Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment
2018; Springer Nature; Volume: 19; Issue: 2 Linguagem: Inglês
10.15252/embr.201745225
ISSN1469-3178
AutoresIsma Bennabi, Isabelle Quéguiner, Agnieszka Kolano, Thomas Boudier, Philippe Mailly, Marie‐Hélène Verlhac, Marie-Émilie Terret,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle12 January 2018Open Access Transparent process Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment Isma Bennabi Isma Bennabi Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Isabelle Quéguiner Isabelle Quéguiner Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Agnieszka Kolano Agnieszka Kolano International Institute of Molecular and Cell Biology, Warsaw, Poland Search for more papers by this author Thomas Boudier Thomas Boudier orcid.org/0000-0002-0148-7733 Université Pierre et Marie Curie, Sorbonne Universités, Paris, France Search for more papers by this author Philippe Mailly Philippe Mailly Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Marie-Hélène Verlhac Corresponding Author Marie-Hélène Verlhac [email protected] orcid.org/0000-0001-8377-9010 Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Marie-Emilie Terret Corresponding Author Marie-Emilie Terret [email protected] orcid.org/0000-0001-5843-925X Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Isma Bennabi Isma Bennabi Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Isabelle Quéguiner Isabelle Quéguiner Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Agnieszka Kolano Agnieszka Kolano International Institute of Molecular and Cell Biology, Warsaw, Poland Search for more papers by this author Thomas Boudier Thomas Boudier orcid.org/0000-0002-0148-7733 Université Pierre et Marie Curie, Sorbonne Universités, Paris, France Search for more papers by this author Philippe Mailly Philippe Mailly Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Marie-Hélène Verlhac Corresponding Author Marie-Hélène Verlhac [email protected] orcid.org/0000-0001-8377-9010 Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Marie-Emilie Terret Corresponding Author Marie-Emilie Terret [email protected] orcid.org/0000-0001-5843-925X Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France Search for more papers by this author Author Information Isma Bennabi1, Isabelle Quéguiner1, Agnieszka Kolano2, Thomas Boudier3, Philippe Mailly1, Marie-Hélène Verlhac *,1,‡ and Marie-Emilie Terret *,1,‡ 1Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Equipe labellisée FRM, Paris, France 2International Institute of Molecular and Cell Biology, Warsaw, Poland 3Université Pierre et Marie Curie, Sorbonne Universités, Paris, France ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +33144271082; E-mail: [email protected] *Corresponding author. Tel: +33144271692; E-mail: [email protected] EMBO Reports (2018)19:368-381https://doi.org/10.15252/embr.201745225 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitotic spindles assemble from two centrosomes, which are major microtubule-organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an "inside-out" mechanism, ending with establishment of the poles. We used HSET (kinesin-14) as a tool to shift meiotic spindle assembly toward a mitotic "outside-in" mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic-like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique "inside-out" mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes. Synopsis Meiotic spindles in mouse oocytes form 'inside-out', ending with a barrel-shaped pole. Shifting spindles towards a mitotic-like 'outside-in' mode leads to severe chromosome alignment defects, indicating that 'inside-out' spindle assembly is essential to prevent aneuploid gametes. Moderate overexpression of the microtubule crosslinking motor HSET forces meiotic spindles to become mitotic-like. Mitotic-like spindles show rapid bipolarization and pole assembly coupled with focused poles. Switching spindle morphogenesis leads to severe chromosome alignment and segregation defects. Introduction Animal cells generally assemble mitotic spindles using an "outside-in" mechanism that relies on centrosomes acting as dominant microtubule-nucleating centers (MTOCs). The two centrosomes define the spindle poles and thus the spindle axis along which chromosome segregation will take place at anaphase 12. Oocytes however lack canonical centrosomes, the centrioles being lost before the meiotic divisions occur 34. Interestingly, it was shown in Drosophila that maintaining functional supernumerary centrioles during female meiotic divisions leads to abnormal meiosis and aborted