Genetic knockdown of genes that are obscure, conserved and essential using CRISPR interference methods in the fission yeast S. pombe.

Characterizing functions of essential genes is challenging, as perturbing them is generally lethal. Conditional gene perturbation, including use of temperature-sensitive mutants, has been widely utilized to reveal functions of essential genes in the fission yeast Schizosaccharomyces pombe. However, recently we implemented a systematic and less time-consuming knockdown method, CRISPR interference (CRISPRi), in this organism using catalytically inactive Cas9 (dCas9). This technology has been expected to facilitate characterization of essential genes in S. pombe, although this still has not occurred. Here, CRISPRi was harnessed to study uncharacterized essential genes that are evolutionally conserved from yeasts to mammals. Transcription of these genes, which we call conserved essential obscure (ceo) genes, was repressed using conventional dCas9-mediated CRISPRi and by implementing technologies that enhance repression efficiency or alleviate limitations on small guide RNA (sgRNA) design. These CRISPRi methods successfully reduced transcription of target genes and allowed us to characterize resulting phenotypes. Knockdown of ceo genes inhibited cell proliferation and altered cellular morphology. Thus, dCas9-based CRISPRi methods utilized in this study enhanced accessibility of genetic analyses targeting essential genes in S. pombe.

Growth conditions inducing G1 cell cycle arrest enhance lipid production in the oleaginous yeast Lipomyces starkeyi.

Lipid droplets are cytoplasmic organelles that store lipids for energy and membrane synthesis. The oleaginous yeast Lipomyces starkeyi is one of the most promising lipid producers and has attracted attention as a biofuel source. It is known that the expansion of lipid droplets is enhanced under nutrient-poor conditions. Therefore, we prepared a novel nitrogen-depleted medium (N medium) in which to culture L. starkeyi cells. Lipid accumulation was rapidly induced, and this was reversed by the addition of ammonium. In this condition, cell proliferation stopped, and cells with giant lipid droplets were arrested in G1 phase. We investigated whether cell cycle arrest at a specific phase is required for lipid accumulation. Lipid accumulation was repressed in hydroxyurea-synchronized S phase cells and was increased in nocodazole-arrested G2/M phase cells. Moreover, the enrichment of G1 phase cells seen upon rapamycin treatment induced massive lipid accumulation. From these results, we conclude that L. starkeyi cells store lipids from G2/M phase and then arrest cell proliferation in the subsequent G1 phase, where lipid accumulation is enhanced. Cell cycle control is an attractive approach for biofuel production.

TORC2 inhibition of α-arrestin Aly3 mediates cell surface persistence of S. pombe Ght5 glucose transporter in low glucose.

In the fission yeast, Schizosaccharomyces pombe, the high-affinity hexose transporter, Ght5, must be transcriptionally upregulated and localized to the cell surface for cell division under limited glucose. Although cell-surface localization of Ght5 depends on Target of rapamycin complex 2 (TORC2), the molecular mechanisms by which TORC2 ensures proper localization of Ght5 remain unknown. We performed genetic screening for gene mutations that restore Ght5 localization on the cell surface in TORC2-deficient mutant cells, and identified a gene encoding an uncharacterized α-arrestin-like protein, Aly3/SPCC584.15c. α-arrestins are thought to recruit a ubiquitin ligase to membrane-associated proteins. Consistently, Ght5 is ubiquitylated in TORC2-deficient cells, and this ubiquitylation is dependent on Aly3. TORC2 supposedly enables cell-surface localization of Ght5 by preventing Aly3-dependent ubiquitylation and subsequent ubiquitylation-dependent translocation of Ght5 to vacuoles. Surprisingly, nitrogen starvation, but not glucose depletion, triggers Aly3-dependent transport of Ght5 to vacuoles in S. pombe, unlike budding yeast hexose transporters, vacuolar transport of which is initiated upon changes in hexose concentration. This study provides new insights into the molecular mechanisms controlling the subcellular localization of hexose transporters in response to extracellular stimuli.

