Targeting the adaptability of heterogeneous aneuploids.

Aneuploid genomes, characterized by unbalanced chromosome stoichiometry (karyotype), are associated with cancer malignancy and drug resistance of pathogenic fungi. The phenotypic diversity resulting from karyotypic diversity endows the cell population with superior adaptability. We show here, using a combination of experimental data and a general stochastic model, that the degree of phenotypic variation, thus evolvability, escalates with the degree of overall growth suppression. Such scaling likely explains the challenge of treating aneuploidy diseases with a single stress-inducing agent. Instead, we propose the design of an "evolutionary trap" (ET) targeting both karyotypic diversity and fitness. This strategy entails a selective condition "channeling" a karyotypically divergent population into one with a predominant and predictably drugable karyotypic feature. We provide a proof-of-principle case in budding yeast and demonstrate the potential efficacy of this strategy toward aneuploidy-based azole resistance in Candida albicans. By analyzing existing pharmacogenomics data, we propose the potential design of an ET against glioblastoma.

Proteome plasticity in response to persistent environmental change.

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

The role of gene dosage in budding yeast centrosome scaling and spontaneous diploidization.

Ploidy is the number of whole sets of chromosomes in a species. Ploidy is typically a stable cellular feature that is critical for survival. Polyploidization is a route recognized to increase gene dosage, improve fitness under stressful conditions and promote evolutionary diversity. However, the mechanism of regulation and maintenance of ploidy is not well characterized. Here, we examine the spontaneous diploidization associated with mutations in components of the Saccharomyces cerevisiae centrosome, known as the spindle pole body (SPB). Although SPB mutants are associated with defects in spindle formation, we show that two copies of the mutant in a haploid yeast favors diploidization in some cases, leading us to speculate that the increased gene dosage in diploids 'rescues' SPB duplication defects, allowing cells to successfully propagate with a stable diploid karyotype. This copy number-based rescue is linked to SPB scaling: certain SPB subcomplexes do not scale or only minimally scale with ploidy. We hypothesize that lesions in structures with incompatible allometries such as the centrosome may drive changes such as whole genome duplication, which have shaped the evolutionary landscape of many eukaryotes.

The SUN protein Mps3 is required for spindle pole body insertion into the nuclear membrane and nuclear envelope homeostasis.

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition.

A molecular cell biology toolkit for the study of meiosis in the silkworm Bombyx mori.

Meiosis is usually described as 4 essential and sequential processes: (1) homolog pairing; (2) synapsis, mediated by the synaptonemal complex; (3) crossing over; and (4) segregation. In this canonical model, the maturation of crossovers into chiasmata plays a vital role in holding homologs together and ensuring their segregation at the first meiotic division. However, Lepidoptera (moths and butterflies) undergo 3 distinct meiotic processes, only one of which is canonical. Lepidoptera males utilize 2 meiotic processes: canonical meiosis that produces nucleated fertile sperm, and a noncanonical meiosis that produces anucleated nonfertile sperm which are nonetheless essential for reproduction. Lepidoptera females, which carry heteromorphic sex chromosomes, undergo a completely achiasmate (lacking crossovers) meiosis, thereby requiring an alternative mechanism to ensure proper homolog segregation. Here, we report that the development of a molecular cell biology toolkit designed to properly analyze features of meiosis, including the synaptonemal complex structure and function, in the silkworm Bombyx mori. In addition to standard homology searches to identify Bombyx orthologs of known synaptonemal complex encoding genes, we developed an ortholog discovery app (Shinyapp) to identify Bombyx orthologs of proteins involved in several meiotic processes. We used this information to clone genes expressed in the testes and then created antibodies against their protein products. We used the antibodies to confirm the localization of these proteins in normal male spermatocytes, as well as using in vitro assays to confirm orthologous interactions. The development of this toolkit will facilitate further study of the unique meiotic processes that characterize meiosis in Lepidoptera.

Diverse mating phenotypes impact the spread of wtf meiotic drivers in Schizosaccharomyces pombe.

