scholarly journals Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors

2018 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoaki Yamauchi ◽  
Ken-ichi Kimura ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Cancer Cells ◽  
Yeast Cells ◽  
Transformed Cells ◽  
Functional Assay ◽  
Spindle Poles ◽  
Multipolar Spindles ◽  
Protein Overproduction ◽  
Bipolar Spindles

ABSTRACTMany cancer cells contain more than two centrosomes, yet these cancer cells can form bipolar spindles and appear to proliferate normally, instead of committing lethal mitoses with multipolar spindles. It is shown that extra centrosomes are clustered into two pseudo-bipolar spindle poles, thereby escaping from multipolarity. Human kinesin-14 (HSET or KIFC1), a minus end-directed motor, plays a crucial role in centrosome clustering and as such, HSET is essential for cell viability only in cancer cells with supernumerary centrosomes, but not in non-transformed cells. Accordingly, HSET is deemed to be an efficient chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity, efficacy and off-target cytotoxicity have not been evaluated rigorously. Here we show that these inhibitors on their own are cytotoxic to fission yeast, suggesting that they have other targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize overproduced HSET and partially rescue its lethality. This methodology of protein overproduction in fission yeast provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest.

scholarly journals In Vivo Functional Assay of a Recombinant Aquaporin in Pichia pastoris

2006 ◽  
Vol 72 (2) ◽  
pp. 1507-1514 ◽  
Author(s):  
Mark J. Daniels ◽  
Malcolm R. Wood ◽  
Mark Yeager
Keyword(s):  
Pichia Pastoris ◽  
Linear Relationship ◽  
Osmotic Shock ◽  
Water Channel ◽  
Yeast Cells ◽  
Mercury Chloride ◽  
Functional Assay ◽  
Channel Activity ◽  
Wild Type

ABSTRACT The water channel protein PvTIP3;1 (α-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.

scholarly journals Ase1/Prc1-dependent spindle elongation corrects merotely during anaphase in fission yeast

2009 ◽  
Vol 187 (3) ◽  
pp. 399-412 ◽  
Author(s):  
Thibault Courtheoux ◽  
Guillaume Gay ◽  
Yannick Gachet ◽  
Sylvie Tournier
Keyword(s):  
Fission Yeast ◽  
Mechanical Model ◽  
Elongation Rate ◽  
Force Balance ◽  
Balance Model ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Spindle Elongation ◽  
Dependent Mechanism

Faithful segregation of sister chromatids requires the attachment of each kinetochore (Kt) to microtubules (MTs) that extend from opposite spindle poles. Merotelic Kt orientation is a Kt–MT misattachment in which a single Kt binds MTs from both spindle poles rather than just one. Genetic induction of merotelic Kt attachment during anaphase in fission yeast resulted in intra-Kt stretching followed by either correction or Kt disruption. Laser ablation of spindle MTs revealed that intra-Kt stretching and merotelic correction were dependent on MT forces. The presence of multiple merotelic chromosomes linearly antagonized the spindle elongation rate, and this phenomenon could be solved numerically using a simple force balance model. Based on the predictions of our mechanical model, we provide in vivo evidence that correction of merotelic attachment in anaphase is tension dependent and requires an Ase1/Prc1-dependent mechanism that prevents spindle collapse and thus asymmetric division and/or the appearance of the cut phenotype.

scholarly journals Kinetochore-mediated outward force promotes spindle pole separation in fission yeast

2019 ◽  
Vol 30 (22) ◽  
pp. 2802-2813 ◽  
Author(s):  
Yutaka Shirasugi ◽  
Masamitsu Sato
Keyword(s):  
Fission Yeast ◽  
Motor Proteins ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Double Knockout ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Bipolar Spindles ◽  
Meiosis I

Bipolar spindles are organized by motor proteins that generate microtubule-­dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

scholarly journals Centriole number and the reproductive capacity of spindle poles.

