Fission Yeast System Research Articles

Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends

Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.

Fission Yeast Schizosaccharomyces pombe: A Unicellular “Micromammal” Model Organism

The fission yeast Schizosaccharomyces pombe is a rod-shaped unicellular eukaryote, well known for its contributions as a model organism for our understanding of regulation and conservation of the eukaryotic cell cycle. As a yeast divergent from the budding yeast Saccharomyces cerevisiae, S. pombe shares more common features with humans including gene structures, chromatin dynamics, and the prevalence of introns, as well as the control of gene expression through pre-mRNA splicing, epigenetic gene silencing, and RNAi pathways. With the advent of new methodologies for research, S. pombe has become an increasingly used model to investigate various molecular and cellular processes over the last 50 years. Also, S. pombe serves as an excellent system for undergraduate students to obtain hands-on research experience. Versatile experimental approaches are amenable using the fission yeast system due to its relative ease of maintenance, its inherent cellular properties, its power in classic and molecular genetics, and its feasibility in genomics and proteomics analyses. This article provides an overview of S. pombe's rise as a valuable model organism and presents examples to highlight the significance of S. pombe as a unicellular "micromammal" in investigating biological questions. We especially focus on the advantages of and the advancements in using fission yeast for studying biological processes that are characteristic of metazoans to decipher the underlining molecular mechanisms fundamental to all eukaryotes. © 2021 Wiley Periodicals LLC.

Kinetochores, cohesin, and DNA breaks: Controlling meiotic recombination within pericentromeres.

In meiosis, DNA break formation and repair are essential for the formation of crossovers between homologous chromosomes. Without crossover formation, faithful meiotic chromosome segregation and sexual reproduction cannot occur. Crossover formation is initiated by the programmed, meiosis‐specific introduction of numerous DNA double‐strand breaks, after which specific repair pathways promote recombination between homologous chromosomes. Despite its crucial nature, meiotic recombination is fraud with danger: When positioned or repaired inappropriately, DNA breaks can have catastrophic consequences on genome stability of the resulting gametes. As such, DNA break formation and repair needs to be carefully controlled. Within centromeres and surrounding regions (i.e., pericentromeres), meiotic crossover recombination is repressed in organisms ranging from yeast to humans, and a failure to do so is implicated in chromosome missegregation and developmental aneuploidy. (Peri)centromere sequence identity and organization diverge considerably across eukaryotes, yet suppression of meiotic DNA break formation and repair appear universal. Here, we discuss emerging work that has used budding and fission yeast systems to study the mechanisms underlying pericentromeric suppression of DNA break formation and repair. We particularly highlight a role for the kinetochore, a universally conserved, centromere‐associated structure essential for chromosome segregation, in suppressing (peri)centromeric DNA break formation and repair. We discuss the current understanding of kinetochore‐associated and chromosomal factors involved in this regulation and suggest future avenues of research.

CPn0572, the C. pneumoniae ortholog of TarP, reorganizes the actin cytoskeleton via a newly identified F-actin binding domain and recruitment of vinculin.

Chlamydia pneumoniae is one of the two major species of the Chlamydiaceae family that have a profound effect on human health. C. pneumoniae is linked to a number of severe acute and chronic diseases of the upper and lower respiratory tract including pneumonia, asthma, bronchitis and infection by the pathogen might play a role in lung cancer. Following adhesion, Chlamydiae secrete effector proteins into the host cytoplasm that modulate the actin cytoskeleton facilitating internalization and infection. Members of the conserved TarP protein family comprise such effector proteins that polymerize actin, and in the case of the C. trachomatis TarP protein, has been shown to play a critical role in pathogenesis. In a previous study, we demonstrated that, upon bacterial invasion, the C. pneumoniae TarP family member CPn0572 is secreted into the host cytoplasm and recruits and associates with actin via an actin-binding domain conserved in TarP proteins. We have now extended our analysis of CPn0572 and found that the CPn0572 actin binding and modulating capability is more complex. With the help of the fission yeast system, a second actin modulating domain was identified independent of the actin binding domain. Microscopic analysis of HEp-2 cells expressing different CPn0572 deletion variants mapped this domain to the C-terminal part of the protein as CPn0572536-755 binds F-actin in vitro and colocalizes with aberrantly thickened actin cables in vivo. Finally, microscopic and bioinformatic analysis revealed the existence of a vinculin binding sequence in CPn0572. Our findings contribute to the understanding of the function of the TarP family and underscore the existence of several actin binding domains and a vinculin binding site for host actin modulation.