embryonic development 5, highlighting the fact that centriole loss is essential for successful sexual reproduction. In mouse oocytes, microtubules are nucleated from chromatin and multiple acentriolar microtubule-organizing centers (aMTOCs) composed of pericentriolar material 6789. These aMTOCs are perinuclear before meiotic divisions and fragment at NEBD (nuclear envelope breakdown) to become evenly distributed around chromatin 1011. Following NEBD, microtubules become nucleated and stabilized first around chromatin, forming a microtubule ball, and then organized into a stable central array via microtubule motors and microtubule-associated proteins, which sort and orient the microtubules 121314151617. aMTOCs are then progressively sorted along this central array 16. Following spindle bipolarity setup, the aMTOCs become clustered to establish the spindle poles 17. Meiotic spindles in oocytes are thus assembled "inside-out". Spindle assembly in oocytes is a very slow process. Spindle bipolarization is achieved by 4 h in mice 1213 and by around 7 h in humans 18, thus occupying about half the transition time from NEBD to anaphase in these species. It mirrors the long duration of the first meiotic division, as meiosis I requires 8–12 h in mice and more than 20 h in humans 18. In addition, whereas spindle poles are organized by two centrosomes in mitosis, pole formation is different in meiosis. In mouse oocytes, poles are organized by multiple aMTOCs. Thus, meiotic spindle poles are often less focused than mitotic ones, having this typical barrel-shaped aspect. Are these unique "inside-out" spindle assembly and organization required for meiotic spindle function, that is, segregating chromosomes? To answer this question, we switched meiotic spindle assembly toward a more mitotic-like mode, with rapid bipolarity and focused pole assembly, and looked at chromosome alignment and segregation. To do so, deregulation of HSET levels was used as a tool to alter early stages of spindle morphogenesis. The kinesin-14 HSET is a minus-end-directed microtubule cross-linking motor important for regulating spindle assembly, spindle length, and pole organization 19202122232425. During mitosis, HSET can slide anti-parallel microtubules apart and sort them into parallel bundles 262728. In contrast, when the orientation of two opposing microtubules is parallel, HSET cross-links them and transports them to the poles 2326. We show here that a slight increase in HSET levels accelerates spindle formation, in particular spindle bipolarization and aMTOCs clustering. Importantly, this leads to severe chromosome alignment abnormalities. In an unexpected manner, the unusual length of meiosis I (8 h) is not sufficient to correct early spindle morphogenesis defects, contributing to chromosome misalignment and mis-segregation. Thus, the unique "inside-out" spindle assembly and organization prevent aneuploidy in female gametes. Results Altering the timing of spindle bipolarization To modify spindle morphogenesis, we developed an HSET gain-of-function approach. The localization of endogenous HSET was first analyzed in mouse oocytes, by performing immunofluorescence experiments on fixed oocytes. We found that endogenous HSET is localized on the spindle in meiosis I (Fig EV1A, left panel). HSET dynamics and localization were followed in living oocytes, by expressing an exogenous GFP-tagged HSET wild-type (WT) construct. Our exogenous GFP-HSET WT probe displayed the same spindle localization as endogenous HSET (Fig EV1A, middle panel, immunofluorescence) and remained associated with the spindle throughout meiosis I (Fig EV1B, live microscopy). However, HSET WT exogenous expression must be tightly controlled, since too much of it induced spindle collapse and mono-aster formation (see Materials and Methods). We therefore performed experiments with a maximum HSET WT overexpression of 1.6-fold in the whole oocyte (Fig EV1C, immunofluorescence quantification) corresponding to a 4.2-fold accumulation of HSET in the spindle (Fig EV1D, immunofluorescence quantification). Meiotic spindle assembly in the context of an HSET WT overexpression was analyzed by time-lapse spinning disk microscopy. In controls, microtubules formed bipolar spindles within ~4 h after NEBD (Fig 1A, upper panel). In contrast, spindle bipolarization took place much more rapidly in oocytes overexpressing HSET WT (Fig 1A, lower panel and B), skipping the microtubule ball stage described in Ref. 13. Indeed, the average time of bipolarization setup was achieved in 4 h and 3 min in controls compared to 1 h and 19 min in