Haspin kinase regulates microtubule-organizing center clustering and stability through Aurora kinase C in mouse oocytes

Abstract

Meiotic oocytes lack classic centrosomes and, therefore, bipolar spindle assembly depends on clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. However, the molecular mechanism regulating MTOC assembly into two poles is not fully understood. The kinase haspin (also known as GSG2) is required to regulate Aurora kinase C (AURKC) localization at chromosomes during meiosis I. Here, we show that inhibition of haspin perturbed MTOC clustering into two poles and the stability of the clustered MTOCs. Furthermore, we show that AURKC localizes to MTOCs in mouse oocytes. Inhibition of haspin perturbed the localization of AURKC at MTOCs, and overexpression of AURKC rescued the MTOC-clustering defects in haspin-inhibited oocytes. Taken together, our data uncover a role for haspin as a regulator of bipolar spindle assembly by regulating AURKC function at acentriolar MTOCs in oocytes.

Keywords: Aurora kinase; Haspin; MTOC; Oocyte; Spindle.

Fig. 1.

Haspin inhibition causes unfocused metaphase I spindle poles. (A–E) Fully grown, prophase-I-arrested oocytes were in vitro matured in the presence of ethanol (EtOH) or 500 nM of 5-Iodotubercidin (5-Itu) to metaphase I (7 h) prior to fixation and detection of MTOCs (γ-tubulin; red in merge), spindle microtubules (α-tubulin; green in merge) and DNA (DAPI; blue). (B) Quantification of the percentage of oocytes analyzed in A with unfocused spindle poles. (C) Quantification of the number of γ-tubulin-positive poles analyzed per oocyte in A. These experiments were repeated five times and the total numbers of oocytes examined are indicated above the graph bars. (D) Prior to fixation detection of MTOCs (γ-tubulin; green in merge), spindle microtubules (α-tubulin; gray in merge), kinetochores (CREST; red in merge) and DNA (DAPI; blue), oocytes were exposed to ice-cold medium to allow visualization of kinetochore-attached microtubules. The optical zooms were taken from the indicated boxes and show two functional poles at the same end of the spindle. Arrows indicate microtubules making a connection to kinetochores. These experiments were repeated twice, and 25 and 30 oocytes were examined in the control and 5-Itu-treated groups, respectively. (E,F) Half of the oocytes were injected with non-degradable GFP-tagged Cyclin B cRNA (Ccnb1-Δ90-Gfp) whereas control oocytes were injected with Gfp control cRNA, and all the oocytes were in vitro matured in the presence of EtOH or 500 nM of 5-Itu to metaphase I (7 h) prior to fixation and confocal microscopy. Arrows highlight unclustered MTOCs. (F) Quantification of the percentage of oocytes analyzed in E with unfocused spindle poles. These experiments were repeated twice and the total numbers of oocytes examined are indicated above the graph bars. The data are expressed as mean±s.e.m. Scale bars: 10 µm. *P<0.05; **P<0.01 [Student's t-test (B,C) or one-way ANOVA (F)].

Fig. 2.

Haspin-inhibited oocytes fail to cluster MTOCs. (A) Time-lapse imaging of live mouse oocytes expressing mEGFP–CDK5RAP2 (green, MTOCs) and H2B–mCherry (red, chromosomes) stained with SiR–tubulin (microtubules, gray). Mouse oocytes were treated with either ethanol (control, upper panel) or 1 µM 5-Iodotubercidin (5-Itu) (middle and lower panels). A maximum intensity z-projection of 16 sections of selected times are presented. White and yellow arrowheads indicate MTOCs outside of the clustered spindle pole; yellow arrows indicate microtubules that originate at spindle poles but do not make contact with chromosomes or the metaphase plate. The upper panel corresponds to Movie 1, the middle panel corresponds to Movie 2 and the lower panel corresponds to Movie 3. Time points are indicated relative to the time after nuclear envelope breakdown (hh: min). Scale bar: 10 µm. (B) Distribution of MTOCs (green) and chromosomes (red) along the spindle axis at 3 h after NEBD and at metaphase I in control and 5-Itu-treated oocytes. Distribution is expressed relative to the total area, and the length of spindle axis is arbitrarily set to 1. Data for individual representative oocytes are shown. (C) Quantification of the percentage of filmed oocytes with defective MTOC clustering. (D) Quantification of the percentage of filmed oocytes with chromosome segregation errors (n=16 for EtOH, and 12 for 5-Itu). *P<0.05; **P<0.01 (Fisher's exact test).

Fig. 3.

Haspin inhibition at metaphase I causes MTOC instability. (A) Schematic to illustrate the timing of haspin inhibition as it correlates with the cell cycle stages. Meiotic resumption occurs at the start of the experiment when milrinone is washed from the maturation medium. Nuclear envelope breakdown, NEBD; prometaphase I, Pro-Met I; metaphase I, Met I. (B,C) Fully grown prophase-I-arrested oocytes were in vitro matured for 5 h prior to fixation (Pro-Met I) or followed by an additional maturation for 2.5 h in the presence of ethanol (EtOH) or 1 µM of 5-Iodotubercidin (5-Itu) prior to fixation. MTOCs (γ-tubulin; red in merge), spindle microtubules (α-tubulin; green in merge) and DNA (DAPI; blue in merge) were detected by performing confocal microscopy. Shown are representative z-projections and the arrows illustrate the multiple poles. (C) Quantification of the percentage of oocytes with multipolar spindles. The data are expressed as mean±s.e.m. The experiment was repeated three times and the total numbers of oocytes examined are indicated above the graph bars. **P<0.01 (one-way ANOVA). (D) Time-lapse imaging of live mouse oocytes expressing mEGFP–CDK5RAP2 (green, MTOCs) and H2B–mCherry (red, chromosomes) stained with SiR–tubulin (microtubules, gray). Mouse oocytes were treated with either ethanol (control, upper panel; n=15) or 1 µM 5-Iodotubercidin (5-Itu; n=15) (lower panel). Maximum intensity z-projection of 16 sections of selected times are presented. The white arrowheads show MTOCs that are no longer clustered and the white arrows indicate microtubules that emanate from a MTOC but do not make contact with a chromosome. Time points are indicated relative to the time after addition of 5-Itu (hh:min), which was added in late prometaphase I (5 h). The upper panel corresponds to Movie 4 and the lower panel corresponds to Movie 5. Scale bars: 10 µm.

