Biology subjects

Gould, K. L.

Publications and source records attributed to Gould, K. L..

11 recordsLinked to original sources

bioRxiv2026

A multivalent docking platform and Rcn1-mediated inhibition control the extent of calcineurin recruitment to the cell division site for the dephosphorylation of multiple cytokinetic proteins

Cytokinesis requires coordinated signaling to ensure the accurate physical separation of daughter cells. Calcineurin (CN), a conserved Ca2+/calmodulin-dependent phosphatase, is required for cytokinesis in organisms ranging from yeast to humans, yet how CN is regulated at the division site and the substrates through which it promotes cell division remain poorly understood. Here we use the fission yeast Schizosaccharomyces pombe, which display striking cell division defects in the absence of CN, to define how CN is anchored at the cell division site. We show that CN recruitment to the cytokinetic ring (CR) requires its PxIxIT- and LxVP-binding surfaces and is mediated by multivalent interactions with the CR components paxillin-like Pxl1 and the F-BAR protein Cdc15. Disrupting these interactions nearly eliminates CN from the CR and causes gross cytokinetic defects similar to complete loss of CN function. Cell cycle stage-specific quantitative phosphoproteomics combined with proximity labeling-based proteomics were used to identify candidate CN substrates involved in cytokinesis. Validation of a cohort of these proteins localizing to the CR, including the F-BAR protein Rga7, the actin regulator Aim21 and three protein kinases, revealed that CN targets a broad network of structural and signaling components involved in cell division. We also identify the conserved CN inhibitor Rcn1 as a CN substrate and show that Rcn1 restricts CN accumulation at the CR to provide an additional layer of spatial regulation. Thus, spatial control of CN enables proper protein dephosphorylation for successful cytokinesis.

cell biology↗

bioRxiv2026

A Novel Eukaryotic Ribosome Factor Enables Translation Restart Following Cellular Dormancy

Dormancy is a survival strategy employed by all domains of life to withstand prolonged nutrient deprivation and environmental stress that is marked by a global shutdown of protein synthesis. However, the molecular mechanisms driving ribosome inactivation and reactivation during and after dormancy in eukaryotes remain poorly understood. Here, we identify SNOR, a novel SBDS-like ribosome-associated factor in Schizosaccharomyces pombe, that is upregulated and associates with ribosomes during induced dormancy triggered by glucose depletion. SNOR contributes to protein synthesis repression by binding the ribosome to probe the peptidyl transferase center (PTC), block tRNA-binding sites, and cap the polypeptide exit tunnel (PET). Importantly, we show that SNOR is essential for the restart of protein synthesis upon glucose reintroduction and exit from dormancy. SNOR is evolutionarily conserved and specifically upregulated in response to glucose stress in fungi. These findings reveal a previously unrecognized ribosome-associated factor that links glucose stress and cellular dormancy to surveillance of protein synthesis and highlight the power of in situ structural biology to uncover stress-responsive regulators of translation.

biophysics↗

bioRxiv2026

A Glucan Synthase-Remodeler Module Organizes Branched Glucan Assembly in the Fungal Cell Wall

The fungal cell wall is an essential extracellular matrix that underpins growth, morphogenesis, and pathogenesis. Cell wall construction requires numerous enzymes that synthesize and remodel extracellular polymers, yet the principles governing their spatial and functional organization remain unclear. In the fission yeast Schizosaccharomyces pombe, we identify Ghs2, a predicted glycoside hydrolase 16 (GH16) domain-containing transmembrane protein, as an obligate binding partner of the {beta}-1,3-glucan synthase Bgs3. Ghs2 and Bgs3 co-localize at sites of polarized growth and physically associate in vivo. Structure-guided modeling positions the Ghs2 GH16 domain proximal to the predicted Bgs3 glucan extrusion pore, suggesting coordinated polymer synthesis and remodeling. Solid-state NMR analyses demonstrate that both Ghs2 and Bgs3 are required for the proper accumulation of branched {beta}-1,3-glucan. Together with genetic and cell biological evidence, these findings support a model in which Bgs3 synthesizes linear {beta}-1,3-glucan and Ghs2 subsequently introduces {beta}-1,6-linked branch points onto the nascent polymer. More broadly, we propose that synthase-modifier pairs may act together to shape polymer architecture during cell wall assembly.