P-E1 A novel way to test and discover new inhibitors against drug resistant HIV-1 proteases

HIV-1 protease (PR) inhibitor (PI) is one of the most potent anti-HIV drugs. When it is used in combination with other drugs, if could suppress HIV to an undetectable level. However, successful treatment is often threaten by emergence of viral drug resistance of PRs (vdrPRs). Three vdrPRs were isolated from HIV-infected patients that carry 7 (M7PR), 10 (M10PR) and 11 (M11PR) PR gene mutations, respectively. They were expressed in a gene-inducible fission yeast system to allow the measurement of PR-specific activities. All 3 vdrPRs proteolyzed natural HIV viral substrates and conferred drug resistance to Indinavir in the fission yeast, suggesting they maintained the same proteolytic and drug resistant activities in the fission yeast as in mammalian cells. Moreover, the viral enzymatic activities of these vdrPRs coupled with the induction of growth inhibition and cell death, which could potentially be used as endpoints to test the efficacy of PI activities. In this study, 5 investigational PIs were used to test the utility of the PR-producing yeast system with Darunavir (DRV) as a control. All 6 compounds suppressed the wildtype PR and the M7PR-mediated activities. However, none of them suppressed activities conferred by M10PR or M11PR. The fact that M10PR and M11PR were resistant to all of the existing PI drugs including DRV, underscores the importance of continued searching for new PIs against vdrPRs. The described fission yeast cell-based system might be suitable for future testing or discovery of new PIs through high-throughput drug screening. Because this yeast cell-based method is function-driven. It has no presumption of what kind of PI will be found. It has the potential to uncover novel PIs.

The Yeast Genomes in Three Dimensions: Mechanisms and Functions.

The three-dimensional (3D) genome structure is highly ordered by a hierarchy of organizing events ranging from enhancer-promoter or gene-gene contacts to chromosomal territorial arrangement. It is becoming clear that the cohesin and condensin complexes are key molecular machines that organize the 3D genome structure. These complexes are highly conserved from simple systems, e.g., yeast cells, to the much more complex human system. Therefore, knowledge from the budding and fission yeast systems illuminates highly conserved molecular mechanisms of how cohesin and condensin establish the functional 3D genome structures. Here I discuss how these complexes are recruited across the yeast genomes, mediate distinct genome-organizing events such as gene contacts and topological domain formation, and participate in important nuclear activities including transcriptional regulation and chromosomal dynamics.

Big data mining powers fungal research: recent advances in fission yeast systems biology approaches

Biology research has entered into big data era. Systems biology approaches therefore become the powerful tools to obtain the whole landscape of how cell separate, grow, and resist the stresses. Fission yeast Schizosaccharomyces pombe is wonderful unicellular eukaryote model, especially studying its division and metabolism can facilitate to understanding the molecular mechanism of cancer and discovering anticancer agents. In this perspective, we discuss the recent advanced fission yeast systems biology tools, mainly focus on metabolomics profiling and metabolic modeling, protein-protein interactome and genetic interaction network, DNA sequencing and applications, and high-throughput phenotypic screening. We therefore hope this review can be useful for interested fungal researchers as well as bioformaticians.

