Proliferation Analyses of Conditional Knockdown Strains Using CRISPR Interference in Fission Yeast.

CRISPR interference is a method to conditionally inhibit transcription of an arbitrary target gene. This is useful to study the functions of essential genes, which are required for cellular viability. Although many conditional gene perturbation techniques are available for Schizosaccharomyces pombe, CRISPRi facilitates construction of a large number of knockdown strains because of its systematic, simple procedure.

Arrayed CRISPRi library to suppress genes required for Schizosaccharomyces pombe viability.

The fission yeast, Schizosaccharomyces pombe, is an excellent eukaryote model organism for studying essential biological processes. Its genome contains ∼1,200 genes essential for cell viability, most of which are evolutionarily conserved. To study these essential genes, resources enabling conditional perturbation of target genes are required.

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.

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 , we screened 457 deletion () mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.

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.

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).

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.

Characterization of hexose transporter genes in the views of the chronological life span and glucose uptake in fission yeast.

Fission yeast, Schizosaccharomyces pombe, possesses eight hexose transporters, Ght1~8. In order to clarify the role of each hexose transporter on glucose uptake, a glucose uptake assay system was established and the actual glucose uptake activity of each hexose transporter-deletion mutant was measured. Under normal growth condition containing 2% glucose, ∆ght5 and ∆ght2 mutants showed large and small decrease in glucose uptake activity, respectively.

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, , 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.

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.

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 , 770 proteins encoded by chromosomal genes are located in mitochondria.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Epigenetic inactivation and subsequent heterochromatinization of a centromere stabilize dicentric chromosomes.

Background: The kinetochore is a multiprotein complex that forms on a chromosomal locus designated as the centromere, which links the chromosome to the spindle during mitosis and meiosis. Most eukaryotes, with the exception of holocentric species, have a single distinct centromere per chromosome, and the presence of multiple centromeres on a single chromosome is predicted to cause breakage and/or loss of that chromosome. However, some stably maintained non-Robertsonian translocated chromosomes have been reported, suggesting that the excessive centromeres are inactivated by an as yet undetermined mechanism.

Specific biomarkers for stochastic division patterns and starvation-induced quiescence under limited glucose levels in fission yeast.

Glucose as a source of energy is centrally important to our understanding of life. We investigated the cell division-quiescence behavior of the fission yeast Schizosaccharomyces pombe under a wide range of glucose concentrations (0-111 mM). The mode of S.

Hsk1- and SCF(Pof3)-dependent proteolysis of S. pombe Ams2 ensures histone homeostasis and centromere function.

Schizosaccharomyces pombe GATA factor Ams2 is responsible for cell cycle-dependent transcriptional activation of all the core histone genes peaking at G1/S phase. Intriguingly, its own protein level also fluctuates concurrently. Here, we show that Ams2 is ubiquitylated and degraded through the SCF (Skp1-Cdc53/Cullin-1-F-box) ubiquitin ligase, in which F box protein Pof3 binds this protein.

CENP-A reduction induces a p53-dependent cellular senescence response to protect cells from executing defective mitoses.

Cellular senescence is an irreversible growth arrest and is presumed to be a natural barrier to tumor development. Like telomere shortening, certain defects in chromosome integrity can trigger senescence; however, the roles of centromere proteins in regulating commitment to the senescent state remains to be established. We examined chromatin structure in senescent human primary fibroblasts and found that CENP-A protein levels are diminished in senescent cells.

Dual regulation of Mad2 localization on kinetochores by Bub1 and Dam1/DASH that ensure proper spindle interaction.

The spindle assembly checkpoint monitors the state of spindle-kinetochore interaction to prevent premature onset of anaphase. Although checkpoint proteins, such as Mad2, are localized on kinetochores that do not interact properly with the spindle, it remains unknown how the checkpoint proteins recognize abnormalities in spindle-kinetochore interaction. Here, we report that Mad2 localization on kinetochores in fission yeast is regulated by two partially overlapping but distinct pathways: the Dam1/DASH and the Bub1 pathways.