The putative ceramide-conjugation protein Cwh43 regulates G0 quiescence, nutrient metabolism and lipid homeostasis in fission yeast.

Cellular nutrient states control whether cells proliferate, or whether they enter or exit quiescence. Here, we report characterizations of fission yeast temperature-sensitive (ts) mutants of the evolutionarily conserved transmembrane protein Cwh43, and explore its relevance to utilization of glucose, nitrogen source and lipids. GFP-tagged Cwh43 localizes at ER associated with the nuclear envelope and the plasma membrane, as in budding yeast. We found that cwh43 mutants failed to divide in low glucose and lost viability during quiescence under nitrogen starvation. In cwh43 mutants, comprehensive metabolome analysis demonstrated dramatic changes in marker metabolites that altered under low glucose and/or nitrogen starvation, although cwh43 cells apparently consumed glucose in the culture medium. Furthermore, we found that cwh43 mutant cells had elevated levels of triacylglycerols (TGs) and coenzyme A, and that they accumulated lipid droplets. Notably, TG biosynthesis was required to maintain cell division in the cwh43 mutant. Thus, Cwh43 affects utilization of glucose and nitrogen sources, as well as storage lipid metabolism. These results may fit a notion developed in budding yeast stating that Cwh43 conjugates ceramide to glycosylphosphatidylinositol (GPI)-anchored proteins and maintains integrity of membrane organization.

Identification of 15 New Bypassable Essential Genes of Fission Yeast.

Every organism has a different set of genes essential for its viability. This indicates that an organism can become tolerant to the loss of an essential gene under certain circumstances during evolution, via the manifestation of 'masked' alternative mechanisms. In our quest to systematically uncover masked mechanisms in eukaryotic cells, we developed an extragenic suppressor screening method using haploid spores deleted of an essential gene in the fission yeast Schizosaccharomyces pombe. We screened for the 'bypass' suppressors of lethality of 92 randomly selected genes that are essential for viability in standard laboratory culture conditions. Remarkably, extragenic mutations bypassed the essentiality of as many as 20 genes (22%), 15 of which have not been previously reported. Half of the bypass-suppressible genes were involved in mitochondria function; we also identified multiple genes regulating RNA processing. 18 suppressible genes were conserved in the budding yeast Saccharomyces cerevisiae, but 13 of them were non-essential in that species. These trends suggest that essentiality bypass is not a rare event and that each organism may be endowed with secondary or backup mechanisms that can substitute for primary mechanisms in various biological processes. Furthermore, the robustness of our simple spore-based methodology paves the way for genome-scale screening.Key words: Schizosaccharomyces pombe, extragenic suppressor screening, bypass of essentiality (BOE), cut7 (kinesin-5), hul5 (E3 ubiquitin ligase).

Selenium deposition in an atypically disintegrated hoof wall in a Thoroughbred racehorse with alkali disease: Proof by energy-dispersive X-ray fluorescence analysis.

From summer 2018 to summer 2019, several Thoroughbred racehorses held at the Miho Training Centre of the Japan Racing Association inadvertently ingested excessive amounts of sodium selenite, resulting in typical chronic selenium (Se) poisoning - the so-called alkali disease. The typical abnormality was a hoof wall disorder with a circumferentially deep ring and/or transverse hoof wall cracks parallel to the coronet on all feet and appearing after excessive ingestion. One affected Thoroughbred male was unique in that all the hooves had a rough surface with a very fragile hoof wall, but no wall rings or transverse cracking. This horse was euthanized because of dysstasia due to the permanent foot pain associated with hoof wall deformities in the front feet. To detect Se deposition in the hooves, we used energy-dispersive X-ray fluorescence (EDXRF) analysis to measure the Se signal intensity of each lesion. Characteristic Se-kα signals were emitted from the areas of histologically damaged hoof wall at 33.76 ± 11.78 (mean ± SD) counts per second (cps)/mm2. In contrast, the signal from the uninjured proximal hoof wall was 1.43 ± 0.14 cps/mm2 and that from the uninjured distal hoof wall was 1.51 ± 0.23 cps/mm2. The much greater Se deposition in the injured hoof walls suggests that their disintegration was caused by alkali disease. These results indicate that atypical hoof wall abnormalities due to alkali disease can be diagnosed by EDXRF analysis.

