Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

Generation and characterization of temperature-sensitive alleles encoding GPI anchored proteins Psu1 and Dfg502 in Schizosaccharomyces pombe.

Glycosyl-phosphatidylinositol (GPI) anchored proteins are implicated in remodeling of the yeast cell wall during growth and division. Schizosaccharomyces pombe proteins, Psu1 , Dfg501 , and Dfg502 are predicted GPI anchored proteins with likely cell wall modifying activity. Here, we isolated and characterized null and temperature-sensitive alleles that will allow further analysis of the function of these proteins and S. pombe cell wall formation. Our data confirm that Psu1 is necessary for cell separation, maintaining proper cell shape, and viability. Additionally, we found that Dfg501 and Dfg502 share a redundant and essential function necessary for cell separation and viability.

MAPK-dependent control of mitotic progression in S. pombe.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress. RESULTS: We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20Slp1 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20Slp1 N-terminus through a canonical MAPK docking site. Most important, the Cdc20Slp1 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit. CONCLUSIONS: Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20Slp1 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.

Generation and characterization of temperature-sensitive alleles of the glucanosyltransferase Gas1 in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe Gas family of ß-1,3-glucanosyltransferases modify the cell wall by elongating ß-1,3-glucan chains. While gas1Δ cells are inviable under standard laboratory growth conditions, they are viable in the presence of an osmotic stabilizer. Even under these conditions however, gas1Δ cells are slow-growing and display cell separation and morphology defects. Here, we isolated and characterized two gas1 temperature-sensitive alleles. Our data support that Gas1 is the primary S. pombe ß-1,3-glucanosyltransferase important for cell separation and cell viability and provide useful tools for further analysis of S. pombe cell wall formation.

The Cdc14 phosphatase, Clp1, does not affect genome expression.

Schizosaccharomyces pombe Clp1 is a Cdc14-family phosphatase that reverses mitotic Cdk1 phosphorylation. Despite evolutionary conservation, Clp1 's mammalian orthologs do not share this function. Rather, higher eukaryotic Cdc14 enzymes act in DNA repair, ciliogenesis, and gene regulation. To examine if Clp1 regulates gene expression, we compared the transcriptional profiles of cells lacking Clp1 function to that of wildtype. Because clp1∆ cells are sensitive to the actin depolymerizing drug, LatrunculinA, we also investigated whether a transcriptional response was involved. Our results indicate that Clp1 does not detectably affect gene expression and highlight the organism-specific functions of this conserved phosphatase family.

The Greatwall-Endosulfine-PP2A/B55 pathway controls entry into quiescence by promoting translation of Elongator-tuneable transcripts.

Quiescent cells require a continuous supply of proteins to maintain protein homeostasis. In fission yeast, entry into quiescence is triggered by nitrogen stress, leading to the inactivation of TORC1 and the activation of TORC2. Here, we report that the Greatwall-Endosulfine-PPA/B55 pathway connects the downregulation of TORC1 with the upregulation of TORC2, resulting in the activation of Elongator-dependent tRNA modifications essential for sustaining the translation programme during entry into quiescence. This process promotes U34 and A37 tRNA modifications at the anticodon stem loop, enhancing translation efficiency and fidelity of mRNAs enriched for AAA versus AAG lysine codons. Notably, some of these mRNAs encode inhibitors of TORC1, activators of TORC2, tRNA modifiers, and proteins necessary for telomeric and subtelomeric functions. Therefore, we propose a novel mechanism by which cells respond to nitrogen stress at the level of translation, involving a coordinated interplay between the tRNA epitranscriptome and biased codon usage.

From goal to outcome: Analyzing the progression of biomedical sciences PhD careers in a longitudinal study using an expanded taxonomy.