oocytes overexpressing HSET WT (Figs 1B and EV2A). Click here to expand this figure. Figure EV1. Endogenous HSET and slightly overexpressed exogenous HSET localize to the spindle throughout meiosis I Immunofluorescence showing endogenous HSET (left panel), exogenous HSET WT (middle panel), and exogenous HSET N593K (right panel). Endogenous and exogenous HSET display the same localization: they all localize on the spindle, as observed here on fixed oocytes at NEBD+4h30 (HSET antibody: green; DNA: blue). Note that the spindle is elongated in HSET WT expressing oocytes. Scale bar 10 μm. Time-lapse spinning disk confocal microscopy of an oocyte expressing GFP-HSET WT (green) and Histone-RFP (blue). GFP-HSET WT localizes on the spindle throughout meiosis I. The white asterisk marks a chromosome outside of the metaphase plate. Scale bar 5 μm. HSET total fluorescence intensity measured in the whole cell for fixed oocytes at NEBD+4h30 expressing HSET WT or not (Ctrl). Data are represented as mean ± SD. The ratio of total HSET WT overexpression is 1.6. HSET normalized fluorescence intensity measured on the spindle of fixed oocytes at NEBD+4h30 expressing HSET WT or not (Ctrl). Data are represented as mean ± SD. The ratio of HSET OE on the spindle is 4.2. Time-lapse spinning disk confocal microscopy of an oocyte expressing GFP-HSET N593K (green). GFP-HSET N593K displays a localization similar to endogenous or GFP-HSET WT on the spindle throughout meiosis I. Scale bar 10 μm. GFP total fluorescence intensity measured in the whole cell for live oocytes at NEBD+7h expressing GFP-HSET WT or GFP-HSET N593K. Data are represented as mean ± SD. Statistical significance of differences is assessed with a t-test: **P-value = 0.004. Download figure Download PowerPoint Figure 1. Modification of the timing of spindle bipolarization Time-lapse spinning disk confocal microscopy of oocytes expressing GFP-EB3 (gray) alone (Ctrl, upper panel) or together with HSET WT (lower panel). Spindle bipolarization is advanced in HSET WT expressing oocytes compared to controls. Scale bar 10 μm. Graph showing the kinetics of spindle bipolarization in controls (gray squares) vs. HSET WT oocytes (blue dots). The kinetics of bipolarization is accelerated in oocytes overexpressing HSET WT compared to controls. Time-lapse spinning disk confocal microscopy of oocytes expressing GFP-EB3 (gray) treated (HSET Inh, lower panel) or not (Ctrl, upper panel) with the HSET inhibitor AZ82. Spindle bipolarization is delayed in oocytes inhibited for HSET. Scale bar 10 μm. Graph showing the kinetics of spindle bipolarization in controls (gray squares) vs. oocytes inhibited for HSET (purple diamonds). The kinetics of bipolarization is delayed in oocytes inhibited for HSET compared to controls. Time-lapse spinning disk confocal microscopy of control oocytes (Ctrl, upper panel) and oocytes expressing HSET N593K (HSET N593K, lower panel). Spindle bipolarization is slightly advanced in oocytes overexpressing HSET N593K compared to controls. All oocytes were incubated with SiR-Tubulin (gray). Scale bar 10 μm. Graph showing the kinetics of spindle bipolarization in controls (gray squares) vs. HSET N593K oocytes (dark blue dots). The kinetics of bipolarization is modestly affected in oocytes overexpressing HSET N593K compared to controls. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. HSET levels control the timing of spindle bipolarization Histogram showing the mean time of bipolarization setup (data are represented as mean ± SD). Bipolarity was scored when two poles were distinguishable. The mean time of bipolarization setup for oocytes overexpressing HSET WT is 1 h and 19 min (blue bar), compared to 4 h and 3 min for controls (gray bar), ***P-value < 0.0001, compared to 6 h and 55 min for oocytes inhibited for HSET (purple bar), ***P-value < 0.0001. Statistical significance of differences is assessed with a Mann–Whitney test. Histogram showing the mean time of bipolarization setup in controls vs. HSET N593K expressing oocytes (data are represented as mean ± SD). Bipolarity was scored when two poles were distinguishable. The mean time of bipolarization setup for oocytes overexpressing HSET N593K is 2 h and 36 min (dark blue bar), compared to 3 h and 10 min for the controls (gray bar). Statistical significance of differences is assessed with a t-test: *P-value = 0.034. Graph representing the kinetics of spindle bipolarization in controls (gray) vs. oocytes inhibited for HSET with AZ82 (purple) or CW069 (violet). The number of oocytes analyzed is written in parentheses. Histogram showing the mean time of