1985 ◽  
Vol 100 (3) ◽  
pp. 887-896 ◽  
Author(s):  
G Sluder ◽  
C L Rieder
Keyword(s):  
Spindle Pole ◽  
Reproductive Capacity ◽  
Centriole Duplication ◽  
Voltage Electron ◽  
Spindle Poles ◽  
Mother And Daughter ◽  
Pericentriolar Material ◽  
History Of ◽  
Bipolar Spindles

The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.

Identification of RNA sequences and structural elements required for assembly of fission yeast SRP54 protein with signal recognition particle RNA

1993 ◽  
Vol 13 (3) ◽  
pp. 1353-1362
Author(s):  
D Selinger ◽  
P Brennwald ◽  
X Liao ◽  
J A Wise
Keyword(s):  
Fission Yeast ◽  
Base Pair ◽  
Structural Integrity ◽  
Signal Recognition Particle ◽  
Yeast Cells ◽  
Growth Defect ◽  
Signal Recognition ◽  
Nucleotide Substitutions ◽  
Srp Rna

Signal recognition particle (SRP) is a ribonucleoprotein composed of six polypeptides and a single RNA molecule. SRP RNA can be divided into four structural domains, the last of which is the most highly conserved and, in Schizosaccharomyces pombe, is the primary location to which deleterious mutations map. The ability of mammalian SRP54 protein (SRP54p) to bind Escherichia coli 4.5S RNA, a homolog of SRP RNA which contains only domain IV, suggested that SRP54p might interact directly with this region. To determine whether domain IV is critical for SRP54p binding in fission yeast cells, we used a native immunoprecipitation-RNA sequencing assay to test 13 mutant SRP RNAs for the ability to associate with the protein in vivo. The G156A mutation, which alters the 5' residue of the noncanonical first base pair of the domain IV terminal helix and confers a mild conditional growth defect, reduces assembly of the RNA with SRP54p. Mutating either of the two evolutionarily invariant residues in the bulged region 5' to G156 is more deleterious to growth and virtually abolishes SRP54p binding. We conclude that the conservation of nucleotides 154 to 156 is likely to be a consequence of their role as a sequence-specific recognition element for the SRP54 protein. We also tested a series of mutants with nucleotide substitutions in the conserved tetranucleotide loop and adjoining stem of domain IV. Although tetraloop mutations are deleterious to growth, they have little effect on SRP54p binding. Mutations which disrupt the base pair flanking the tetraloop result in conditional growth defects and significantly reduce association with SRP54p. Disruption of the other two base pairs in the short stem adjacent to the tetranucleotide loop has similar but less dramatic effects on SRP54p binding. These data provide the first evidence that both sequence-specific contacts and the structural integrity of domain IV of SRP RNA are important for assembly with SRP54p.

scholarly journals Cid1, a Fission Yeast Protein Required for S-M Checkpoint Control when DNA Polymerase δ or ɛ Is Inactivated

2000 ◽  
Vol 20 (9) ◽  
pp. 3234-3244 ◽  
Author(s):  
Shao-Win Wang ◽  
Takashi Toda ◽  
Robert MacCallum ◽  
Adrian L. Harris ◽  
Chris Norbury
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Dna Polymerase ◽  
Intracellular Signaling ◽  
Yeast Cells ◽  
Checkpoint Control ◽  
Checkpoint Signaling ◽  
Intracellular Signaling Pathway ◽  
Dna Polymerase Δ

