An evolutionary perspective on meiotic kinetochore architecture and function in oocytes – EvoMeioForce

To answer these questions, we study kinetochore attachments in detail by high and super resolution microscopy techniques, determine the tension applied on mono-or bioriented kinetochores with FRET sensors, and determine the segregation pattern by latest state-of-the-art live imaging, and when kinetochore structure is changed.
Even though two different model systems are used by the two partners, both rely on latest state of the art imaging techniques of both fixed and live samples, single cell analysis and genetics to answer the questions raised such as planned.

The Wassmann team has been able to address the main question, namely whether kinetochore structure determines the segregation pattern of the genetic material in oocyte meiosis. More specifically, the team was able to show that sister kinetochores, which appear fused up to metaphase in meiosis I, individualize into two distinguishable entities in anaphase I, even though they are still mono-oriented. Importantly, they could show that this kinetochore individualization is required in the subsequent second meiotic division, to be able to separate sister chromatids. The kinetochore individualization requires the cleavage activity of the protease Separase. In absence of Separase, as the team could show by using oocytes derived of conditional knock-out mice, kinetochore individualization does not take place in meiosis I, and oocytes are unable to segregate sister chromatids in the following meiosis II.

The Dumont lab is in the process of addressing the main question of this ANR project. We are currently analyzing kinetochore formation in chromosomes from a series of meiosis mutants that display abnormal chromosomal organization. Our results so far suggest that kinetochores are defined “by default” on any exposed chromatin surface in the C. elegans oocyte. In parallel, we are finalizing a manuscript that directly relates to the question of kinetochore assembly and function and thus to the present project, and that aims at defining the precise function of some kinetochore components in the segregation of meiotic chromosomes. More specifically, we dissected a kinetochore pathway that controls kinetochore microtubule dynamics and we show that perturbing this pathway leads to severe meiotic chromosome segregation defects and to oocyte aneuploidy. We anticipate that this manuscript will be submitted for publication within the next few weeks, providing that the country is not confined again and our institute shut down.

The work for up to now shows that the largely accepted model for sister chromatid segregation, namely the need of bipolar tension tearing sister chromatids apart in meiosis II, is wrong. Overall, the team has already answered the main question the coordinator was asking in the proposal, namely whether the pattern of chromosome segregation is determined by the cell cycle stage, or the chromosome itself. Sister kinetochore fusion or individualization is the crucial element that determines the segregation pattern, hence the information is intrinsic to the chromosome itself.For the remaining time of the project the Wassmann team will progress as planned by determining kinetochore attachment and tension applied by microtubules, using FRET sensors that are currently being established, and super resolution microscopy. Currently, the team is establishing a protocol for imaging mouse oocytes with expansion microscopy. Acquisitions will be done with the newly acquired super resolution microscope at the IBPS. Their work opens up new scientific questions, such as to identify the Separase substrate that has to be cleaved in meiosis I, for sister chromatids to segregate in meiosis II. The Dumont team will hopefully progress as planned and finalize a second story that aims at defining the precise function of each kinetochore sub-complex in the C. elegans oocyte using high-resolution microscopy.

Two manuscripts have been published by the Wassmann team (one in the Embo Journal), and two more manuscripts are in preparation. The Dumont team is currently writing up a manuscript for publication, and expecting to submit another publication later this year.

Cell division is crucial for the development of complex organisms, for the homeostasis of tissues, and for the reproductive capacity of individuals. While most somatic cells are diploid and proliferate through mitosis, multiplication of sexually reproducing species relies on haploid gametes that are generated through a specialized cell division process called meiosis. To achieve this reduction in ploidy, two rounds of chromosome segregation follow a single phase of genome replication. Inaccuracy in this process leads to gametes that carry an incorrect number of chromosomes and to aneuploid embryos after fertilization. In their vast majority, these are non-viable and lead to spontaneous abortion: defective meiotic division is therefore a major obstacle in achieving reproduction. However, the key principles that drive this process are still poorly understood, one main reason being the diversity of the molecular scenarios that have been adopted across evolution to regulate oocyte chromosome segregation.

Unlike any other type of cell division, reductional meiosis I leads to the segregation of chromosomes and not sister chromatids. This requires sister kinetochores, the macromolecular assemblies that link each chromosome to spindle microtubules, to function as a single unit. How this meiotic adaptation, which is essential for successful gamete production and reproduction, is achieved remains unclear. To dissect the key components of successful oocyte meiotic chromosome segregation, we propose to carry out a multi-disciplinary approach, combining two powerful model organisms, the nematode Caenorhabditis elegans and mice, with the use of cutting edge high-resolution live and electron microscopy methods. C. elegans and mice both produce haploid oocytes, but display drastically different chromosomal and kinetochore architectures. Our common project should therefore also provide an evolutionary perspective on oocyte meiosis. For this, we will:

(AIM1) analyse meiotic kinetochore assembly and composition by immunostaining, live cell imaging and super resolution microscopy. Microtubule attachment sites will be identified by 3D-electron tomography, and the site and amplitude of forces applied to meiotic kinetochores in meiosis I and II will be determined with FRET-based tension sensors.
(AIM 2) we will then assess whether a structure physically fusing sister kinetochores can be detected by performing Serial Block Face-Scanning Electron Microscopy (SBF-SEM) on high pressure frozen oocytes to reconstruct the whole meiosis I kinetochore in 3D. We will ask whether co-orientation and fusion of sister kinetochores depend on centromere-localized cohesion, on recombination, and/or on a protein that was previously proposed to fuse kinetochores together in mouse oocyte meiosis I.
(AIM 3) finally, we will determine whether the meiotic segregation pattern is determined intrinsically by the chromosomes and/or by the cell cycle stage of the oocyte.

Functional data will be obtained by employing a large panel of mouse and C. elegans genetic tools that are already available in the two partner groups, and sophisticated multimodal and latest state of the art. The combined expertise of the two partners and the large panel of tools already generated by both teams should allow successful implementation of the project to generate results that will have important implications for our understanding of meiotic cell division in oocytes.