bipolarization setup. Bipolarity was scored when two poles were distinguishable. Data are represented as mean ± SD. The mean time of bipolarization setup for controls (gray bar) is 4 h and 18 min, compared to 6 h and 55 min for oocytes inhibited for HSET with AZ82 (purple bar), compared to 7 h and 0 min for oocytes inhibited for HSET with CW069 (violet bar). Statistical significance of differences is assessed with a Mann–Whitney test: **P-value = 0.007, ***P-value < 0.0001, not significant (n.s.) P-value = 0.929. The number of oocytes analyzed is written in parentheses. Download figure Download PowerPoint The gain-of-function analysis was complemented with an HSET loss-of-function approach. To do so, oocytes were treated with AZ82, a small molecule inhibitor of HSET 2930, and meiotic spindle assembly was followed using time-lapse spinning disk microscopy. Spindle bipolarization was delayed in HSET-inhibited oocytes (HSET Inh) compared to controls (Ctrl, Fig 1C), requiring 6 h and 55 min in these oocytes (Figs 1D and EV2A). The delay of spindle bipolarization observed with AZ82 could be phenocopied using another allosteric inhibitor of HSET, CW069 (Fig EV2C and D), structurally unrelated to AZ82 31. Taken together, and in contrast to previously published observations 20, these results suggest that HSET levels modulate the timing of meiotic spindle bipolarity in meiosis I. To understand how HSET drastically impacts the timing of spindle bipolarization, we took advantage of a GFP-HSET mutant N593K (HSET N593K) that can cross-link but does not slide microtubules 23. GFP-HSET N593K localized on the spindle (Fig EV1A, right panel, immunofluorescence) and had similar distribution along the spindle as GFP-HSET WT (compare Fig EV1B and EV1E, live microscopy). It reached even higher expression levels at NEBD+7h (Fig EV1F, quantification of live microscopy). The timing of spindle bipolarization was only slightly advanced in oocytes overexpressing HSET N593K compared to controls (Figs 1E and F, and EV2B). This suggests that, for the most part, changes in the timing of spindle bipolarization require microtubule sliding by HSET. Accelerating spindle pole assembly Because spindle bipolarization occurs precociously in oocytes overexpressing HSET WT, we next analyzed the consequences of its overexpression on sorting of the aMTOCs. This process occurs concomitant with spindle bipolarization and is followed by aMTOC clustering, which allows spindle pole focusing. To do this, the behavior of aMTOCs was followed by time-lapse microscopy, using mCherry-Plk4 (Polo-like kinase 4) as a marker 32. We performed an automated 3D analysis of aMTOCs within the spindle. For that, we developed a Fiji plug-in that converts images obtained using live microcopy to binary images and in 3D finds the spindle poles and calculates the distance of each aMTOC to the closest pole (see Materials and Methods; Fig EV3A). In addition, this plug-in allows extraction of the number and distribution of the aMTOCs together with spindle measurements (length, central width, spindle pole width). The measurements were performed at three time points during meiosis I, spanning the critical steps of spindle morphogenesis in controls (Figs 2A and EV3C, middle panels). Click here to expand this figure. Figure EV3. Spindle morphogenesis after perturbation of HSET levels Principle of the automated 3D analysis of aMTOCs within the spindle. We developed a Fiji plug-in that converts images from spinning disk confocal live microcopy (here a spindle region magnification of an oocyte expressing GFP-EB3 (green) and mCherry-Plk4 (red) at NEBD+6h30, left panel) to binary images (middle panel), and then to 3D images (right panel). aMTOCs sorting in controls (gray dots) and oocytes overexpressing HSET WT (blue dots) at NEBD+1h30, +4h30, and +6h30. Each dot is one aMTOC. The vertical axis plots the aMTOCs volume, and the horizontal axis represents an hemi-spindle starting from the central spindle to the pole (as written on the scheme). The distance of aMTOCs to the closest spindle pole is normalized by the spindle length. Binary images corresponding to Fig 2A. Scale bar 10 μm. Quantification in 3D of the spindle length in controls (gray dots) and oocytes inhibited for HSET (purple dots) at NEBD+1h30, +4h30, and +6h30. Each dot represents an oocyte, the number of oocytes analyzed is written in parentheses. Statistical significance of differences is assessed with a Mann–Whitney test: *P-value = 0.017, **P-value = 0.003, ***P-value < 0.0001. Download figure Download PowerPoint Figure 2. Acceleration of aMTOCs sorting