Switching the centromeres on and off: epigenetic chromatin alterations provide plasticity in centromere activity stabilizing aberrant dicentric chromosomes.

The kinetochore, which forms on a specific chromosomal locus called the centromere, mediates interactions between the chromosome and the spindle during mitosis and meiosis. Abnormal chromosome rearrangements and/or neocentromere formation can cause the presence of multiple centromeres on a single chromosome, which results in chromosome breakage or cell cycle arrest. Analyses of artificial dicentric chromosomes suggested that the activity of the centromere is regulated epigenetically; on some stably maintained dicentric chromosomes, one of the centromeres no longer functions as a platform for kinetochore formation, although the DNA sequence remains intact. Such epigenetic centromere inactivation occurs in cells of various eukaryotes harbouring 'regional centromeres', such as those of maize, fission yeast and humans, suggesting that the position of the active centromere is determined by epigenetic markers on a chromosome rather than the nucleotide sequence. Our recent findings in fission yeast revealed that epigenetic centromere inactivation consists of two steps: disassembly of the kinetochore initiates inactivation and subsequent heterochromatinization prevents revival of the inactivated centromere. Kinetochore disassembly followed by heterochromatinization is also observed in normal senescent human cells. Thus epigenetic centromere inactivation may not only stabilize abnormally generated dicentric chromosomes, but also be part of an intrinsic mechanism regulating cell proliferation.

Transcriptional Regulation Technology for Gene Perturbation in Fission Yeast.

Isolation and introduction of genetic mutations is the primary approach to characterize gene functions in model yeasts. Although this approach has proven very powerful, it is not applicable to all genes in these organisms. For example, introducing defective mutations into essential genes causes lethality upon loss of function. To circumvent this difficulty, conditional and partial repression of target transcription is possible. While transcriptional regulation techniques, such as promoter replacement and 3' untranslated region (3'UTR) disruption, are available for yeast systems, CRISPR-Cas-based technologies have provided additional options. This review summarizes these gene perturbation technologies, including recent advances in methods based on CRISPR-Cas systems for Schizosaccharomyces pombe. We discuss how biological resources afforded by CRISPRi can promote fission yeast genetics.

In fission yeast, 65 non-essential mitochondrial proteins related to respiration and stress become essential in low-glucose conditions.

Mitochondria perform critical functions, including respiration, ATP production, small molecule metabolism, and anti-oxidation, and they are involved in a number of human diseases. While the mitochondrial genome contains a small number of protein-coding genes, the vast majority of mitochondrial proteins are encoded by nuclear genes. In fission yeast Schizosaccharomyces pombe, we screened 457 deletion (del) mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.03-0.1%; low-glucose sensitive, lgs), but that proliferate in regular 2-3% glucose medium. Sixty-five (14%) of the 457 deletion mutants displayed the lgs phenotype. Thirty-three of them are defective either in dehydrogenases, subunits of respiratory complexes, the citric acid cycle, or in one of the nine steps of the CoQ10 biosynthetic pathway. The remaining 32 lgs mutants do not seem to be directly related to respiration. Fifteen are implicated in translation, and six encode transporters. The remaining 11 function in anti-oxidation, amino acid synthesis, repair of DNA damage, microtubule cytoskeleton, intracellular mitochondrial distribution or unknown functions. These 32 diverse lgs genes collectively maintain mitochondrial functions under low (1/20-1/60× normal) glucose concentrations. Interestingly, 30 of them have homologues associated with human diseases.

