Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes.

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

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.

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.

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.

A cross-institutional analysis of the effects of broadening trainee professional development on research productivity.

PhD-trained scientists are essential contributors to the workforce in diverse employment sectors that include academia, industry, government, and nonprofit organizations. Hence, best practices for training the future biomedical workforce are of national concern. Complementing coursework and laboratory research training, many institutions now offer professional training that enables career exploration and develops a broad set of skills critical to various career paths. The National Institutes of Health (NIH) funded academic institutions to design innovative programming to enable this professional development through a mechanism known as Broadening Experiences in Scientific Training (BEST). Programming at the NIH BEST awardee institutions included career panels, skill-building workshops, job search workshops, site visits, and internships. Because doctoral training is lengthy and requires focused attention on dissertation research, an initial concern was that students participating in additional complementary training activities might exhibit an increased time to degree or diminished research productivity. Metrics were analyzed from 10 NIH BEST awardee institutions to address this concern, using time to degree and publication records as measures of efficiency and productivity. Comparing doctoral students who participated to those who did not, results revealed that across these diverse academic institutions, there were no differences in time to degree or manuscript output. Our findings support the policy that doctoral students should participate in career and professional development opportunities that are intended to prepare them for a variety of diverse and important careers in the workforce.

Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment.

The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast Schizosaccharomyces pombe. Because cell cycle-regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog Cdc15 controls Cdc15 oligomerization state, localization and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR, with the bulk of phosphorylation being constitutive. In vitro, the casein kinase 1 (CK1) isoforms Hhp1 and Hhp2 can phosphorylate 17 sites, and Cdk1 (also known as Cdc2) can phosphorylate the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 accumulation at the contractile ring (CR). Mutations that eliminate CK1 phosphorylation sites allow CR sliding, and phosphomimetic substitutions at these sites reduce Imp2 protein levels and slow CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive, with milder effects on localization and function. This article has an associated First Person interview with the first author of the paper.

Phosphoregulation of the cytokinetic protein Fic1 contributes to fission yeast growth polarity establishment.

Cellular polarization underlies many facets of cell behavior, including cell growth. The rod-shaped fission yeast Schizosaccharomyces pombe is a well-established, genetically tractable system for studying growth polarity regulation. S. pombe cells elongate at their two cell tips in a cell cycle-controlled manner, transitioning from monopolar to bipolar growth in interphase when new ends established by the most recent cell division begin to extend. We previously identified cytokinesis as a critical regulator of new end growth and demonstrated that Fic1, a cytokinetic factor, is required for normal polarized growth at new ends. Here, we report that Fic1 is phosphorylated on two C-terminal residues, which are each targeted by multiple protein kinases. Endogenously expressed Fic1 phosphomutants cannot support proper bipolar growth, and the resultant defects facilitate the switch into an invasive pseudohyphal state. Thus, phosphoregulation of Fic1 links the completion of cytokinesis to the re-establishment of polarized growth in the next cell cycle. These findings broaden the scope of signaling events that contribute to regulating S. pombe growth polarity, underscoring that cytokinetic factors constitute relevant targets of kinases affecting new end growth.This article has an associated First Person interview with Anthony M. Rossi, joint first author of the paper.

Fission yeast Opy1 is an endogenous PI(4,5)P2 sensor that binds to the phosphatidylinositol 4-phosphate 5-kinase Its3.

Phosphoinositides (PIPs) are a dynamic family of lipids that execute diverse roles in cell biology. PIP levels are regulated by numerous enzymes, but our understanding of how these enzymes are controlled in space and time is incomplete. One role of the PIP phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is to anchor the cytokinetic ring (CR) to the plasma membrane (PM) in Schizosaccharomyces pombe While examining potential PI(4,5)P2-binding proteins for roles in CR anchoring, we identified the dual pleckstrin homology (PH) domain-containing protein Opy1. Although related proteins are implicated in PIP regulation, we found no role for S. pombe Opy1 in CR anchoring, which would be expected if it modulated PM PI(4,5)P2 levels. Our data indicate that although Opy1 senses PM PI(4,5)P2 levels and binds to the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, Opy1 does not regulate Its3 kinase activity or PM PI(4,5)P2 levels, a striking difference from its Saccharomyces cerevisiae homolog. However, overexpression of Opy1 resulted in cytokinesis defects, as might be expected if it sequestered PI(4,5)P2 Our results highlight the evolutionary divergence of dual PH domain-containing proteins and the need for caution when interpreting results based on their overexpression.This article has an associated First Person interview with the first author of the paper.