and clustering Spinning disk confocal microscopy images showing spindle region magnifications of oocytes overexpressing HSET WT, controls and oocytes inhibited for HSET at NEBD+1h30, +4h30, and +6h30. All oocytes express GFP-EB3 (green) and mCherry-Plk4 (red). Scale bar 10 μm. aMTOC sorting in oocytes overexpressing HSET WT (blue dots) and controls (gray dots) at NEBD+1h30, +4h30, and +6h30. The dot plot represents the standard deviation of the repartition of aMTOCs along the axis of the spindle for each oocyte analyzed. Each dot represents an oocyte; the number of oocytes analyzed is written in parentheses. Statistical significance of differences is assessed with a t-test with Welch correction where needed: *P-value = 0.018, **P-value = 0.002. As shown on the scheme, when aMTOCs are not sorted, the standard deviation is high; in contrast, when aMTOCs are sorted to the poles, the standard deviation is low. aMTOCs clustering in oocytes overexpressing HSET WT (blue dots) and controls (gray dots) at NEBD+1h30, +4h30, and +6h30. The dot plot represents the number of aMTOCs per oocyte. Each dot represents an oocyte; the number of oocytes analyzed for each condition is written in parentheses. Statistical significance of differences is assessed with a t-test with Welch correction: *P-value = 0.011, **P-value = 0.003, ***P-value < 0.0001. Super resolution images of aMTOCs using SIM, in fixed controls and HSET WT expressing oocytes (pericentrin antibody: gray). Scale bar 5 and 2 μm. Quantification of aMTOCs volume from SIM super-resolution images. Control oocytes gray dots and HSET WT expressing oocytes blue dots. Statistical significance of differences is assessed with a t-test with Welch correction: *P-value = 0.0453. FRAP analysis of SiR-Tubulin in controls (gray) and in oocytes overexpressing HSET WT (blue) at NEBD+6h30. SiR-Tubulin was photobleached at spindle poles, and its fluorescence recovery was followed. The SiR-Tubulin fluorescence intensity was normalized so that 1 corresponds to the prebleached value and 0 corresponds to the value at the first time point after bleaching. For a single exponential recovery model, the halftime to fluorescence recovery in controls oocytes is t1/2 = 62 s compared to t1/2 = 55 s for oocytes overexpressing HSET WT. Data are represented as mean ± SD. Statistical significance of differences for the t1/2 is assessed with a Mann–Whitney test: P-value = 0.87. Download figure Download PowerPoint At NEBD+1h30, microtubules form a ball, with aMTOCs dispersed around it 13. At NEBD+4h30, spindle bipolarization is achieved and a robust central array of microtubules allows the progressive sorting of aMTOCs to the poles 16. At NEBD+6h30, the spindle poles begin to focus following clustering of the aMTOCs. In oocytes inhibited for HSET by treatment with AZ82, the spindle was not yet bipolar at NEBD+6h30 (Figs 2A and EV3C, right panels). Instead, these spindles remained in a ball-shape, as quantified in Fig EV3D. The diameter of the microtubule mass even decreased slightly between the first and last time points in the HSET-inhibited oocytes (Fig EV3D, purple dots, 25 μm at NEBD+1h30 vs. 22 μm at NEBD+6h30), whereas in control oocytes, the spindle elongated (Fig EV3D, gray dots, 26 μm at NEBD+1h30 vs. 33 μm at NEBD+6h30). Therefore, measurements of aMTOCs sorting and clustering were not relevant in oocytes inhibited for HSET and we focused our analysis on oocytes overexpressing HSET WT where spindle bipolarization is advanced. We first analyzed aMTOC sorting in controls and HSET WT oocytes (Figs 2A and EV3C, middle and left panels). To do so, the distribution of the aMTOCs was measured in 3D along the long axis of the spindle at the time points where the spindle is bipolar (Fig EV3B, each dot corresponds to one aMTOC, the horizontal axis represents an hemi-spindle from the central spindle to the pole, the distance of aMTOCs to the nearest spindle pole is normalized by the spindle length, and no measurements were conducted at NEBD+1h30 in controls since at that stage spindles are not yet bipolar). In controls, the spindle was bipolar at NEBD+4h30 and the aMTOCs were scattered along the spindle's long axis (Fig EV3B, upper panel, all the gray dots are homogeneously distributed along the hemi-spindle). At NEBD+6h30, the aMTOCs were partially sorted and began to accumulate at spindle poles (Fig EV3B, upper panel, gray dots). We also plotted the standard deviation of the distribution of aMTOCs along the axis of the spindle for each oocyte analyzed (Fig 2B, each dot represents one oocyte). Before aMTOCs are sorted, the standard