Meiotic drivers are genetic elements that break Mendel's law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing (mating between individuals from distinct lineages) because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe, a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity for cells from distinct clonal lineages to mate varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed levels of preferential mating between cells from the same clonal lineage can slow, but not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs linked to the driver. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

The fission yeast, Schizosaccharomyces pombe, is a haploid organism, meaning it has a single copy of each of its genes. S. pombe cells generally carry one copy of each chromosome and can reproduce clonally by duplicating these chromosomes and then dividing into two cells. However, when the yeast are starving, they can reproduce sexually. This involves two cells mating by fusing together to create a 'diploid zygote', which contains two copies of each gene. The zygote then undergoes 'meiosis', a special type of cell division in which the zygote first duplicates its genome and then divides twice. This results in four haploid spores which are analogous to sperm and eggs in humans that each contain one copy of the genome. The spores will grow and divide normally when conditions improve. The genes carried by each of the haploid spores depend on the cells that formed the zygote. If the two 'parent' yeast had the same version or 'allele' of a gene, all four spores will have it in their genome. However, if the two parents have different alleles, only 50% of the offspring will carry each version. Although this is usually the case, there are certain alleles, called meiotic drivers, that are transmitted to all offspring even in situations where it is only carried by one parent. Meiotic drivers can be found in many organisms, including mammals, but their behavior is easiest to study in yeast. Meiotic drivers known as killers achieve this by disposing of any 'sister' spores that do not inherit the same allele of this gene. This 'killing' can only happen when only one of the 'parents' carries the driver. This scenario is thought to rarely occur in species that inbreed, as inbreeding leads to both gene copies being the same. However, this does not appear to be the case for S. pombe, which contain a whole family of killer meiotic drivers, the wtf genes, despite also being reported to mainly inbreed. To investigate this contradiction, López Hernández et al. isolated several genetically distinct populations of S.pombe. These isolates were grown together to determine how often the each one would outcross (mate with an individual from a different population) or inbreed. The results found that levels of inbreeding varied between isolates. Next, López Hernández et al. used mathematical modelling and experimental evolution analyses to study how wtf drivers spread amongst these populations. This revealed that wtf genes spread faster in populations with more outcrossing. In some instances, the wtf driver was linked to a gene that could harm the population. In these cases, López Hernández et al. found than inbreeding could purge these drivers and stop them from spreading the dangerous alleles through the population. López Hernández et al. establish a simple experimental system to model driver evolution and experimentally demonstrate how key parameters, such as outcrossing rates, affect the spread of these genes. Understanding how meiotic drivers spread is important, as these systems could potentially be used to modify populations important to humans, such as crops or disease vectors.

High-Throughput Identification of Nuclear Envelope Protein Interactions in Schizosaccharomyces pombe Using an Arrayed Membrane Yeast-Two Hybrid Library.

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins from the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two -hybrid system. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nuclear membrane: Cut11/Ndc1, Lem2 and Ima1/Samp1/Net5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11-ts mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death.

Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism to selectively kill meiotic products (spores) that do not inherit wtf4. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate trafficking, as mutations that disrupt either of these processes result in cell death in the presence of the Wtf4 proteins. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy spores.

Meiotic drivers are genes that break the normal rules of inheritance. Usually, a gene has a 50% chance of passing on to the next generation. Meiotic drivers force their way into the next generation by poisoning the gametes (the sex cells that combine to form a zygote) that do not carry them. Harnessing the power of genetic drivers could allow scientists to spread beneficial genes across populations. One group of meiotic drivers found in fission yeast is called the 'with transposon fission yeast', or 'wtf' gene family. The wtf drivers act during the production of spores, which are the fission yeast equivalent of sperm, and they encode both a poison that can destroy the spores and its antidote. The poison spreads through the sac holding the spores, and can affect all of them, while the antidote only protects the spores that make it. This means that the spores carrying the wtf genes survive, while the rest of the spores are killed. To understand whether it is possible to use the wtf meiotic drivers to spread other genes, perhaps outside of fission yeast, scientists must first establish exactly how the proteins coded for by genes behave. To do this, Nuckolls et al. examined a member of the wtf family called wtf4. Attaching a fluorescent label to the poison and antidote proteins produced by wtf4 made it possible to see what they do. This revealed that the poison clumps, forming toxic aggregates that damage yeast spores. The antidote works by mopping up these aggregates and moving them to the cell's main storage compartment, called the vacuole. Mutations that disrupted the ability of the antidote to interact with the poison or its ability to move the poison into storage stopped the antidote from working. Nuckolls et al. also showed that if genetic engineering was used to introduce wtf4 into a distantly related species of budding yeast the effects of this meiotic driver were the same. This suggests that the wtf genes may be good candidates for future genetic engineering experiments. Engineered systems known as 'gene drives' could spread beneficial genetic traits through populations. This could include disease-resistance genes in crops, or disease-preventing genes in mosquitoes. The wtf genes are small and work independently of other genes, making them promising candidates for this type of system. These experiments also suggest that the wtf genes could be useful for understanding why clumps of proteins are toxic to cells. Future work could explore why clumps of wtf poison kill spores, while clumps of poison plus antidote do not. This could aid research into human ailments caused by protein clumps, such as Huntington's or Alzheimer's disease.