Microtubule Dynamics of S. Pombe Tubulin

We have developed a fission yeast system for expression and purification of single isoform tubulin. Replacing the non-essential S. pombe α2 tubulin gene with a second copy of the α1 encoding gene creates a strain expressing single α1 and β tubulin isoforms. We obtain yields of about 10 mg of highly purified (>99%) single isoform tubulin from an 80 l culture. Mass spectroscopy of the purified tubulin detects no post-translational modifications. Dynamic microtubules were nucleated from axonemes, recorded by video enhanced DIC microscopy and microtubule lengths measured. Microtubule growth rates increase linearly with tubulin concentration at both the fast and slow growing microtubule ends. Rate constants were determined using a simple bimolecular model. α1β tubulin has fast end kon 5.5 uM−1 s−1 and koff 6.7 s−1, and slow end kon 3.1 uM−1 s−1 and koff 2.8 s−1. Mixed isoform α1α2β has fast end kon 7.6 uM s−1 and koff 15.1 s−1 and slow end kon 2.6 uM−1 s−1 and koff 5.0 s−1. The Kd for both microtubule ends was ∼2uM for α1α2β and ∼1.1 uM for α1β suggesting similar binding affinities for tubulin heterodimers at fast and slow ends, but different kinetics. Following catastrophe the rate of rapid shrinkage was independent of the free tubulin concentration and about 2x faster at the fast compared to the slow ends: 218 ± 16 s−1 compared to 124 ± 23 s−1 for α1α2β and 263 ± 26 s−1 compared to 111 ± 29 s−1 for α1β. We conclude that S. pombe microtubule dynamics are qualitatively similar to those of brain tubulin microtubules but the kinetic rates are different, consistent with S. pombe tubulin assembling at lower concentrations than brain tubulin. We are now comparing S. pombe microtubule growth with that of brain tubulin at higher resolution.

Fission yeast Schizosaccharomyces pombe as a new system for the investigation of corticosterone methyloxidase deficiency-causing mutations

The aldosterone synthase, CYP11B2, catalyses the conversion of 11-deoxycorticosterone to aldosterone, a process that requires three steps: a hydroxylation at position 11β to form corticosterone, another one at position 18 to produce 18-hydroxycorticosterone, and, finally, an oxidation at position 18 to form aldosterone. Aldosterone synthase deficiency usually finds its expression in infancy as a life-threatening electrolyte imbalance, caused by mutations in the CYP11B2 gene. Therefore, in depth studies of mutations and their enzymatic activities will provide information for the diagnosis and management of hypoaldosteronism caused by CYP11B2 deficiencies. Here, we report the development of a fast and cheap whole-cell technology for the enzymatic characterisation of CYP11B2 mutations. The principle of the new system is the heterologous expression of the mutants of CYP11B2 in fission yeast ( Schizosaccharomyces pombe) followed by steroid bioconversion assays for the enzymatic characterisation of the investigated mutants. The new system was validated and 10 known mutations of CYP11B2 have been investigated, two of them for the first time concerning their effect on the CYP11B2 three-step reaction. The results of the fission yeast system were in good agreement with the cell culture results presenting this new system as an alternative non radioactive method that can be applied for the enzymatic characterisation of CYP11B2 mutations.

Homologous Recombination Restarts Blocked Replication Forks at the Expense of Genome Rearrangements by Template Exchange

Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

The Fission Yeast Inhibitor of Growth (ING) Protein Png1p Functions in Response to DNA Damage

In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.

Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

Rad51 suppresses gross chromosomal rearrangement at centromere in Schizosaccharomyces pombe

Centromere that plays a pivotal role in chromosome segregation is composed of repetitive elements in many eukaryotes. Although chromosomal regions containing repeats are the hotspots of rearrangements, little is known about the stability of centromere repeats. Here, by using a minichromosome that has a complete set of centromere sequences, we have developed a fission yeast system to detect gross chromosomal rearrangements (GCRs) that occur spontaneously. Southern and comprehensive genome hybridization analyses of rearranged chromosomes show two types of GCRs: translocation between homologous chromosomes and formation of isochromosomes in which a chromosome arm is replaced by a copy of the other. Remarkably, all the examined isochromosomes contain the breakpoint in centromere repeats, showing that isochromosomes are produced by centromere rearrangement. Mutations in the Rad3 checkpoint kinase increase both types of GCRs. In contrast, the deletion of Rad51 recombinase preferentially elevates isochromosome formation. Chromatin immunoprecipitation analysis shows that Rad51 localizes at centromere around S phase. These data suggest that Rad51 suppresses rearrangements of centromere repeats that result in isochromosome formation.

Functional screening of salt stress‐related genes fromThellungiella halophilausing fission yeast system

Salinity stress is one of the most serious factors limiting plant growth and threatening global food security. Identification of salt tolerance relevant genes from halophytes can provide more insights into the determinants of salt stress tolerance in plants. In this study, functional screening of genes for the salinity tolerance inThellungiella halophila(salt cress) was carried out using fission yeast as a host organism. A cDNA library of the salt cress, constructed in the yeast vector under the control of the inducible promoter nmt1, was transformed into the fission yeastSchizosaccharomyces pombe.Transgenic yeast cells that showed enhanced salt tolerance were selected. A total of 69 such clones were identified from 106of yeast transformants by one‐time screening. Functional assignment indicates that a large percentage (81%) of these cDNAs encode for homologues of proteins that are known to have roles in plant salt tolerance, while the other portion (19%) codes for unknown proteins. Among the proteins encoded by the known functional genes, the regulatory proteins account for a relatively higher proportion. Northern blot analysis showed that the expression of some isolated genes was salt stress inducible, whereas that of the others was constitutive. These results indicate that fission yeast is a simple and efficient system for functional and large‐scale identification of salt tolerance‐related genes from other organisms.

Mouse and Human La Proteins Differ in Kinase Substrate Activity and Activation Mechanism for tRNA Processing

The La protein interacts with a variety of small RNAs as well as certain growth-associated mRNAs such as Mdm2 mRNA. Human La (hLa) phosphoprotein is so highly conserved that it can replace the tRNA processing function of the fission yeast La protein in vivo. We used this system, which is based on tRNA-mediated suppression (TMS) of ade6-704 in S. pombe, to compare the activities of mouse and human La proteins. Prior studies indicate that hLa is activated by phosphorylation of serine-366 by protein kinase CK2, neutralizing a negative effect of a short basic motif (SBM). First, we report the sequence mapping of the UGA stop codon that requires suppressor tRNA for TMS, to an unexpected site in S. pombe ade6-704. Next, we show that, unlike hLa, native mLa is unexpectedly inactive for TMS, although its intrinsic activity is revealed by deletion of its SBM. We then show that mLa is not phosphorylated by CK2, accounting for the mechanistic difference between mLa and hLa. We found a PKA/PKG target sequence in mLa (S199) that is not present in hLa, and show that PKA/PKG efficiently phosphorylates mLa S199 in vitro. A noteworthy conclusion that comes from this work is that this fission yeast system can be used to gain insight into differences in control mechanisms used by La proteins of different mammalian species. Finally, RNA binding assays indicate that while mutation of mLa S199 has little effect on pre-tRNA binding, it substantially decreases binding to a probe derived from Mdm2 mRNA. In closing, we note that species-specific signaling through La may be relevant to the La-dependent Mdm2 pathways of p53 metabolism and cancer progression in mice and humans.