Fission Yeast TORC2 Signaling Pathway Ensures Cell Proliferation under Glucose-Limited, Nitrogen-Replete Conditions.

Target of rapamycin (TOR) kinases form two distinct complexes, TORC1 and TORC2, which are evolutionarily conserved among eukaryotes. These complexes control intracellular biochemical processes in response to changes in extracellular nutrient conditions. Previous studies using the fission yeast, Schizosaccharomyces pombe, showed that the TORC2 signaling pathway, which is essential for cell proliferation under glucose-limited conditions, ensures cell-surface localization of a high-affinity hexose transporter, Ght5, by downregulating its endocytosis. The TORC2 signaling pathway retains Ght5 on the cell surface, depending on the presence of nitrogen sources in medium. Ght5 is transported to vacuoles upon nitrogen starvation. In this review, we discuss the molecular mechanisms underlying this regulation to cope with nutritional stress, a response which may be conserved from yeasts to mammals.

Implementation of dCas9-mediated CRISPRi in the fission yeast Schizosaccharomyces pombe.

Controllable and reversible transcriptional repression is an essential method to study gene functions. A systematic knock-down method using catalytically inactive Cas9 (dCas9) was originally established in bacteria. dCas9 forms a ribonucleoprotein with a small guide RNA and uses it to recognize a specific DNA sequence via Watson-Crick base-pairing. When specifically bound to a targeted DNA, dCas9 impairs RNA polymerase activity and represses transcription of that target gene. This technology, CRISPRi, has been implemented in several organisms, but not in Schizosaccharomyces pombe using dCas9. Here, we provide a plasmid that expresses dCas9 and sgRNA in fission yeast. With this plasmid, CRISPRi repressed endogenous gene transcription by as much as 87%. This transcriptional repression method is controllable, reversible, and efficient enough to alter cellular phenotypes. Here, we offer a CRISPRi method to choose proper targeting sequences for transcriptional repression in fission yeast. Implementation of CRISPRi will help to reveal gene functions and to develop tools based on dCas9 technology in S. pombe.

Multiple nutritional phenotypes of fission yeast mutants defective in genes encoding essential mitochondrial proteins.

Mitochondria are essential for regulation of cellular respiration, energy production, small molecule metabolism, anti-oxidation and cell ageing, among other things. While the mitochondrial genome contains a small number of protein-coding genes, the great majority of mitochondrial proteins are encoded by chromosomal genes. In the fission yeast Schizosaccharomyces pombe, 770 proteins encoded by chromosomal genes are located in mitochondria. Of these, 195 proteins, many of which are implicated in translation and transport, are absolutely essential for viability. We isolated and characterized eight temperature-sensitive (ts) strains with mutations in essential mitochondrial proteins. Interestingly, they are also sensitive to limited nutrition (glucose and/or nitrogen), producing low-glucose-sensitive and 'super-housekeeping' phenotypes. They fail to produce colonies under low-glucose conditions at the permissive temperature or lose cell viability under nitrogen starvation at the restrictive temperature. The majority of these ts mitochondrial mutations may cause defects of gene expression in the mitochondrial genome. mrp4 and mrp17 are defective in mitochondrial ribosomal proteins. ppr3 is defective in rRNA expression, and trz2 and vrs2 are defective in tRNA maturation. This study promises potentially large dividends because mitochondrial quiescent functions are vital for human brain and muscle, and also for longevity.

Expression of a novel 90-kDa protein, Lsd90, involved in the metabolism of very long-chain fatty acid-containing phospholipids in a mitosis-defective fission yeast mutant.

