An approach from fission yeast to study mammalian meiosis

Motivation: Nowadays, the major cause of human infertility is woman age-related infertility. The genetic quality of female gametes drastically decreases with age due to the increase of aneuploidy, result of a deficient meiosis, which severely impairs embryo development and most frequently leads to miscarriage. Because studying human meiosis is hampered by technical, biological and ethical constraints, research is mainly carried out in various model organisms. Among these, the fission yeast Schizosaccharomyces pombe has served it well, but species-specific differences keep it distant from the human case. Here, a novel fission yeast model whose meiosis approaches to that of mammals, is used to study this process aiming to obtain more representative results that can be better extrapolated to human.Methods: Strains are generated via cross and tetrad analysis and cultured according to standard protocols. Meiosis is monitored via live timelapse fluorescence microscopy, using fluorescently tagged proteins to follow subcellular structures.Results: In this model, virtually all meiotic cells show severe defects in chromosome segregation in both meiosis I and II, leading to disruption of the whole gametogenesis. However, although nuclear divisions are not effective, DNA masses experiment poleward segregating movements, unveiling an insufficient but maintained functionality in this aberrant meiosis.Conclusions: Despite meiosis progression is disrupted in this model, there exists a conservation of certain functionality. Interpreting this meiosis as a hybrid between yeast and mammal cases, these findings reflex the discrepancies between higher and lower eukaryotes meiosis, probably being these mismatches responsible for the observed defects. Also, this scenario opens up the possibility of studying the minimal, basic principles of meiosis in this model to later extrapolate them up to mammals and even humans

Strategies to improve the robustness of acentrosomal spindle formation in female meiosis

In meiosis, centrosomes are so important because they organize microtubules and nucleation of the spindle for a correct chromosomal segregation in eukaryotics cells. Human female oocytes lack centrosomes, so microtubules must self-assembly, which can cause mistakes in the process and diseases to the embryo. To study the molecular mechanisms supporting acentrosomal spindle, we are using the fission yeast Saccharomyces pombe as model scenario. In this organism, spindle pole bodies (SPBs), the functional equivalents of centrosomes, are sitting on the nuclear envelope (NE), which is dissasembled in each cell cycle by activating proteins like Sad1 and Bqt1, that mediate chromosome-NE contacts (Pineda-Santaella & Fernández-Álvarez, 2019). Based on these findings, our aims are making the acentrosomal spindle more robust and minimizing chromosomal segregation errors. In order to get then, we want to analyze the effects of overexpression of Cls1p, a cytoplasmic linker associated protein (CLASP) that stabilizes specific groups of MTs in S. pombe and has two homologous proteins in humans, CLASP1 and CLASP2. They contribute to the formation and maintenance of the spindle midzone by promoting MT rescue events (Al-Bassam et al., 2010). On the other hand, there are another important proteins in this process, like Klp6, a kind of kinesin-8, whose homologous proteins in humans are Kif18A, Kif18B, and Kif19. An in vivo study suggets that Klp6 binds to the tubulin triggering the birth of new MTs and promoting nucleation and catastrophe at the growing MT tip (Erent et al., 2012). Deletion or knockdown of Klp6 leads to longer spindles and defects in its assembly and position in many cases (Gergely et al., 2017), but we suggest that a longer acentrosomal spindle could also be stronger and more stable. So, we also pretend to observe the impact of deletion of klp6 on the spindle behavior and chromosome movements. To perform that experiments, we have obtained two different mutants for klp6 and cls1 in a bqt1Δ sad1.2 background by crossing some strains with these characteristics and we are studying what happens in the cell nucleus by fluorescent microscopy, using a DeltaVision microscope. As a result, we expect that chromosomal segregation in mutants for cls1 and klp6 will be more efficient with respect to the mutant control, which has only bqt1Δ sad1.2, and, ultimately, improve the meiotic process in this context

