Cell division relies on many steps, precisely synchronised, to ensure the fidelity of chromosome segregation. To achieve such complex and multiple functions, cells have evolved mechanisms by which one protein can participate in numerous events on the cell life. Over the past few years, an increasing number of functions have been assigned to the proteins of the nuclear pore complex (NPC) also called nucleoporins. NPCs are large complexes studded in the nuclear envelope, which control the nucleocytoplasmic transport. It is now known that nucleoporins participate in spindle assembly, kinetochore organisation, spindle assembly checkpoint, and all processes important for genome integrity maintenance. This work demonstrates that the nucleoporin ALADIN participates in mitosis, meiosis and in cilia. In both mitosis and meiosis, ALADIN is important for proper spindle assembly. In mitosis, it was also discovered that ALADIN is a novel factor in the spatial regulation of the mitotic regulator Aurora A kinase. Without ALADIN, active Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. Interestingly, mutations in ALADIN causes triple A syndrome and some of the mitotic phenotypes observed after ALADIN depletion also occur in cells from triple A syndrome patients. In meiosis, ALADIN contributes to trigger the resumption of meiosis in female mouse. Impairment of ALADIN from mouse oocyte slows spindle assembly, migration and reduces oocytes ability to extrude polar bodies during meiosis I, which concomitantly affects the robustness of oocyte maturation and impairs mouse embryo development. Nucleoporins were also found at the base of the cilia, a centriole-derived organelle that participates in differentiation, migration, cell growth from development to adulthood. Here it is shown that ALADIN is also localised at the base of the cilia. With this work, new ALADIN’s functions have been identified across cell division, as well as uncovered an unexpected relation between triple A syndrome and cell division.

Meiosis is a highly regulated process, partly due to the need to break and then repair DNA as part of the meiotic program. In this thesis, mechanisms of meiotic regulation are investigated, including the post-translational modification termed SUMOylation, and a potential novel negative regulator of error-prone DNA repair pathways. Post-translational modifications are widely used during meiotic events to regulate steps such as protein complex formation, checkpoint activation, and protein attenuation. In this thesis, we investigate how proteins that are obligatory components of the SUMO pathway, one such post-translational modification, affect the C. elegans germline. We show that UBC-9, the E2 conjugation enzyme, and the C. elegans homolog of SUMO, SMO-1, localized to germline nuclei throughout prophase I. Mutant analysis of smo-1 and ubc-9 revealed increased recombination intermediates throughout the germline, originating during the mitotic divisions. SUMOylation mutants also showed late meiotic defects including defects in the restructuring of oocyte bivalents and endomitotic oocytes (EMO). Increased rates of non-interfering crossovers (COs) were observed in ubc-9 heterozygotes, even though interfering COs were unaffected. We have also identified a physical interaction between UBC-9 and DNA repair protein MRE-11. ubc-9 and mre-11 null mutants exhibited similar phenotypes at germline mitotic nuclei and were synthetically sick. These phenotypes and genetic interactions were specific to MRE-11 null mutants as opposed to RAD-50 or resection-defective MRE-11. We propose that the SUMOylation pathway acts redundantly with MRE-11, and in this process MRE-11 likely plays a structural role. We also found a candidate negative regulator of non-homologous end joining through an RNAi screen using the mre-11(iow1) resection-defective mutant. Mutant analysis did not rescue the resection-defective phenotype when multiple CRISPR-generated nhr-2 deletion alleles were generated. Therefore, nhr-2 does not appear to be directly involved in DNA repair.

The fate of any lineage is contingent on the rate at which its genome changes over time. Genome dynamics are influenced by patterns of mutation and recombination. Mutations as the raw force of variation can be acted on independently during exchanges of homologous genetic regions via meiotic recombination. While molecular evolution in sexual lineages is impacted by both mutation and recombination, asexual lineage fate is primarily influenced by the mutation rate; recombination is often altered or absent in asexuals. Although multiple studies show accelerated mutation accumulation in asexual lineages that have lost recombination, virtually nothing is known about rate patterns when meiosis is retained. Here, I use parasitic wasps in genus Diachasma to investigate genome evolution in a recently-derived asexual lineage. I provide evidence that asexual Diachasma possess a canonical set of meiosis genes as well as high levels of genomic homozygosity. Taken together, these observations support an active, albeit modified, form of meiosis in this asexual lineage. In addition, I present the first documentation of accelerated mutation accumulation in the nuclear genome of a naturally-occurring, meiotically- reproducing organism. If harmful, these mutations could impede asexual lineage persistence and contribute strong support for the long-term benefits of sex.