The fission yeast lsd1/fas2 strain carries a temperature-sensitive mutation of the fatty-acid-synthase alpha-subunit, exhibiting an aberrant mitosis lsd phenotype, with accumulation of very-long-chain fatty-acid-containing phospholipid (VLCFA-PL). A novel 90-kDa protein, Lsd90 (SPBC16E9.16c), was found to be newly expressed in small particle-like structures in lsd1/fas2 cells under restrictive conditions. Two mismatches leading to a double frame shift were found between the sequences of the lsd90(+) gene registered in the genomic database and the sequences determined experimentally at the amino acid, cDNA and genomic DNA levels. Unexpectedly, overexpression and disruption of the lsd90(+) gene in either lsd1/fas2 or wild-type cells did not affect either cell growth or expression of the lsd phenotype. The amounts of VLCFA-PL that accumulated in lsd90-overexpressing lsd1/fas2 cells were significantly lower than those in lsd1/fas2 cells, suggesting the involvement of Lsd90 in the metabolism of VLCFA-PL.

Spindle checkpoint signaling requires the mis6 kinetochore subcomplex, which interacts with mad2 and mitotic spindles.

The spindle checkpoint coordinates cell cycle progression and chromosome segregation by inhibiting anaphase promoting complex/cyclosome until all kinetochores interact with the spindle properly. During early mitosis, the spindle checkpoint proteins, such as Mad2 and Bub1, accumulate at kinetochores that do not associate with the spindle. Here, we assess the requirement of various kinetochore components for the accumulation of Mad2 and Bub1 on the kinetochore in fission yeast and show that the necessity of the Mis6-complex and the Nuf2-complex is an evolutionarily conserved feature in the loading of Mad2 onto the kinetochore. Furthermore, we demonstrated that Nuf2 is required for maintaining the Mis6-complex on the kinetochore during mitosis. The Mis6-complex physically interacts with Mad2 under the condition that the Mad2-dependent checkpoint is activated. Ectopically expressed N-terminal fragments of Mis6 localize along the mitotic spindle, highlighting the potential binding ability of Mis6 not only to the centromeric chromatin but also to the spindle microtubules. We propose that the Mis6-complex, in collaboration with the Nuf2-complex, monitors the spindle-kinetochore attachment state and acts as a platform for Mad2 to accumulate at unattached kinetochores.

BAF53/Arp4 homolog Alp5 in fission yeast is required for histone H4 acetylation, kinetochore-spindle attachment, and gene silencing at centromere.

Nuclear actin-related proteins play vital roles in transcriptional regulation; however, their biological roles remain elusive. Here, we characterize Alp5, fission yeast homolog of Arp4/BAF53. The temperature-sensitive mutant alp5-1134 contains a single amino acid substitution in the conserved C-terminal domain (S402N) and displays mitotic phenotypes, including chromosome condensation and missegregation. Alp5 forms a complex with Mst1-HAT (histone acetyltransferase). Consistently, inhibition of histone deacetylases (HDACs), by either addition of a specific inhibitor or a mutation in HDAC-encoding clr6+ gene, rescues alp5-1134. Immunoblotting with specific antibodies against acetylated histones shows that Alp5 is required for histone H4 acetylation at lysines 5, 8, and 12, but not histone H3 lysines 9 or 14, and furthermore Clr6 plays an opposing role. Mitotic arrest is ascribable to activation of the Mad2/Bub1 spindle checkpoint, in which both proteins localize to the mitotic kinetochores in alp5-1134. Intriguingly, alp5-1134 displays transcriptional desilencing at the core centromere without altering the overall chromatin structure, which also is suppressed by a simultaneous mutation in clr6+. This result shows that Alp5 is essential for histone H4 acetylation, and its crucial role lies in the establishment of bipolar attachment of the kinetochore to the spindle and transcriptional silencing at the centromere.

Extracellular glucose level regulates dependence on GRP78 for cell surface localization of multipass transmembrane proteins in HeLa cells.

Many human-cultured cell lines survive glucose starvation, but the underlying mechanisms remain unclear. Here, we searched for proteins required for cellular adaptation to glucose-limited conditions and identified several endoplasmic reticulum chaperones in the glucose-regulated protein (GRP) family as proteins enriched in the cellular membrane. Surprisingly, these proteins, which are required for cell surface localization of GLUT1 under high-glucose conditions, become dispensable for targeting GLUT1 to the surface upon glucose starvation. In marked contrast, cell surface localization of single-pass transmembrane proteins, such as epidermal growth factor receptor and CD98, is not disturbed by GRP78 depletion regardless of the extracellular glucose level. These results indicate that the extracellular glucose level regulates dependence on the GRPs for cell surface localization of multipass transmembrane proteins.

