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1
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Dendooven T, Yatskevich S, Burt A, Chen ZA, Bellini D, Rappsilber J, Kilmartin JV, Barford D. Structure of the native γ-tubulin ring complex capping spindle microtubules. Nat Struct Mol Biol 2024; 31:1134-1144. [PMID: 38609662 PMCID: PMC11257966 DOI: 10.1038/s41594-024-01281-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2023] [Accepted: 03/19/2024] [Indexed: 04/14/2024]
Abstract
Microtubule (MT) filaments, composed of α/β-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/β-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/β-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.
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Affiliation(s)
| | - Stanislau Yatskevich
- MRC Laboratory of Molecular Biology, Cambridge, UK.
- Genentech, South San Francisco, CA, USA.
| | - Alister Burt
- MRC Laboratory of Molecular Biology, Cambridge, UK
- Genentech, South San Francisco, CA, USA
| | - Zhuo A Chen
- Technische Universität Berlin, Chair of Bioanalytics, Berlin, Germany
| | - Dom Bellini
- MRC Laboratory of Molecular Biology, Cambridge, UK
| | - Juri Rappsilber
- Technische Universität Berlin, Chair of Bioanalytics, Berlin, Germany
- Si-M/'Der Simulierte Mensch', Technische Universität Berlin and Charité, Universitätsmedizin Berlin, Berlin, Germany
- Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK
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2
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Wethekam LC, Moore JK. Tubulin isotype regulation maintains asymmetric requirement for α-tubulin over β-tubulin. J Cell Biol 2023; 222:e202202102. [PMID: 36719400 PMCID: PMC9930134 DOI: 10.1083/jcb.202202102] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Revised: 07/19/2022] [Accepted: 11/14/2022] [Indexed: 02/01/2023] Open
Abstract
How cells regulate α- and β-tubulin to meet the demand for αβ-heterodimers and avoid consequences of monomer imbalance is not understood. We investigate the role of gene copy number and how shifting expression of α- or β-tubulin genes impacts tubulin proteostasis and microtubule function in Saccharomyces cerevisiae. We find that α-tubulin gene copy number is important for maintaining excess α-tubulin protein compared to β-tubulin protein. Excess α-tubulin prevents accumulation of super-stoichiometric β-tubulin, which leads to loss of microtubules, formation of non-microtubule assemblies of tubulin, and disrupts cell proliferation. In contrast, sub-stoichiometric β-tubulin or overexpression of α-tubulin has minor effects. We provide evidence that yeast cells equilibrate α-tubulin protein concentration when α-tubulin isotype expression is increased. We propose an asymmetric relationship between α- and β-tubulins, in which α-tubulins are maintained in excess to supply αβ-heterodimers and limit the accumulation of β-tubulin monomers.
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Affiliation(s)
- Linnea C. Wethekam
- Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA
| | - Jeffrey K. Moore
- Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA
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3
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Bera A, Gupta ML. Microtubules in Microorganisms: How Tubulin Isotypes Contribute to Diverse Cytoskeletal Functions. Front Cell Dev Biol 2022; 10:913809. [PMID: 35865635 PMCID: PMC9294176 DOI: 10.3389/fcell.2022.913809] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2022] [Accepted: 06/15/2022] [Indexed: 11/19/2022] Open
Abstract
The cellular functions of the microtubule (MT) cytoskeleton range from relatively simple to amazingly complex. Assembled from tubulin, a heterodimeric protein with α- and β-tubulin subunits, microtubules are long, hollow cylindrical filaments with inherent polarity. They are intrinsically dynamic polymers that utilize GTP binding by tubulin, and subsequent hydrolysis, to drive spontaneous assembly and disassembly. Early studies indicated that cellular MTs are composed of multiple variants, or isotypes, of α- and β-tubulins, and that these multi-isotype polymers are further diversified by a range of posttranslational modifications (PTMs) to tubulin. These findings support the multi-tubulin hypothesis whereby individual, or combinations of tubulin isotypes possess unique properties needed to support diverse MT structures and/or cellular processes. Beginning 40 years ago researchers have sought to address this hypothesis, and the role of tubulin isotypes, by exploiting experimentally accessible, genetically tractable and functionally conserved model systems. Among these systems, important insights have been gained from eukaryotic microbial models. In this review, we illustrate how using microorganisms yielded among the earliest evidence that tubulin isotypes harbor distinct properties, as well as recent insights as to how they facilitate specific cellular processes. Ongoing and future research in microorganisms will likely continue to reveal basic mechanisms for how tubulin isotypes facilitate MT functions, along with valuable perspectives on how they mediate the range of conserved and diverse processes observed across eukaryotic microbes.