Defects in meiotic prophase I events, resulting in aneuploidy, are a leading cause of birth defects in humans; however, these are difficult to study in mammalian systems due to their occurrence very early in development. The nematode, Caenorhabditis elegans, is an excellent model for prophase I studies as its gonad is temporally and spatially organized around these meiotic events. Homolog pairing, synapsis, meiotic recombination and crossover formation are essential to the proper segregation of chromosomes into the respective gametes, either the egg or sperm. Disturbances in these events leads to missegregation of chromosomes in the gametes in the meiotic divisions. Synapsis is especially critical in meiosis as it precedes and is required for meiotic recombination in C. elegans. The formation of the synaptonemal complex (SC) is fundamental to chromosomal synapsis, yet the molecular mechanisms of synaptonemal complex morphogenesis are largely unknown. The investigations described in this thesis were undertaken to better understand the molecular contributions to synaptonemal complex morphogenesis. Chapter One reviews knowledge of morphogenesis and its relationship to the events of meiotic prophase I. Recent studies in our laboratory have implicated AKIRIN, a nuclear protein with multiple biological functions, as having a role in synaptonemal complex disassembly, specifically preventing the aggregation of synaptonemal proteins (Clemons et al., 2013). As a result of our efforts to discern the mechanism by which AKIRIN regulates disassembly, we found that the highly conserved CSN/COP9 signalosome has a role in SC assembly, leading to defects in prophase I events and in MAPK signaling , leading to the arrest of nuclei in the later stages of meiosis. While the CSN/COP9 signalosome has been implicated in general fertility in C. elegans (Pintard et al., 2003), no role had been defined in earlier meiotic stages until this study. Chapter Two describes an RNAi enhancer/suppressor screen undertaken in the akir-1 mutant background. Several RNAi clones were selected for future study based on a reduction in brood size; one of which, csn-5/, is the focus of the analysis presented in Chapter 3. Chapter Three describes the phenotypic characterization of two CSN/COP9 signalosome subunits, csn-2 and csn-5. Alleles of both genes display synaptonemal complex protein aggregation and defects in mitotic cell proliferation, homologous chromosome pairing, meiotic recombination and crossover formation, leading to an increase in apoptosis. Oocyte maturation is also disrupted by a lack of MAPK signaling, resulting in a lack of viable oocytes, which renders the csnmutant homozygotes sterile. These findings support a model suggesting the CSN/COP9 signalosome has an essential role in regulating meiotic prophase I events and oocyte maturation. Chapter 4 describes the methodology used in this study. Chapter 5 provides a summary of the thesis findings and examines the future directions to extend this work.

The inherent asymmetry of female meiosis presents an opportunity for genetic material to gain an evolutionary advantage during the formation of the egg. Since centromeres mediate chromosomal segregation by forming the bridge between microtubules and chromosomes during cell division, they are loci that can drive, or selfishly evolve, during female meiosis by manipulating the process of entering the egg. Mimulus guttatus, a species of yellow monkeyflowers, has the best documented case of centromeric drive (Fishman and Saunders, 2008). Since homozygotes for drive have decreased pollen viability, lower seed counts, and poor reproductive success, CENH3 —the gene that encodes the H3 histone specific to centromeres— has evolved to suppress centromeric drive. CENH3 is duplicated in Mimulus, and the sequence variation of CENH3_A suggests that this paralog can suppress centromeric drive during female meiosis (Finseth et al. 2015). Our analysis of gene expression levels in meiotic and mitotic tissues indicates that both CENH3_A and CENH3_B are still expressed at similar levels, suggesting that the paralogs have not specialized for different roles in cell divisions. However, qPCR was only performed with nine tissue samples, so further analysis of gene expression with a larger sample set is needed to confirm whether or not the CENH3 paralogs have specialized roles in meiosis and mitosis.

Meiosis is a specialized cellular division occurring in organisms capable of sexual reproduction that leads to the formation of gametes containing half of the original chromosome number. Meiosis involves two cell divisions, the first of which segregates homologous chromosomes to opposite poles, reducing ploidy by half. In most organisms, this segregation requires crossovers, the exchange of DNA sequences between homologous chromosomes, which in turn, is dependent upon stable associations of homologs. In early meiotic prophase I, chromosomes form pairing interactions that bring chromosomes into close physical associations. The process of synapsis then stabilizes these pairing interactions throughout the homolog pair, and is mediated by the synaptonemal complex (SC), a meiosis specific protein complex. Absent or misregulated assembly of the SC prevents the stabilization of pairing interactions that are essential for meiosis, leading to chromosome missegregation. Divided into two main projects, my work aimed to further our understanding of the regulation of synaptonemal complex assembly. One project examined meiotic chromosomal movement by characterizing a relatively unstudied protein in C. elegans, FKB-6. We showed that FKB-6 is important for creating pauses between chromosome movements. These pauses are needed for allowing chromosomes to properly pair and thus allowing for proper SC assembly. In the absence of FKB-6, a decrease in pausing occurs which perturbs chromosome pairing and causes SC assembly defects. A second project examined the role of CUL-4, an E3 ubiquitin ligase, in meiotic prophase I. We show that CUL-4 plays a role in both SC assembly and meiotic recombination. This work exemplifies the multiple levels of control of SC assembly which still require further study.