Glucose restriction induces transient G2 cell cycle arrest extending cellular chronological lifespan.

While glucose is the fundamental source of energy in most eukaryotes, it is not always abundantly available in natural environments, including within the human body. Eukaryotic cells are therefore thought to possess adaptive mechanisms to survive glucose-limited conditions, which remain unclear. Here, we report a novel mechanism regulating cell cycle progression in response to abrupt changes in extracellular glucose concentration. Upon reduction of glucose in the medium, wild-type fission yeast cells undergo transient arrest specifically at G2 phase. This cell cycle arrest is dependent on the Wee1 tyrosine kinase inhibiting the key cell cycle regulator, CDK1/Cdc2. Mutant cells lacking Wee1 are not arrested at G2 upon glucose limitation and lose viability faster than the wild-type cells under glucose-depleted quiescent conditions, suggesting that this cell cycle arrest is required for extension of chronological lifespan. Our findings indicate the presence of a novel cell cycle checkpoint monitoring glucose availability, which may be a good molecular target for cancer therapy.

Adaptive regulation of glucose transport, glycolysis and respiration for cell proliferation.

The cell must utilise nutrients to generate energy as a means of sustaining its life. As the environment is not necessarily abundant in nutrients and oxygen, the cell must be able to regulate energy metabolism to adapt to changes in extracellular and intracellular conditions. Recently, several key regulators of energy metabolism have been reported. This review describes the recent advances in molecular regulation of energy metabolism, focusing mainly on glycolysis and its shunt pathways. Human diseases, such as cancer and neurodegenerative disorders, are also discussed in relation to failure of energy metabolism regulation.

Mechanisms of expression and translocation of major fission yeast glucose transporters regulated by CaMKK/phosphatases, nuclear shuttling, and TOR.

Hexose transporters are required for cellular glucose uptake; thus they play a pivotal role in glucose homeostasis in multicellular organisms. Using fission yeast, we explored hexose transporter regulation in response to extracellular glucose concentrations. The high-affinity transporter Ght5 is regulated with regard to transcription and localization, much like the human GLUT transporters, which are implicated in diabetes. When restricted to a glucose concentration equivalent to that of human blood, the fission yeast transcriptional regulator Scr1, which represses Ght5 transcription in the presence of high glucose, is displaced from the nucleus. Its displacement is dependent on Ca(2+)/calmodulin-dependent kinase kinase, Ssp1, and Sds23 inhibition of PP2A/PP6-like protein phosphatases. Newly synthesized Ght5 locates preferentially at the cell tips with the aid of the target of rapamycin (TOR) complex 2 signaling. These results clarify the evolutionarily conserved molecular mechanisms underlying glucose homeostasis, which are essential for preventing hyperglycemia in humans.

Does a shift to limited glucose activate checkpoint control in fission yeast?

Here we review cell cycle control in the fission yeast, Schizosaccharomyces pombe, in response to an abrupt reduction of glucose concentration in culture media. S. pombe arrests cell cycle progression when transferred from media containing 2.0% glucose to media containing 0.1%. After a delay, S. pombe resumes cell division at a surprisingly fast rate, comparable to that observed in 2% glucose. We found that a number of genes, including zinc-finger transcription factor Scr1, CaMKK-like protein kinase Ssp1, and glucose transporter Ght5, enable rapid cell division in low glucose. In this article, we examine whether cell cycle checkpoint-like control operates during the delay and after resumption of cell division in limited-glucose. Using microarray analysis and genetic screening, we identified several candidate genes that may be involved in controlling this low-glucose adaptation.