Fig. 4.

AURKA localization and activation are not perturbed at MTOCs when haspin is inhibited. (A–D) Fully grown prophase-I-arrested oocytes were in vitro matured in the presence of ethanol (EtOH) or 500 nM of 5-Iodotubercidin (5-Itu) to metaphase I (7 h) prior to fixation and detection of MTOCs (γ-tubulin; red in merge), spindle microtubules (α-tubulin; green in merge) and DNA (DAPI; blue). (B,D) Quantification of the average pixel intensities of AURKA (B) or pAURKA (D) at MTOCs. The values in EtOH-treated oocytes were set to 1. The data are expressed as mean±s.e.m. These experiments were conducted two times and the total numbers of oocytes examined are indicated above the graph bars. There was no significant difference between treated and control oocytes (Student's t-test). Scale bars: 10µm.

Fig. 5.

AURKC colocalizes with MTOCs. (A–C) Fully grown prophase-I-arrested oocytes were in vitro matured for 0 (Pro I, prophase I), 2 (Pro Met I, prometaphase I) or 7 h (Met I, metaphase I) prior to fixation and immunocytochemistry. (A) In two groups, metaphase I oocytes were treated with Nocodozole (Noc) for 10 min or Taxol for 45 min to perturb the spindle. AURKC (green in merge), MTOCs (γ-tubulin; red in merge) and DNA (DAPI; blue) were detected by confocal microscopy; shown are representative z-projections. The arrows indicate co-localization of AURKC and γ-tubulin. 53 oocytes were examined. (B) Representative z-projection, obtained with confocal microscopy, of colocalization of AURKC (green in merge), MTOCs (γ-tubulin; red in merge) and DNA (DAPI; blue) in ethanol (EtOH) or 5-Iodotubercidin (5-Itu) treatment groups at metaphase I. (C) Quantification of the intensity of EtOH- or 5-Itu-treated AURKC at MTOCs in oocytes. The values were normalized to the DNA signal and the average values in EtOH-treated oocytes were set to 1. These experiments were conducted three times and the total numbers of oocytes examined are indicated above the graph bars. The data are expressed as mean±s.e.m. *P<0.05 (Student's t-test). (D) Fully grown prophase-I-arrested oocytes were injected with GFP–haspin cRNA and matured to metaphase I (7 h) prior to fixation and confocal imaging. The brightness of the GFP channel was increased so that the portion of protein at the spindle poles could be visualized, thereby causing saturation on the chromosomes. Twenty oocytes were examined. Scale bars: 10µm.

Fig. 6.

AURKC perturbation causes unclustered metaphase I spindle poles in a CPC-independent manner. (A–F) Fully grown prophase-I-arrested oocytes were in vitro matured to metaphase I (7 h) prior to fixation and detection of MTOCs (γ-tubulin, red in merge) or pINCENP (pink in merge), spindle microtubules (α-tubulin; green in merge) and DNA (DAPI; blue). (A) Oocytes were microinjected with mCherry (control) or Aurkc-L93A-mCherry (Aurka-LA-mCh) cRNA prior to maturation. The arrows indicate multiple MTOCs. (B) Quantification of the percentage of oocytes analyzed in A with unclustered poles. These experiments were conducted four times and the total numbers of oocytes examined are indicated above the graph bars. (C–F) Oocytes were microinjected with a morpholino oligonucleotide of either a scrambled sequence (control) or one designed to target INCENP [INCENP knockdown (KD)]. (D) Quantification of the intensity of pINCENP on chromosomes as shown in C. These experiments were conducted three times and the total numbers of oocytes examined are indicated above the graph bars. (F) Quantification of the percentage of oocytes analyzed in E with clustered MTOCs. These experiments were conducted two times and the total numbers of oocytes examined are indicated above the graph bars. The data are expressed as mean±s.e.m. Scale bars: 10 µm. **P<0.01; ****P<0.0001 (Student's t-test).

Fig. 7.

Haspin regulates bipolar spindle assembly through AURKC. (A,B) Fully grown prophase-I-arrested oocytes were microinjected with the indicated cRNA and matured in the presence EtOH or 500 nM 5-Itu. The GFP channel (green in merge) demonstrates the localization of the GFP-tagged proteins. The arrows indicate MTOCs. (B) Quantification of the percentage of oocytes analyzed in A with unclustered MTOCs. ‘+’ indicates the addition of 500 nM 5-Itu, and a, b, c indicates the isoform of Aurk cRNA used in microinjection. The data are expressed as mean±s.e.m. These experiments were conducted two times and the total numbers of oocytes examined are indicated above the graph bars. Scale bar: 10 µm. *P<0.05; ***P<0.001 (one-way ANOVA).