Drosophila male is an example of achiasmatic meiosis which lacks crossingover and chiasmata during meiosis. Previous studies showed that homologous pairing of both euchromatin and centromeres is lost during middle prophase I, however, homologs are still connected as they form bivalents. The X-Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes specifically targeting heterochromatin regions to assay pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the fourth homologs pair at the heterochromatic region 61 and associate with the X chromosome throughout prophase I. The pairing of the fourth homologs is disrupted in the homolog conjunction complex mutants. Conversely, six tested heterochromatic regions of the major autosomes (second and third chromosomes) have proved to be largely unpaired after early prophase I. This suggests that pairing mechanism of the major autosomes may differ from the sex and fourth chromosomes; stable connections between major autosomal homologs might occur at different sites along chromosomes in different cells by analogy to chiasmata. Moreover, FISH analysis also revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion, suggesting that sister chromatid cohesion is incomplete within heterochromatin but with preferential sites in male meiosis.Modifier of Mdg4 in Meiosis (MNM) and Stromalin in Meiosis (SNM) are components of homolog conjunction complex and essential for homolog pairing and segregation in male meiosis. Using yeast two-hybrid assay and co-immunoprecipitation, we showed that the MNM and SNM interact with each other. Specifically, the BTB domain of MNM is responsible for the interaction with SNM, whereas FLYWCH domain of MNM is crucial for this interaction but does not directly interact with SNM. Additionally, point mutation analysis revealed that L9K replacement of the BTB domain weakened the MNM-SNM interaction and caused high frequencies of chromosome nondisjunction. In conclusion, these results provide a biochemical basis for the mechanism of homolog pairing and support the role of homolog conjunction complex in male meiosis.

The purpose of this project was to create molecular tools for the study of meiosis in Arabidopsis thaliana and to evaluate their effectiveness. Two types of transgenic plants were created with an intron-spliced hairpin RNA (ihpRNA) construct to target the AHP2 gene for RNA silencing. One had a constitutively expressed promoter; the other’s promoter was inducible with dexamethasone (DEX). Transformations with the constitutively expressed construct gave rise to ahp2RNAi plants with reduced AHP2 transcript levels, abnormal meioses and reduced fertility phenotypes. The creation of plants containing the dexamethasone-inducible construct was confirmed via PCR genotyping, and induction with DEX. However, the induction conditions tested do not appear to silence AHP2 as the transgenics had normal meiotic and reproductive phenotypes. Also a triple-locus, three color, fluorescent protein marker-tagged Arabidopsis line was created that will allow calculation of recombination frequencies for two intervals on chromosome 2 in both wild type and mutant plants.

In the presence of a non-fermentable carbon source, nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae undergo a meiotic program called sporulation to form gametes called spores. While four spores are produced under standard laboratory sporulation conditions, spore number is known to be regulated by carbon availability: under carbon-depleted conditions, yeast cells package a portion of the four haploid meiotic nuclei into spores. Our lab has demonstrated that these unpackaged meiotic products undergo programmed nuclear destruction (PND) that is associated with apoptotic-like DNA fragmentation. Nevertheless, the mechanism that mediates PND remained to be elucidated. Here, I describe the execution of PND through an unusual form of autophagy that has not been documented previously in yeast. This form of autophagy is most similar to megaautophagy in plants and lysosomal membrane permeabilization in mammals. My results demonstrate further diversity in cell death programs in unicellular microbes that is potentially conserved across eukaryotes.

The purpose of this project was to create molecular tools for the study of meiosis in Arabidopsis thaliana and to evaluate their effectiveness. Two types of transgenic plants were created with an intron-spliced hairpin RNA (ihpRNA) construct to target the AHP2 gene for RNA silencing. One had a constitutively expressed promoter; the other’s promoter was inducible with dexamethasone (DEX). Transformations with the constitutively expressed construct gave rise to ahp2RNAi plants with reduced AHP2 transcript levels, abnormal meioses and reduced fertility phenotypes. The creation of plants containing the dexamethasone-inducible construct was confirmed via PCR genotyping, and induction with DEX. However, the induction conditions tested do not appear to silence AHP2 as the transgenics had normal meiotic and reproductive phenotypes. Also a triple-locus, three color, fluorescent protein marker-tagged Arabidopsis line was created that will allow calculation of recombination frequencies for two intervals on chromosome 2 in both wild type and mutant plants.