cell biology↗

bioRxiv2025

Solid-State NMR Analysis of Schizosaccharomyces pombe Reveals Role of α-Amylase Family Enzymes in Cell Wall Structure and Function

The fission yeast Schizosaccharomyces pombe is a widely employed model organism for studying the eukaryotic cell cycle. Like plants and bacteria, S. pombe must build a cell wall in concert with its cell cycle, but how cell wall-synthesizing and remodeling enzymes mediate this process remains unclear. Here we characterize the functions of Aah1 and Aah3, two related S. pombe -amylases that are putative members of this evolutionarily conserved family of cell wall-modifying proteins. We found that unlike rod-shaped wildtype S. pombe cells, aah1{Delta} aah3{Delta} cells are nearly spherical, grow slowly, have thickened cell walls, and have severe defects in cell separation following cytokinesis. Solid-state NMR spectroscopy analyses of intact wildtype and aah1{Delta} aah3{Delta} cells revealed that aah1{Delta} aah3{Delta} cell walls are rigidified with a significant reduction in the -glucan matrix, characterized by reduced amounts of the major -1,3-glucan and the minor -1,4-glucan within the rigid and mobile phases; this reduction was compensated for by a two-fold increase in {beta}-glucan content. Indeed, viability of aah1{Delta} aah3{Delta} cells depended on {beta}-glucan upregulation and the cell wall integrity pathway that mediates it. While aah1{Delta} aah3{Delta} cells resemble cells with impaired function of the transglycosylation domain of -glucan synthase 1 (Ags1), increased expression of Aah3 does not compensate for impaired Ags1 function or vice-versa. Overall, our data suggest that Aah1 and Aah3 are required in addition to Ags1, likely downstream, for the transglycosylation of -glucan chains to generate fibers of appropriate dimensions to support proper cell morphology, growth, and division. Significance StatementThis study utilized a range of imaging techniques and high-resolution solid-state NMR spectroscopy of intact S. pombe cells to refine our understanding of S. pombe cell wall composition. This study also determined that two related GPI-anchored -amylase family proteins, Aah1 and Aah3, likely act as transglycosylases non-redundantly with an -glucan synthase in the synthesis of -glucan chains of appropriate content and size to support polarized growth and cell division. Our results also highlight the anti-fungal therapeutic potential of GPI-anchored enzymes acting in concert with glucan synthases.

microbiology↗

bioRxiv2023

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)P2]. 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)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 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. Summary statementFission yeast Pik1 localizes exclusively to the trans-Golgi independently of Ncs1, where it contributes to PI4P but not PI(4,5)P2 synthesis. Pik1 does not affect cytokinesis.

cell biology↗

bioRxiv2023

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 1,452 biomedical sciences PhDs who graduated from Vanderbilt University between 1997-2021. 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 defense, and the reasons why students became more- or less-interested in research-intensive faculty careers. We linked students career goal at defense to whether they did a postdoc, the duration of time between defense and the first non-training position, the career area of the first non-training position, and the career area of the job at ten years after graduation. Finally, we followed individual careers for ten 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 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.

scientific communication and education↗

bioRxiv2023

Fission yeast CK1 promotes DNA double-strand break repair through both homologous recombination and non-homologous end joining

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells activated the DNA damage checkpoint due to persistent double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and non-homologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. In order to understand how Hhp1 and Hhp2 promote DSB repair, we identified new substrates using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 that is important for DSB repair. Our data suggest that Hhp1 and Hhp2 facilitate DSB repair by phosphorylating multiple substrates, including Arp8.