Molecular basis of self-assembled spindles in fission yeast meiosis

Resumen del trabajo presentado en el EMBO workshop Meiosis, celebrado en Pamhagen (Austria), del 18 al 23 de junio de 2023In female meiosis in many metazoans, chromosomes are segregated by acentrosomal spindles, implying that spindle microtubules self-assemble, capture and pull apart chromosomes without the intervention of the centrosomes. Although it is thought that acentrosomal meiosis is not conserved in fungi, we described the formation of self-assembled microtubule arrays able to segregate chromosomes, in a fission yeast scenario where the contribution of the spindle pole bodies (SPBs) was specifically blocked during meiosis. Here, we show that this unexpected microtubule formation represents a bona fide type of self-assembled. Moreover, we compare both self-assembled and SPB-dependent spindles, revealing major similarities and differences. On one side, both spindles share a similar structural polarity and their elongation and structure depend on microtubule crosslinker Ase1/PRC1. On the other side, loss of the gamma-tubulin complex produces only partial defects on self-assembled spindles, whereas it produces a full abolishment of SPB-dependent spindles; further, self-assembled spindles partially depend on microtubule polymerase Alp14/XMAP215, whereas it is crucial for SPB-dependent spindles; plus, formation of self-assembled spindles significantly depends on the RanGTP gradient and kinetochores, while these elements are dispensable for SPB-dependent spindles. Our results reveal common features between fission yeast self-assembled and metazoan acentrosomal spindles, opening the possibility to harness the experimental advantages of fission yeast to get insight into the molecular basis of female gametogenesis

Spindle assembly without spindle pole body insertion into the nuclear envelope in fission yeast meiosis

Centrosomes represent the major microtubule organizing center (MTOC) in eukaryotic cells and are responsible for nucleation of the spindle, the vehicle of chromosome segregation. In human female meiosis, however, spindle assembly occurs in the absence of centrosomes or other MTOCs and microtubules are nucleated around chromosomes. In yeast, spindle formation in mitosis and meiosis depends on the activity of spindle pole bodies (SPBs), the functional equivalents of centrosomes; thus, SPBs and centrosomes use similar machineries to assemble spindles. Here, we develop a system to explore the molecular mechanisms supporting acentrosomal spindle formation using fission yeast meiosis as a model scenario. We achieve this situation by removing access of the SPBs to the nucleus after their duplication. Under these conditions, we observe self-assembly-based spindle formation in the nuclear environment, conferring an ability to segregate chromosomes independently of the SPBs. Our results open the possibility to utilize the experimental advantages of fission yeast for insights into the molecular basis of acentrosomal spindle formation in meiosis.This work was supported by Spanish Government (Ramon y Cajal program, RyC-2016-19659) to AF-A; AP-S by Pablo de Olavide University Fellowship (PPI1807

Analyzing self-assembled spindle dynamics in fission yeast meiosis using in vivo fluorescence imaging

Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles. The analysis of the dynamics of self-assembled spindles is a challenge due to the low availability of oocytes. Here, we present a protocol for analyzing self-assembled spindle dynamics in fission yeast meiosis using in vivo fluorescence imaging. We describe steps for starter culture preparation, meiosis induction, and sample preparation. We then detail procedures for acquisition and analysis of images of self-assembled spindles. For complete details on the use and execution of this protocol, please refer to Pineda-Santaella and Fernández-Álvarez (2019)1 and Pineda-Santaella et al. (2021).2.We thank all lab members for critical comments on the manuscript. This work was supported by PID2021-127232NB-I00 funded by the MCIN/ AEI /10.13039/501100011033 and by the “FEDER, Una manera de hacer Europa”, awarded to A.F.-A. The Institute of Functional Biology and Genomics (IBFG) has received funding through the program “Escalera de Excelencia” of the Regional Government of Castile and Leon (ref.: CLU-2017-03) and co-financed by the P.O. FEDER of Castile and León 14–20, and the Internationalization Project “CL-EI-2021-08-IBFG Unit of Excellence” of the Spanish National Research Council (CSIC), funded by the Regional Government of Castile and Leon and co-financed by the European Regional Development Fund (ERDF “Europe drives our growth”)

Centromere positioining governs telomere clustering

Trabajo presentado en el Gordon Conference Chromosome Dynamics: Chromosome Biology From Cellular, Molecular and Physical Perspectives, celebrado en Lucca, Barga (Italia), del 25 al 30 de junio de 202

Loss of kinesin-8 improves the robustness of the self-assembled spindle in Schizosaccharomyces pombe

Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles, the existence of which implies that microtubule spindles self-assemble without the participation of the centrosomes. Although it is thought that acentrosomal meiosis is not conserved in fungi, we recently reported the formation of self-assembled microtubule arrays, which were able to segregate chromosomes, in fission yeast mutants, in which the contribution of the spindle pole body (SPB; the centrosome equivalent in yeast) was specifically blocked during meiosis. Here, we demonstrate that this unexpected microtubule formation represents a bona fide type of acentrosomal spindle. Moreover, a comparative analysis of these self-assembled spindles and the canonical SPB-dependent spindle reveals similarities and differences; for example, both spindles have a similar polarity, but the location of the γ-tubulin complex differs. We also show that the robustness of self-assembled spindles can be reinforced by eliminating kinesin-8 family members, whereas kinesin-8 mutants have an adverse impact on SPB-dependent spindles. Hence, we consider that reinforced self-assembled spindles in yeast will help to clarify the molecular mechanisms behind acentrosomal meiosis, a crucial step towards better understanding gametogenesis.This work was supported by Ministerio de Ciencia e Innovación, Plan Nacional PGC2018-098118-A-I00 and Ramon y Cajal programme RyC-2016-19659 to A.F.-A.; and by Pablo de Olavide University ‘Ayuda Puente Predoctoral’ fellowship (PPI1803) to A.P.-S