Biomedical sciences PhDs pursue a wide range of careers inside and outside academia. However, there is little data regarding how career interests of PhD students relate to the decision to pursue postdoctoral training or to their eventual career outcomes. Here, we present the career goals and career outcomes of 1452 biomedical sciences PhDs who graduated from Vanderbilt University between 1997 and 2021. We categorized careers using an expanded three-tiered taxonomy and flags that delineate key career milestones. We also analyzed career goal changes between matriculation and doctoral defense, and the reasons why students became more- or less-interested in research-intensive faculty careers. We linked students' career goal at doctoral defense to whether they did a postdoc, the duration of time between doctoral defense and the first non-training position, the career area of the first non-training position, and the career area of the job at 10 years after graduation. Finally, we followed individual careers for 10 years after graduation to characterize movement between different career areas over time. We found that most students changed their career goal during graduate school, declining numbers of alumni pursued postdoctoral training, many alumni entered first non-training positions in a different career area than their goal at doctoral defense, and the career area of the first non-training position was a good indicator of the job that alumni held 10 years after graduation. Our findings emphasize that students need a wide range of career development opportunities and career mentoring during graduate school to prepare them for futures in research and research-related professions.

Characterization of Pik1 function in fission yeast reveals its conserved role in lipid synthesis and not cytokinesis.

Phosphatidylinositol (PI)-4-phosphate (PI4P) is a lipid found at the plasma membrane (PM) and Golgi in cells from yeast to humans. PI4P is generated from PI by PI4-kinases and can be converted into PI-4,5-bisphosphate [PI(4,5)P2]. Schizosaccharomyces pombe have two essential PI4-kinases - Stt4 and Pik1. Stt4 localizes to the PM, and its loss from the PM results in a decrease of PM PI4P and PI(4,5)P2. As a result, cells divide non-medially due to disrupted cytokinetic ring-PM anchoring. However, the localization and function of S. pombe Pik1 has not been thoroughly examined. Here, we found that Pik1 localizes exclusively to the trans-Golgi and is required for Golgi PI4P production. We determined that Ncs1 regulates Pik1, but unlike in other organisms, it is not required for Pik1 Golgi localization. When Pik1 function was disrupted, PM PI4P but not PI(4,5)P2 levels were reduced, a major difference compared with Stt4. We conclude that Stt4 is the chief enzyme responsible for producing the PI4P that generates PI(4,5)P2. Also, that cells with disrupted Pik1 do not divide asymmetrically highlights the specific importance of PM PI(4,5)P2 for cytokinetic ring-PM anchoring.

Elevated levels of sphingolipid MIPC in the plasma membrane disrupt the coordination of cell growth with cell wall formation in fission yeast.

Coupling cell wall expansion with cell growth is a universal challenge faced by walled organisms. Mutations in Schizosaccharomyces pombe css1, which encodes a PM inositol phosphosphingolipid phospholipase C, prevent cell wall expansion but not synthesis of cell wall material. To probe how Css1 modulates cell wall formation we used classical and chemical genetics coupled with quantitative mass spectrometry. We found that elevated levels of the sphingolipid biosynthetic pathway's final product, mannosylinositol phosphorylceramide (MIPC), specifically correlated with the css1-3 phenotype. We also found that an apparent indicator of sphingolipids and a sterol biosensor accumulated at the cytosolic face of the PM at cell tips and the division site of css1-3 cells and, in accord, the PM in css1-3 was less dynamic than in wildtype cells. Interestingly, disrupting the protein glycosylation machinery recapitulated the css1-3 phenotype and led us to investigate Ghs2, a glycosylated PM protein predicted to modify cell wall material. Disrupting Ghs2 function led to aberrant cell wall material accumulation suggesting Ghs2 is dysfunctional in css1-3. We conclude that preventing an excess of MIPC in the S. pombe PM is critical to the function of key PM-localized proteins necessary for coupling growth with cell wall formation.

Revised fission yeast gene and allele nomenclature guidelines for machine readability.