deviation is high; in contrast, once they are sent to the poles, the standard deviation is low (Fig 2B, scheme). In controls, the difference between NEBD+4h30 and +6h30 was small, highlighting the fact that aMTOC sorting is a long and progressive process (Fig 2B, gray dots). In oocytes overexpressing HSET, the spindle was already bipolar at NEBD+1h30 and aMTOCs were scattered along its long axis (Fig EV3B, lower panel, blue dots are homogeneously distributed along the hemi-spindle), resembling the NEBD+4h30 time point in controls. At NEBD+4h30, the aMTOCs were partially sorted as indicated by their substantial accumulation at spindle poles (Fig EV3B, lower panel, blue dots), resembling the NEBD+6h30 time point in controls. By NEBD+6h30, aMTOCs were further sorted (Fig EV3B, lower panel, blue dots). The standard deviation of the distribution of aMTOCs along the axis of the spindle for each oocyte showed the same behavior (Fig 2B, blue dots): The standard deviation at NEBD+1h30 in oocytes overexpressing HSET WT was comparable to the standard deviation at NEBD+4h30 in the controls, and at NEBD+4h30 and 6h30, it was smaller than in the controls. Altogether, these results show that aMTOC sorting takes place precociously in oocytes overexpressing HSET WT. We then analyzed aMTOC clustering in controls and oocytes overexpressing HSET WT (Figs 2A and EV3C, middle and left panels). To do so, the number of aMTOCs per oocyte was counted in 3D (Fig 2C, each dot represents one oocyte). In controls, the number of aMTOCs diminished in parallel with meiosis I progression (Fig 2C, gray dots). This shows that aMTOCs tend to fuse and cluster during meiosis I. In oocytes overexpressing HSET WT, this process started earlier when the spindle bipolarized around NEBD+1h30, as evidenced by a reduced number of aMTOCs (Fig 2C, compare blue and gray dots). Later during meiosis I, the clustering of aMTOCs continued to be enhanced compared to controls (Fig 2C, compare blue and gray dots). Interestingly, aMTOCs were also more compact in oocytes overexpressing HSET WT compared to controls (Fig 2A and D). First, their organization was different: in controls, aMTOCs formed a typical O-shaped structure circumscribing the poles 9, whereas in oocytes overexpressing HSET WT, they formed a single round entity (Fig 2A and D). Second, they occupied a smaller volume as quantified from the N-SIM super-resolution images (Fig 2E). This suggests that HSET may play a role in the spacing of aMTOCs at spindle poles. We next assessed whether microtubule dynamics was altered in the hyper-clustered spindle poles of oocytes overexpressing HSET WT. To compare microtubule dynamics, we performed FRAP of SiR-Tubulin at spindle poles at NEBD+6h30 (Fig 2F). Essentially identical recovery curves were observed in oocytes overexpressing HSET WT and controls, indicating that microtubule dynamics at spindle poles was similar in the two groups. This strongly suggests that changes in microtubule nucleation or stability are not the root of the difference in spindle pole focusing. These results thus show that the timing of spindle morphogenesis is accelerated in oocytes overexpressing HSET WT: Spindle bipolarization is established precociously together with more efficient sorting and clustering of aMTOCs, markers of spindle pole assembly. We then analyzed the impact of accelerated kinetics on spindle shape. Shifting meiotic spindle morphology toward mitotic-like morphology To determine whether accelerating bipolarization and spindle pole formation affected global spindle shape, spindle length, central spindle width, and spindle pole width were measured at the same time points used to analyze aMTOC behavior. In oocytes overexpressing HSET WT, the spindle was already bipolar at NEBD+1h30. Strikingly, the spindle at this stage was extraordinarily long (Fig 3A, live microscopy), with a mean length of 36 μm and reaching a maximum of 54 μm (Fig 3B). As previously shown, this effect of HSET overexpression on spindle length required microtubule sliding 23, as HSET N593K expressing oocytes displayed spindle lengths similar to controls (Fig EV4A). However, in oocytes overexpressing HSET WT, the spindles progressively shortened (Fig 3B) to reach a size comparable to controls by NEBD+7h (Fig 3D). Figure 3. Turning meiosis I spindles into more mitotic ones A. Spinning disk confocal microscopy images showing spindle region magnifications of oocytes expressing GFP-EB3 (green), mCherry-Plk4 (red), and HSET WT at NEBD+1h30, +4h30, and +6h30. Scale bar 10 μm. B. Quantification in 3D of spindle length in oocytes ex
Referência(s)