Plasticity in spindle assembly during fission yeast meiosis

Resumen del trabajo presentado en el 2022 Gordon Research Conference on Meiosis celebrado en New London (US) del 5 al 10 de Junio de 2022Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles, the existence of which implies that microtubule spindles self-assemble without the participation of the centrosomes. Although it is thought that acentrosomal meiosis is not conserved in fungi, we recently reported the formation of a bona fide type of acentrosomal spindle, which were able to segregate chromosomes, in fission yeast mutants, in which the contribution of the spindle pole body (SPB; the centrosome equivalent in yeast) was specifically blocked during meiosis. Here, we have performed a comparative quantitative analysis of these self-assembled spindles and the canonical SPB-dependent spindle identifying similarities and differences between them; for example, both spindles have a similar polarity, but the location and function of the gamma-tubulin complex differs. We also show that the robustness of self-assembled spindles can be reinforced by eliminating kinesin-8 family members, whereas kinesin-8 mutants have an adverse impact on SPB-dependent spindles. The role of other kinesin proteins on the robustness of the self-assembled spindles in fission yeast is also exploring in our work. Hence, we consider that reinforced self-assembled spindles in yeast mediating by artificially manipulating the kinesin-8 family proteins will help to clarify the molecular mechanisms behind acentrosomal meiosis, a crucial step towards better understanding gametogenesis

Centromere positioning governs the telomere clustering

Resumen del trabajo presentado en el 11th International Fission Yeast Meeting, celebrado en Hiroshima (Japón), del 28 de mayo al 2 de junio de 2023Faithful meiotic progression in fission yeast requires the formation of the telomere bouquet, the 3D chromosome conformation where telomeres cluster at the spindle pole body (SPB) and centromeres dissociate from the nuclear envelope. Telomere clustering is supported by the LINC complex, the meiosis-specific proteins Bqt1 and Bqt2 together with the activity of the cytoplasmic dynein and the telocentrosome, a meiosis-specific microtubule-organising center [1,2,3]. However, the role of centromeres for the induction of telomere clustering has received little attention, being considered passive elements during the bouquet formation. In this work, we will present data supporting that one of the first signals to induce telomere clustering at the SPB comes unexpectedly from the centromeres. In fact, disruption of centromere positioning is sufficient to induce telomere bouquet-like conformations even in mitotic cells. Furthermore, in this scenario, cells undergo transcriptional reprogramming similar to that in the early stages of meiosis. Therefore, our data suggest that the centromeres harbour one of the first signals that trigger the formation of the telomere bouquet

The Rabl chromosome configuration masks a kinetochore reassembly mechanism in yeast mitosis

During cell cycle progression in metazoans, the kinetochore is assembled at mitotic onset and disassembled during mitotic exit. Once assembled, the kinetochore complex attached to centromeres interacts directly with the spindle microtubules, the vehicle of chromosome segregation. This reassembly program is assumed to be absent in budding and fission yeast, because most kinetochore proteins are stably maintained at the centromeres throughout the entire cell cycle. Here, we show that the reassembly program of the outer kinetochore at mitotic onset is unexpectedly conserved in the fission yeast Schizosaccharomyces pombe. We identified this behavior by removing the Rabl chromosome configuration, in which centromeres are permanently associated with the nuclear envelope beneath the spindle pole body during interphase. In addition to having evolutionary implications for kinetochore reassembly, our results aid the understanding of the molecular processes responsible for kinetochore disassembly and assembly during mitotic entry.This work was supported by the Spanish government, Plan Nacional project PGC2018-098118-A-I00, Ramon y Cajal program, RyC-2016-19659 to A.F.-A. and the program “Escalera de Excelencia” of the Junta de Castilla y León Ref. CLU-2017-03, co-funded by the P.O. FEDER of Castilla y León 14-20 and by the Pablo de Olavide University “Ayuda Puente Predoctoral” fellowship (PPI1803) to A.P.-S., and by the Spanish Education and Professional Formation Ministry, Research Collaboration Grant to D.L.-P