Standardized nomenclature for genes, gene products, and isoforms is crucial to prevent ambiguity and enable clear communication of scientific data, facilitating efficient biocuration and data sharing. Standardized genotype nomenclature, which describes alleles present in a specific strain that differ from those in the wild-type reference strain, is equally essential to maximize research impact and ensure that results linking genotypes to phenotypes are Findable, Accessible, Interoperable, and Reusable (FAIR). In this publication, we extend the fission yeast clade gene nomenclature guidelines to support the curation efforts at PomBase (www.pombase.org), the Schizosaccharomyces pombe Model Organism Database. This update introduces nomenclature guidelines for noncoding RNA genes, following those set forth by the Human Genome Organisation Gene Nomenclature Committee. Additionally, we provide a significant update to the allele and genotype nomenclature guidelines originally published in 1987, to standardize the diverse range of genetic modifications enabled by the fission yeast genetic toolbox. These updated guidelines reflect a community consensus between numerous fission yeast researchers. Adoption of these rules will improve consistency in gene and genotype nomenclature, and facilitate machine-readability and automated entity recognition of fission yeast genes and alleles in publications or datasets. In conclusion, our updated guidelines provide a valuable resource for the fission yeast research community, promoting consistency, clarity, and FAIRness in genetic data sharing and interpretation.

Polarity kinases that phosphorylate F-BAR protein Cdc15 have unique localization patterns during cytokinesis and contributions to preventing tip septation in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe F-BAR protein, Cdc15, facilitates the linkage between the cytokinetic ring and the plasma membrane. Cdc15 is phosphorylated on many sites by four polarity kinases and this antagonizes membrane interaction. Dephosphorylation of Cdc15 during mitosis induces its phase separation, allowing oligomerization, membrane association, and protein partner binding. Here, using live cell imaging we examined whether spatial separation of Cdc15 from its four identified kinases potentially explains their diverse effects on tip septation and the mitotic Cdc15 phosphorylation state. We identified a correlation between kinase localization and their ability to antagonize Cdc15 cytokinetic ring and membrane localization.

Isolation of mutant alleles of the U6 snRNA m 6 A methyltransferase Mtl16 and characterization of their genetic interactions with splicing mutants in Schizosaccharomyces pombe.

Schizosaccharomyces pombe Dim1 is a conserved essential component of the U4/U6.U5 tri-snRNP complex essential for pre-mRNA splicing. In a synthetic lethal screen with the temperature-sensitive dim1-35 mutant, we isolated multiple alleles of non-essential mtl16 that encodes the U6 snRNA m 6 A methyltransferase. Further genetic analysis revealed strong and specific negative genetic interactions between mtl16 and a mutation in the Dim1 binding partner, Prp31, and between dim1-35 and a mutation in the Prp31 binding partner, Prp6. Our work provides additional tools to study pre-mRNA splicing in S. pombe and biological confirmation of the importance of the Prp6-Prp31-Dim1-U6 snRNA interactions for pre-mRNA splicing.

Characterization of Pik1 function in fission yeast reveals its conserved role in lipid synthesis and not cytokinesis.

Phosphatidylinositol (PI)-4-phosphate (PI4P) is a lipid found at the plasma membrane (PM) and Golgi in cells from yeast to humans. PI4P is generated from PI by PI4-kinases and can be converted to PI-4,5-bisphosphate [PI(4,5)P 2 ]. Schizosaccharomyces pombe have 2 essential PI4-kinases: Stt4 and Pik1. Stt4 localizes to the PM and its loss from the PM results in a decrease of PM PI4P and PI(4,5)P 2 . As a result, cells divide non-medially due to disrupted cytokinetic ring-PM anchoring. However, the localization and function of S. pombe Pik1 has not been thoroughly examined. Here, we found that Pik1 localizes exclusively to the trans-Golgi and is required for Golgi PI4P production. We determined that Ncs1 regulates Pik1, but unlike in other organisms, it is not required for Pik1 Golgi localization. When Pik1 function was disrupted, PM PI4P but not PI(4,5)P 2 levels were reduced, a major difference with Stt4. We conclude that Stt4 is the chief enzyme responsible for producing the PI4P that generates PI(4,5)P 2 . Also, that cells with disrupted Pik1 do not divide asymmetrically highlights the specific importance of PM PI(4,5)P 2 for cytokinetic ring-PM anchoring. Summary statement: Fission yeast Pik1 localizes exclusively to the trans-Golgi independently of Ncs1, where it contributes to PI4P but not PI(4,5)P 2 synthesis. Pik1 does not affect cytokinesis.