ABSTRACT The S-M checkpoint is an intracellular signaling pathway that ensures that mitosis is not initiated in cells undergoing DNA replication. We identified cid1, a novel fission yeast gene, through its ability when overexpressed to confer specific resistance to a combination of hydroxyurea, which inhibits DNA replication, and caffeine, which overrides the S-M checkpoint. Cid1 overexpression also partially suppressed the hydroxyurea sensitivity characteristic of DNA polymerase δ mutants and mutants defective in the “checkpoint Rad” pathway. Cid1 is a member of a family of putative nucleotidyltransferases including budding yeast Trf4 and Trf5, and mutation of amino acid residues predicted to be essential for this activity resulted in loss of Cid1 function in vivo. Two additional Cid1-like proteins play similar but nonredundant checkpoint-signaling roles in fission yeast. Cells lacking Cid1 were found to be viable but specifically sensitive to the combination of hydroxyurea and caffeine and to be S-M checkpoint defective in the absence of Cds1. Genetic data suggest that Cid1 acts in association with Crb2/Rhp9 and through the checkpoint-signaling kinase Chk1 to inhibit unscheduled mitosis specifically when DNA polymerase δ or ɛ is inhibited.

Centrosomenfreie Spindelpole in Tipuliden-Spermatocyten

1959 ◽  
Vol 14 (12) ◽  
pp. 749-752b ◽  
Author(s):  
Roland Dietz
Keyword(s):  
Chromosome Movement ◽  
Experimental Conditions ◽  
Size And Shape ◽  
Spindle Poles ◽  
Multipolar Spindles ◽  
Stable Orientation ◽  
Bipolar Spindles

In spermatocytes I of the crane-fly Pales crocata under experimental conditions the normal relation between spindle poles and centrosomes can be disturbed. Bipolar spindles can be formed without participation of one or both centrosomes. Multipolar spindles often arise by formation of acentric (centrosomeless) poles in addition to the two centric ones. The effect of centric and acentric poles on chromosome movement is identical. Co-oriented bivalents which (in contrast to univalents and trivalents) exhibit stable orientation in normal spindles, are able to re-orient in multipolar figures. Size and shape of monocentric, bipolar spindles often do not differ from normal bicentric spindles and chromosome movement in them also is normal. Sometimes the spindles are not pointed at the acentric pole and occasionally fan-shaped monocentric, monopolar spindles are formed. Centrosomes which remain unconnected with the spindle keep more or less their diacinetic appearance.These and other results demonstrate that in tipuline spermatocytes the spindle is—at least mainly—organized by the chromosomes, the centrosomes having at best a subsidiary function in focussing the spindle poles.

scholarly journals A Family of Glycosylated Macrolides Selectively Target Energetic Vulnerabilities in Leukemia

2021 ◽  
Author(s):  
Benjamin J Reisman ◽  
Hui Guo ◽  
Haley E Ramsey ◽  
Madison T Wright ◽  
Bradley I Reinfeld ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Molecular Mechanism ◽  
Atp Synthase ◽  
Mechanism Of Action ◽  
Transformed Cells ◽  
Resistance Mutations ◽  
Selective Cytotoxicity ◽  
Binding Interface ◽  
Mitochondrial Atp Synthase

Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F1 subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. CryoEM of apoptolidin and ammocidin-ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR-Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence define the mechanism of action of apoptolidin family glycomacrolides and establish a path to address OXPHOS-dependent cancers.

scholarly journals Mitotic Chromosome Biorientation in Fission Yeast Is Enhanced by Dynein and a Minus-end–directed, Kinesin-like Protein

2007 ◽  
Vol 18 (6) ◽  
pp. 2216-2225 ◽  
Author(s):  
Ekaterina L. Grishchuk ◽  
Ilia S. Spiridonov ◽  
J. Richard McIntosh
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Spindle Pole ◽  
Ultrastructural Analysis ◽  
Yeast Cells ◽  
Mitotic Spindles ◽  
Wild Type ◽  
Spindle Poles ◽  
Spindle Microtubules

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end–directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole.

scholarly journals Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

2020 ◽  
Vol 48 (21) ◽  
pp. 12234-12251
Author(s):  
Torkild Visnes ◽  
Carlos Benítez-Buelga ◽  
Armando Cázares-Körner ◽  
Kumar Sanjiv ◽  
Bishoy M F Hanna ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Oxidative Dna Damage ◽  
Excision Repair ◽  
Therapeutic Index ◽  
Replication Stress ◽  
Transformed Cells ◽  
Wide Range

Abstract Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

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