Genomic Sequencing of sod1D Yeast That Escape Spore Death

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that impacts nerve cells and the spinal cord, which in some cases are linked to mutations in the Superoxide Dismutase 1 (SOD1) gene. Sod1 is an antioxidant within cells that converts reactive oxygen from superoxide into water using a copper and zinc ion to deactivate the oxygen. When the SOD1 gene is deleted, yeast cells are still able to undergo meiotic divisions and generate four spores, but the spores that are produced are inviable. However, we see that randomly, sod1∆ spores can grow on rich media. This leads us to hypothesize that somewhere in the genome, there is a suppressor mutation that allows these cells to grow. We tested this hypothesis by preparing samples for whole genome sequencing. By comparing the genomic sequences from our suppressor mutants to wild-type controls, we’re able to identify a single point mutation within a gene called NCA2, which codes for a protein that regulates expression of Fo-F1 ATP synthase subunits 6 and 8. Given this result, we are now working to try and understand the relationship between the sod1∆ spore death phenotype and the modulation of ATP synthase activity. In summary, the results from our work have the potential to further help us understand what role Sod1 plays in yeast meiosis and may be able to give us a deeper understanding for ALS cases that are linked to Sod1.

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.

Programmed cortical ER collapse drives selective ER degradation and inheritance in yeast meiosis

The endoplasmic reticulum (ER) carries out essential and conserved cellular functions, which depend on the maintenance of its structure and subcellular distribution. Here, we report developmentally regulated changes in ER morphology and composition during budding yeast meiosis, a conserved differentiation program that gives rise to gametes. A subset of the cortical ER collapses away from the plasma membrane at anaphase II, thus separating into a spatially distinct compartment. This programmed collapse depends on the transcription factor Ndt80, conserved ER membrane structuring proteins Lnp1 and reticulons, and the actin cytoskeleton. A subset of ER is retained at the mother cell plasma membrane and excluded from gamete cells via the action of ER–plasma membrane tethering proteins. ER remodeling is coupled to ER degradation by selective autophagy, which relies on ER collapse and is regulated by timed expression of the autophagy receptor Atg40. Thus, developmentally programmed changes in ER morphology determine the selective degradation or inheritance of ER subdomains by gametes.

Linear elements are stable structures along the chromosome axis in fission yeast meiosis

The structure of chromosomes dramatically changes upon entering meiosis to ensure the successful progression of meiosis-specific events. During this process, a multilayer proteinaceous structure called a synaptonemal complex (SC) is formed in many eukaryotes. However, in the fission yeast Schizosaccharomyces pombe, linear elements (LinEs), which are structures related to axial elements of the SC, form on the meiotic cohesin-based chromosome axis. The structure of LinEs has been observed using silver-stained electron micrographs or in immunofluorescence-stained spread nuclei. However, the fine structure of LinEs and their dynamics in intact living cells remain to be elucidated. In this study, we performed live cell imaging with wide-field fluorescence microscopy as well as 3D structured illumination microscopy (3D-SIM) of the core components of LinEs (Rec10, Rec25, Rec27, Mug20) and a linE-binding protein Hop1. We found that LinEs form along the chromosome axis and elongate during meiotic prophase. 3D-SIM microscopy revealed that Rec10 localized to meiotic chromosomes in the absence of other LinE proteins, but shaped into LinEs only in the presence of all three other components, the Rec25, Rec27, and Mug20. Elongation of LinEs was impaired in double-strand break-defective rec12− cells. The structure of LinEs persisted after treatment with 1,6-hexanediol and showed slow fluorescence recovery from photobleaching. These results indicate that LinEs are stable structures resembling axial elements of the SC.

Dynamic Histone H3 Modifications Regulate Meiosis Initiation via Respiration

Meiosis is essential for genetic stability and diversity during sexual reproduction in most eukaryotes. Chromatin structure and gene expression are drastically changed during meiosis, and various histone modifications have been reported to participate in this unique process. However, the dynamic of histone modifications during meiosis is still not well investigated. Here, by using multiple reaction monitoring (MRM) based LC-MS/MS, we detected dynamic changes of histone H3 lysine post-translational modifications (PTMs). We firstly quantified the precise percentage of H3 modifications on different lysine sites during mouse and yeast meiosis, and found H3 acetylation and methylation were dramatically changed. To further study the potential functions of H3 acetylation and methylation in meiosis, we performed histone H3 lysine mutant screening in yeast, and found that yeast strains lacking H3K18 acetylation (H3K18ac) failed to initiate meiosis due to insufficient IME1 expression. Further studies showed that the absence of H3K18ac impaired respiration, leading to the reduction of Rim101p, which further upregulated a negative regulator of IME1 transcription, Smp1p. Together, our studies reveal a novel meiosis initiation pathway mediated by histone H3 modifications.

Meiotic regulation of the Ndc80 complex composition and function

This review describes the current models for how the subunit abundance of the Ndc80 complex, a key kinetochore component, is regulated in budding yeast and metazoan meiosis. The past decades of kinetochore research have established the Ndc80 complex to be a key microtubule interactor and a central hub for regulating chromosome segregation. Recent studies further demonstrate that Ndc80 is the limiting kinetochore subunit that dictates the timing of kinetochore activation in budding yeast meiosis. Here, we discuss the molecular circuits that regulate Ndc80 protein synthesis and degradation in budding yeast meiosis and compare the findings with those from metazoans. We envision the regulatory principles discovered in budding yeast to be conserved in metazoans, thereby providing guidance into future investigations on kinetochore regulation in human health and disease.

Programmed ER fragmentation drives selective ER inheritance and degradation in budding yeast meiosis

The endoplasmic reticulum (ER) is a membrane-bound organelle with diverse, essential functions that rely on the maintenance of membrane shape and distribution within cells. ER structure and function are remodeled in response to changes in cellular demand, such as the presence of external stressors or the onset of cell differentiation, but mechanisms controlling ER remodeling during cell differentiation are not well understood. Here, we describe a series of developmentally regulated changes in ER morphology and composition during budding yeast meiosis, a conserved differentiation program that gives rise to gametes. During meiosis, the cortical ER undergoes fragmentation before collapsing away from the plasma membrane at anaphase II. This programmed collapse depends on the meiotic transcription factor Ndt80, conserved ER membrane structuring proteins Lnp1 and reticulons, and the actin cytoskeleton. A subset of ER is retained at the mother cell plasma membrane and excluded from gamete cells via the action of ER-plasma membrane tethering proteins. ER remodeling is coupled to ER degradation by selective autophagy, which is regulated by the developmentally timed expression of the autophagy receptor Atg40. Autophagy relies on ER collapse, as artificially targeting ER proteins to the cortically retained ER pool prevents their degradation. Thus, developmentally programmed changes in ER morphology determine the selective degradation or inheritance of ER subdomains by gametes.