Molecular form and function of the cytokinetic ring.

Animal cells, amoebas and yeast divide using a force-generating, actin- and myosin-based contractile ring or 'cytokinetic ring' (CR). Despite intensive research, questions remain about the spatial organization of CR components, the mechanism by which the CR generates force, and how other cellular processes are coordinated with the CR for successful membrane ingression and ultimate cell separation. This Review highlights new findings about the spatial relationship of the CR to the plasma membrane and the arrangement of molecules within the CR from studies using advanced microscopy techniques, as well as mechanistic information obtained from in vitro approaches. We also consider advances in understanding coordinated cellular processes that impact the architecture and function of the CR.

The S. pombe adaptor protein Bbc1 regulates localization of Wsp1 and Vrp1 during endocytic actin patch assembly.

Arp2/3 complex-nucleated branched actin networks provide the key force necessary for endocytosis. The Arp2/3 complex is activated by nucleation-promoting factors including the Schizosaccharomyces pombe Wiskott-Aldrich syndrome protein (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. Here, we used quantitative live-cell imaging to determine the function of the uncharacterized S. pombe protein Bbc1. We discovered that Myo1 interacts with and recruits Bbc1 to sites of endocytosis. Bbc1 competes with the verprolin Vrp1 for localization to patches and association with Myo1, thus releasing Vrp1 and its binding partner Wsp1 from Myo1. Normally Myo1 remains at the base of the endocytic invagination and Vrp1-Wsp1 internalizes 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 structures internalize twice as far. We propose that Bbc1 disrupts a transient interaction of Myo1 with Vrp1 and Wsp1 and thereby limits Arp2/3 complex-mediated nucleation of actin branches at the plasma membrane.This article has an associated First Person interview with the first author of the paper.

SIN-dependent phosphoinhibition of formin multimerization controls fission yeast cytokinesis.

Many eukaryotes accomplish cell division by building and constricting a medial actomyosin-based cytokinetic ring (CR). In Schizosaccharomyces pombe, a Hippo-related signaling pathway termed the septation initiation network (SIN) controls CR formation, maintenance, and constriction. However, how the SIN regulates integral CR components was unknown. Here, we identify the essential cytokinetic formin Cdc12 as a key CR substrate of SIN kinase Sid2. Eliminating Sid2-mediated Cdc12 phosphorylation leads to persistent Cdc12 clustering, which prevents CR assembly in the absence of anillin-like Mid1 and causes CRs to collapse when cytokinesis is delayed. Molecularly, Sid2 phosphorylation of Cdc12 abrogates multimerization of a previously unrecognized Cdc12 domain that confers F-actin bundling activity. Taken together, our findings identify a SIN-triggered oligomeric switch that modulates cytokinetic formin function, revealing a novel mechanism of actin cytoskeleton regulation during cell division.

Spatiotemporal regulation of the Dma1-mediated mitotic checkpoint coordinates mitosis with cytokinesis.

During cell division, the timing of mitosis and cytokinesis must be ordered to ensure that each daughter cell receives a complete, undamaged copy of the genome. In fission yeast, the septation initiation network (SIN) is responsible for this coordination, and a mitotic checkpoint dependent on the E3 ubiquitin ligase Dma1 and the protein kinase CK1 controls SIN signaling to delay cytokinesis when there are errors in mitosis. The participation of kinases and ubiquitin ligases in cell cycle checkpoints that maintain genome integrity is conserved from yeast to human, making fission yeast an excellent model system in which to study checkpoint mechanisms. In this review, we highlight recent advances and remaining questions related to checkpoint regulation, which requires the synchronized modulation of protein ubiquitination, phosphorylation, and subcellular localization.

Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site.

Cytokinesis in Schizosaccharomyces pombe requires the function of Cdc15, the founding member of the pombe cdc15 homology (PCH) family of proteins. As an early, abundant contractile ring component with multiple binding partners, Cdc15 plays a key role in organizing the ring. We demonstrate that Cdc15 phosphorylation at many sites generates a closed conformation, inhibits Cdc15 assembly at the division site in interphase, and precludes interaction of Cdc15 with its binding partners. Cdc15 dephosphorylation induces an open conformation, oligomerization, and scaffolding activity during mitosis. Cdc15 mutants with reduced phosphorylation precociously appear at the division site in filament-like structures and display increased association with protein partners and the membrane. Our results indicate that Cdc15 phosphoregulation impels both assembly and disassembly of the contractile apparatus and suggest a regulatory strategy that PCH family and BAR superfamily members might broadly employ to achieve temporal specificity in their roles as linkers between membrane and cytoskeleton.

Structural organization of membrane-inserted hexamers formed by Helicobacter pylori VacA toxin.

Helicobacter pylori colonizes the human stomach and is a potential cause of peptic ulceration or gastric adenocarcinoma. H. pylori secretes a pore-forming toxin known as vacuolating cytotoxin A (VacA). The 88 kDa secreted VacA protein, composed of an N-terminal p33 domain and a C-terminal p55 domain, assembles into water-soluble oligomers. The structural organization of membrane-bound VacA has not been characterized in any detail and the role(s) of specific VacA domains in membrane binding and insertion are unclear. We show that membrane-bound VacA organizes into hexameric oligomers. Comparison of the two-dimensional averages of membrane-bound and soluble VacA hexamers generated using single particle electron microscopy reveals a structural difference in the central region of the oligomers (corresponding to the p33 domain), suggesting that membrane association triggers a structural change in the p33 domain. Analyses of the isolated p55 domain and VacA variants demonstrate that while the p55 domain can bind membranes, the p33 domain is required for membrane insertion. Surprisingly, neither VacA oligomerization nor the presence of putative transmembrane GXXXG repeats in the p33 domain is required for membrane insertion. These findings provide new insights into the process by which VacA binds and inserts into the lipid bilayer to form membrane channels.

The origin and implementation of the Broadening Experiences in Scientific Training programs: an NIH common fund initiative.

Recent national reports and commentaries on the current status and needs of the U.S. biomedical research workforce have highlighted the limited career development opportunities for predoctoral and postdoctoral trainees in academia, yet little attention is paid to preparation for career pathways outside of the traditional faculty path. Recognizing this issue, in 2013, the U.S. National Institutes of Health (NIH) Common Fund issued a request for application titled "NIH Director's Biomedical Research Workforce Innovation Award: Broadening Experiences in Scientific Training (BEST)." These 5-yr 1-time grants, awarded to 17 single or partnering institutions, were designed to develop sustainable approaches to broaden graduate and postgraduate training, aimed at creating training programs that reflect the range of career options that trainees may ultimately pursue. These institutions have formed a consortium in order to work together to develop, evaluate, share, and disseminate best practices and challenges. This is a first report on the early experiences of the consortium and the scope of participating BEST programs. In this report, we describe the state of the U.S. biomedical workforce and development of the BEST award, variations of programmatic approaches to assist with program design without BEST funding, and novel approaches to engage faculty in career development programs. To test the effectiveness of these BEST programs, external evaluators will assess their outcomes not only over the 5 yr grant period but also for an additional 10 yr beyond award completion.

A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival.