Distribution of Proteins at the Inner Nuclear Membrane Is Regulated by the Asi1 E3 Ligase in Saccharomyces cerevisiae.

Inner nuclear membrane (INM) protein composition regulates nuclear function, affecting processes such as gene expression, chromosome organization, nuclear shape, and stability. Mechanisms that drive changes in the INM proteome are poorly understood, in part because it is difficult to definitively assay INM composition rigorously and systematically. Using a split-GFP complementation system to detect INM access, we examined the distribution of all C-terminally tagged Saccharomyces cerevisiae membrane proteins in wild-type cells and in mutants affecting protein quality control pathways, such as INM-associated degradation (INMAD), ER-associated degradation, and vacuolar proteolysis. Deletion of the E3 ligase Asi1 had the most specific effect on the INM compared to mutants in vacuolar or ER-associated degradation pathways, consistent with a role for Asi1 in the INMAD pathway. Our data suggest that Asi1 not only removes mistargeted proteins at the INM, but also controls the levels and distribution of native INM components, such as the membrane nucleoporin Pom33 Interestingly, loss of Asi1 does not affect Pom33 protein levels but instead alters Pom33 distribution in the nuclear envelope through Pom33 ubiquitination, which drives INM redistribution. Taken together, our data demonstrate that the Asi1 E3 ligase has a novel function in INM protein regulation in addition to protein turnover.

Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast.

Aneuploidy and epigenetic alterations have long been associated with carcinogenesis, but it was unknown whether aneuploidy could disrupt the epigenetic states required for cellular differentiation. In this study, we found that ~3% of random aneuploid karyotypes in yeast disrupt the stable inheritance of silenced chromatin during cell proliferation. Karyotype analysis revealed that this phenotype was significantly correlated with gains of chromosomes III and X. Chromosome X disomy alone was sufficient to disrupt chromatin silencing and yeast mating-type identity as indicated by a lack of growth response to pheromone. The silencing defect was not limited to cryptic mating type loci and was associated with broad changes in histone modifications and chromatin localization of Sir2 histone deacetylase. The chromatin-silencing defect of disome X can be partially recapitulated by an extra copy of several genes on chromosome X. These results suggest that aneuploidy can directly cause epigenetic instability and disrupt cellular differentiation.

Mms21 SUMO Ligase Activity Promotes Nucleolar Function in Saccharomyces cerevisiae.

The budding yeast E3 SUMO ligase Mms21, also known as Nse2, is a component of the Smc5/6 complex, which regulates sister chromatid cohesion, DNA replication, and repair. Our study shows that the mms21RINGΔ mutant exhibits (1) reduced ribosomal RNA production; (2) nuclear accumulation of ribosomal proteins; (3) elevated Gcn4 translation, indicating translational stress; and (4) upregulation of Gcn4 targets. Genes involved in ribosome biogenesis and translation are downregulated in the mms21RINGΔ mutant. We identified RPL19A as a novel genetic suppressor of the mms21RINGΔ mutant. Deletion of RPL19A partially suppresses growth defects in both smc5-6 and mms21RINGΔ mutants as well as nuclear accumulation of ribosome subunits in the mms21RINGΔ mutant. Deletion of a previously identified strong suppressor, MPH1, rescues both the accumulation of ribosome subunits and translational stress. This study suggests that the Smc5/6 complex supports nucleolar function.

Analysis of membrane proteins localizing to the inner nuclear envelope in living cells.

Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain. By pairing split-GFP with fluorescence correlation spectroscopy, we compared the composition of complexes at the INM and ER, finding that at least one is unique: Sbh2, but not Sbh1, has access to the INM. Collectively, our work provides a comprehensive analysis of transmembrane protein localization to the INM and paves the way for further research into INM composition and function.