Functional identification of cytokinesis-related genes from tobacco BY-2 cells

The molecular mechanisms controlling cytokinesis in plant cell division cycle remains largely unknown. In this study, a functional approach was taken to identify genes that may play roles in cytokinesis in tobacco BY-2 cells, using fission yeast as the host organism. A total of 22 BY-2 genes that perturbed the terminal stage of cell division when ectopically expressed in yeast cells were isolated, among which, several encode for uncharacterized genes. Additionally, RT-PCR analysis indicated that four of the isolated genes were expressed in a cell cycle-dependent manner. Our results demonstrate that fission yeast system can be efficiently used to identify the genes that may function, either positively or negatively, in the regulation of cytokinesis. More importantly, the candidate genes we have isolated in this work can provide useful information for unraveling the regulators controlling cell separation at the late stage of BY-2 cell division.

Overexpression Phenotypes of Plk1 and Ndrg2 in Schizosaccharomyces Pombe: Fission Yeast System for Mammalian Gene Study

The fission yeast, Schizosaccharomyces pombe is a single-celled free-living fungus that shares many features with cells of more complicated eukaryotes. Many of the genes required for the cell-cycle control, proteolysis, protein modification, and RNA splicing are highly conserved with those of higher eukaryotes. Moreover, fission yeast has the merit of genetics and its genetic system is already well characterized. As such, the current study evaluated the use of a fission yeast system as a tool for the functional study of mammalian genes and attempted to set up an assay system for novel genes. Since the phenotypes of a deletion mutant and the overexpression of a gene are generally analyzed for a functional study of specific genes in yeast, the present study used overexpression phenotypes to study the functions of mammalian genes. Therefore, based on using a thiamine-repressive promoter, two mammalian genes were expressed in fission yeast, and their overexpressed phenotypes compared with those in mammalian cells. The phenotypes resulting from overexpression were analyzed using a FACS, which analyzes the DNA contents, and a microscope. One of the selected genes was the mammalian Polo-like kinase 1 (Plk1), which is activated and plays a role in the mitotic phase of the cell division cycle. The overexpression of various constructs of Plk1 in the HeLa cells caused cell cycle defects, suggesting that the ectopic Plk1s blocked the endogenous Plk1 in the cells. As expected, when the constructs were overexpressed in the fission yeast system, the cells were arrested in mitosis and defected at the end of mitosis. As such, this data suggests that the Plk1-overexpressed phenotypes were similar in the mammalian cells and the fission yeast, thereby enabling the mammalian Plk1 functions to be approximated in the fission yeast. The other selected gene was the N-Myc downstream-regulated gene 2 (ndrg2), which is upregulated during cell differentiation, yet still not well characterized. When the ndrg2 gene was overexpressed in the fission yeast, the cells contained multi-septa. The septa were positioned well, yet their number increased per cell. Therefore, this gene was speculated to block cell division in the last stage of the cell cycle, making the phenotype potentially useful for explaining cell growth and differentiation in mammalian cells. Accordingly, fission yeast is demonstrated to be an appropriate species for the functional study of mammalian genes.

The Schizosaccharomyces pombe mgU6-47 gene is required for 2'-O-methylation of U6 snRNA at A41

Through a computer search of DNA databases, we have identified the homologs of the mgU6-47 snoRNA gene from the yeast Schizosaccharomyces pombe, the fly Drosophila melanogaster and human. The three box C/D-containing snoRNA genes showed no significant similarity in their sequences except for an 11 nt long complementarity to U6 snRNA, suggesting that the mechanism of snoRNA guided snRNA methylation is conserved from mammals to yeast. The corresponding snoRNAs have been positively detected by reverse transcription and northern blotting. Taking advantage of the fission yeast system, we have disrupted the yeast mgU6-47 gene and demonstrated that it is absolutely required for site-specific 2'-O-methylation of U6 at position A41. No growth differences between mgU6-47 gene-disrupted and wild-type cells were observed, suggesting that the mgU6-47 gene, as for most rRNA methylation guides, is dispensable in yeast. Nevertheless, it was revealed by temperature shift assay that abolition of A41 methylation in yeast U6 snRNA might cause a small decrease in mRNA splicing efficiency. The timing of S.pombe U6 pre-RNA transport in the nucleus for splicing and methylation was also analyzed and is described.