Deubiquitinating enzymes (DUBs), cysteine or metallo- proteases that cleave ubiquitin chains or protein conjugates, are present in nearly every cellular compartment, with overlapping protein domain structure, localization, and functions. We discovered a cohort of DUBs that are involved in membrane trafficking (ubp4, ubp5, ubp9, ubp15, and sst2) and found that loss of all five of these DUBs but not loss of any combination of four, significantly impacted cell viability in the fission yeast Schizosaccharomyces pombe (1). Here, we delineate the collective and individual functions and activities of these five conserved DUBs using comparative proteomics, biochemistry, and microscopy. We find these five DUBs are degenerate rather than redundant at the levels of cell morphology, substrate selectivity, ubiquitin chain specificity, and cell viability under stress. These studies reveal the complexity of interplay among these enzymes, providing a foundation for understanding DUB biology and providing another example of how cells utilize degeneracy to improve survival.

Discovery of genes involved in mitosis, cell division, cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains.

The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the Sz. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini-chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of non-essential gene deletions will add to the toolkits available for the study of biological processes in Sz. pombe. Copyright © 2016 John Wiley & Sons, Ltd.

Dma1 ubiquitinates the SIN scaffold, Sid4, to impede the mitotic localization of Plo1 kinase.

Proper cell division requires strict coordination between mitotic exit and cytokinesis. In the event of a mitotic error, cytokinesis must be inhibited to ensure equal partitioning of genetic material. In the fission yeast, Schizosaccharomyces pombe, the checkpoint protein and E3 ubiquitin ligase, Dma1, delays cytokinesis by inhibiting the septation initiation network (SIN) when chromosomes are not attached to the mitotic spindle. To elucidate the mechanism by which Dma1 inhibits the SIN, we screened all SIN components as potential Dma1 substrates and found that the SIN scaffold protein, Sid4, is ubiquitinated in vivo in a Dma1-dependent manner. To investigate the role of Sid4 ubiquitination in checkpoint function, a ubiquitination deficient sid4 allele was generated and our data indicate that Sid4 ubiquitination by Dma1 is required to prevent cytokinesis during a mitotic checkpoint arrest. Furthermore, Sid4 ubiquitination delays recruitment of the Polo-like kinase and SIN activator, Plo1, to spindle pole bodies (SPBs), while at the same time prolonging residence of the SIN inhibitor, Byr4, providing a mechanistic link between Dma1 activity and cytokinesis inhibition.

Comprehensive proteomics analysis reveals new substrates and regulators of the fission yeast clp1/cdc14 phosphatase.

The conserved family of Cdc14 phosphatases targets cyclin-dependent kinase substrates in yeast, mediating late mitotic signaling events. To discover substrates and regulators of the Schizosaccharomyces pombe Cdc14 phosphatase Clp1, TAP-tagged Clp1, and a substrate trapping mutant (Clp1-C286S) were purified from asynchronous and mitotic (prometaphase and anaphase) cells and binding partners were identified by 2D-LC-MS/MS. Over 100 Clp1-interacting proteins were consistently identified, over 70 of these were enriched in Clp1-C286S-TAP (potential substrates) and we and others detected Cdk1 phosphorylation sites in over half (44/73) of these potential substrates. According to GO annotations, Clp1-interacting proteins are involved in many essential cellular processes including mitosis, cytokinesis, ribosome biogenesis, transcription, and trafficking among others. We confirmed association and dephosphorylation of multiple candidate substrates, including a key scaffolding component of the septation initiation network called Cdc11, an essential kinase of the conserved morphogenesis-related NDR kinase network named Shk1, and multiple Mlu1-binding factor transcriptional regulators. In addition, we identified Sal3, a nuclear ß-importin, as the sole karyopherin required for Clp1 nucleoplasmic shuttling, a key mode of Cdc14 phosphatase regulation. Finally, a handful of proteins were more abundant in wild type Clp1-TAP versus Clp1-C286S-TAP, suggesting that they may directly regulate Clp1 signaling or serve as scaffolding platforms to localize Clp1 activity.

Cytokinesis-based constraints on polarized cell growth in fission yeast.

The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state.