signal on the central spindle was weak at early anaphase, decreased further at late anaphase and dropped below detectable levels on the midbody during telophase and cytokinesis. (c) Fluorescence intensity (arbitrary units) of pAurora throughout M-phase in wild-type versus INCENPb RNAi embryos. SE bars are shown for n = 3 replicates. (d) INCENPb knockdown did not affect the localization of Plk1 or centrosome integrity. Active Plk1 localized normally on centromeres at metaphase, the central spindle at anaphase, the midbody at telophase, and on centrosomes during all phases. γ-tubulin staining showed normal integrity of centrosomes and clear centriole disengagement at telophase. (e) Exogenous RNAi-resistant INCENPb cmRNA fully rescued the division defects caused by endogenous INCENPb knockdown. Right panel: in INCENPb RNAi group, more than 95% of embryos experienced cleavage furrow regression defects and arrested as aneuploid; in the rescue group, ~90% of INCENPb RNAi embryos progressed normally through embryogenesis, similar to the control group. Mean values of 3 independent experiments are shown with SE, and the number of embryos evaluated in the INCENPb knockdown, rescue and control groups indicated directly on the histogram bars. CFR, cleavage furrow regression; UF, unfertilized; AC, abnormal cleavage; D, developed normally. Left panel: Aurora B signal was restored on the central spindle during anaphase and on the midbody during telophase. Bars, 2 µm.

Figure 8.

INCENP paralog switching model. Localization of the CPC at each mitotic phase is indicated in the dark middle bar. Major mitotic events are indicated by arrows. Levels of Aurora B activity are represented by degree of red shading for CPCa (top) and CPCb (bottom). CDK1 phosphorylation of (SS₂₀₉P) in INCENPa and subsequent recruitment of Plk1 are shown. INCENPa modulates Aurora B activity during early mitosis, which is critical for H3S10/S28 phosphorylation, chromosome condensation, Aurora B centromeric localization, spindle assembly, and Aurora B transfer from centromeres to the central spindle. INCENPb appears on centromeres during prometaphase, then INCENPb-Aurora B translocates to the central spindle at anaphase onset. INCENPb becomes essential for localization and activity of Aurora B on the central spindle at late anaphase and on the midbody during telophase, cytokinesis and completion of abscission.