cell biology↗

bioRxiv2023

The fission yeast cytokinetic ring component Fic1 promotes septum formation

In Schizosaccharomyces pombe septum formation is coordinated with cytokinetic ring constriction but the mechanisms linking these events are unclear. In this study, we explored the role of the cytokinetic ring component Fic1, first identified by its interaction with the F-BAR protein Cdc15, in septum formation. We found that the fic1 phospho-ablating mutant, fic1-2A, is a gain-of-function allele that suppresses myo2-E1, the temperature-sensitive allele of the essential type-II myosin, myo2. This suppression is achieved by the promotion of septum formation and required Fic1s interaction with the F-BAR proteins Cdc15 and Imp2. Additionally, we found that Fic1 interacts with Cyk3 and that this interaction was likewise required for Fic1s role in septum formation. Fic1, Cdc15, Imp2, and Cyk3 are the orthologs of the Saccharomyces cerevisiae ingression progression complex, which stimulates the chitin synthase Chs2 to promote primary septum formation. However, our findings indicate that Fic1 promotes septum formation and cell abscission independently of the S. pombe Chs2 ortholog. Thus, while similar complexes exist in the two yeasts that each promote septation, they appear to have different downstream effectors. Summary StatementThe S. pombe cytokinetic ring protein Fic1 promotes septum formation in a manner dependent on interactions with the cytokinetic ring components Cdc15, Imp2, and Cyk3.

cell biology↗

bioRxiv2019

Autophosphorylation of the CK1 kinase domain regulates enzyme activity and function

CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. Here, we describe a new mechanism of CK1 autoregulation that is conserved in CK1 enzymes from yeast to human - the autophosphorylation of a threonine in the mobile L-EF loop proximal to the active site. Phosphorylation at this site inhibits kinase activity, in contrast to well-characterized T-loop autophosphorylation in other kinase families. Consequently, yeast and human enzymes with phosphoablating mutations at this site are hyperactive. In S. pombe, hyperactive CK1 causes defects in cell growth and morphology at a high level but protection from heat shock at a low level, highlighting the necessity of regulated CK1 function. We propose that phosphorylation on the L-EF loop prevents substrate docking with the kinase domain by shielding the positively charged binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.\n\nSignificance StatementKinases in the CK1 family are important signaling enzymes, and they function in multiple pathways within the same cell. Misregulation of CK1 activity contributes to human disease, including cancer, neurodegenerative disease, and sleep phase disorders, yet the mechanisms that control CK1 activity are not well understood. We have identified a conserved autophosphorylation site in the CK1 kinase domain that inhibits substrate phosphorylation. We hypothesize that by using kinase domain autophosphorylation in combination with other regulatory mechanisms, CK1 enzymes can coordinate the phosphorylation of their substrates in different pathways.

biochemistry↗

bioRxiv2018

Adaptor protein Bbc1 regulates localization of Wsp1 and Vrp1 during endocytic actin patch assembly

Arp2/3 complex-nucleated branched actin networks provide the force necessary for endocytosis. The Arp2/3 complex is activated by Nucleation Promoting Factors (NPFs) including the Schizosaccharomyces pombe proteins WASp Wsp1 and myosin-1 Myo1. There are >40 known yeast endocytic proteins with distinct spatial and temporal localizations and functions; however, it is still unclear how these proteins work together to drive endocytosis. We used quantitative live cell imaging to determine the function of the uncharacterized S. pombe protein Bbc1. We discovered Myo1 interacts with and recruits Bbc1 to sites of endocytosis. Bbc1 competes with verprolin Vrp1 for Myo1 binding, thus releasing Vrp1 and its binding partner Wsp1 from Myo1. Normally Myo1 remains at the base of the endocytic invagination and Vrp1-Wsp1 internalize with the endocytic vesicle; however, in the absence of Bbc1, a portion of Vrp1-Wsp1 remains with Myo1 at the base of the invagination and endocytic invaginations are twice as long. We propose that Bbc1 disrupts a transient Myo1-Vrp1-Wsp1 interaction and limits Arp2/3 complex-nucleation of actin branches at the plasma membrane.

cell biology↗

bioRxiv2018

Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein, Sid4, at the SPB, and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Polo-like kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive auto-ubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

cell biology↗