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4
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Ti SC. Reconstituting Microtubules: A Decades-Long Effort From Building Block Identification to the Generation of Recombinant α/β-Tubulin. Front Cell Dev Biol 2022; 10:861648. [PMID: 35573669 PMCID: PMC9096264 DOI: 10.3389/fcell.2022.861648] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Accepted: 04/18/2022] [Indexed: 11/13/2022] Open
Abstract
Microtubules are cytoskeletal filaments underlying the morphology and functions of all eukaryotic cells. In higher eukaryotes, the basic building blocks of these non-covalent polymers, ɑ- and β-tubulins, are encoded by expanded tubulin family genes (i.e., isotypes) at distinct loci in the genome. While ɑ/β-tubulin heterodimers have been isolated and examined for more than 50 years, how tubulin isotypes contribute to the microtubule organization and functions that support diverse cellular architectures remains a fundamental question. To address this knowledge gap, in vitro reconstitution of microtubules with purified ɑ/β-tubulin proteins has been employed for biochemical and biophysical characterization. These in vitro assays have provided mechanistic insights into the regulation of microtubule dynamics, stability, and interactions with other associated proteins. Here we survey the evolving strategies of generating purified ɑ/β-tubulin heterodimers and highlight the advances in tubulin protein biochemistry that shed light on the roles of tubulin isotypes in determining microtubule structures and properties.
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5
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Park K, Hoff KJ, Wethekam L, Stence N, Saenz M, Moore JK. Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Specific Brain Malformations. Front Cell Dev Biol 2021; 9:765992. [PMID: 34869359 PMCID: PMC8637541 DOI: 10.3389/fcell.2021.765992] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2021] [Accepted: 10/29/2021] [Indexed: 11/13/2022] Open
Abstract
Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different β-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in β-tubulins and located in the exchangeable GTP-binding pocket of β-tubulin. To determine the impact of T178M on β-tubulin function we created an analogous mutation in the β-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of β-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin’s GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.
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Affiliation(s)
- Kristen Park
- Department of Pediatrics and Neurology, Children's Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO, United States
| | - Katelyn J Hoff
- Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States
| | - Linnea Wethekam
- Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States
| | - Nicholas Stence
- Section of Pediatric Radiology, Department of Radiology, Children's Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO, United States
| | - Margarita Saenz
- Section of Genetics, Department of Pediatrics, Children's Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO, United States
| | - Jeffrey K Moore
- Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States
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6
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Roll-Mecak A. The Tubulin Code in Microtubule Dynamics and Information Encoding. Dev Cell 2020; 54:7-20. [PMID: 32634400 PMCID: PMC11042690 DOI: 10.1016/j.devcel.2020.06.008] [Citation(s) in RCA: 157] [Impact Index Per Article: 31.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2019] [Revised: 05/08/2020] [Accepted: 06/03/2020] [Indexed: 01/05/2023]
Abstract
Microtubules are non-covalent mesoscale polymers central to the eukaryotic cytoskeleton. Microtubule structure, dynamics, and mechanics are modulated by a cell's choice of tubulin isoforms and post-translational modifications, a "tubulin code," which is thought to support the diverse morphology and dynamics of microtubule arrays across various cell types, cell cycle, and developmental stages. We give a brief historical overview of research into tubulin diversity and highlight recent progress toward uncovering the mechanistic underpinnings of the tubulin code. As a large number of essential pathways converge upon the microtubule cytoskeleton, understanding how cells utilize tubulin diversity is crucial to understanding cellular physiology and disease.
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Affiliation(s)
- Antonina Roll-Mecak
- Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA; Biochemistry and Biophysics Center, National Heart Lung and Blood Institute, Bethesda, MD 20892, USA.
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7
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Lee SC, Kim SH, Hoffmeister RA, Yoon MY, Kim SK. Novel Peptide-Based Inhibitors for Microtubule Polymerization in Phytophthora capsici. Int J Mol Sci 2019; 20:ijms20112641. [PMID: 31146360 PMCID: PMC6600545 DOI: 10.3390/ijms20112641] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2019] [Revised: 05/22/2019] [Accepted: 05/27/2019] [Indexed: 12/31/2022] Open
Abstract
The plant disease Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is responsible for major economic losses in pepper production. Microtubules have been an attractive target for many antifungal agents as they are involved in key cellular events such as cell proliferation, signaling, and migration in eukaryotic cells. In order to design a novel biocompatible inhibitor, we screened and identified inhibitory peptides against alpha- and beta-tubulin of P. capsici using a phage display method. The identified peptides displayed a higher binding affinity (nanomolar range) and improved specificity toward P. capsici alpha- and beta-tubulin in comparison to Homo sapiens tubulin as evaluated by fluorometric analysis. One peptide demonstrated the high inhibitory effect on microtubule formation with a nanomolar range of IC50 values, which were much lower than a well-known chemical inhibitor—benomyl (IC50 = 500 µM). Based on these results, this peptide can be employed to further develop promising candidates for novel antifungal agents against Phytophthora blight.
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Affiliation(s)
- Sang-Choon Lee
- Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
| | - Sang-Heon Kim
- Department of Chemistry and Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea.
| | - Rachel A Hoffmeister
- Department of Natural Sciences, Northeastern State University, Tahlequah, OK 74464, USA.
| | - Moon-Young Yoon
- Department of Chemistry and Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea.
| | - Sung-Kun Kim
- Department of Natural Sciences, Northeastern State University, Tahlequah, OK 74464, USA.
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8
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Abstract
Microtubules are dynamic polymers of αβ-tubulin that are essential for intracellular organization, organelle trafficking and chromosome segregation. Microtubule growth and shrinkage occur via addition and loss of αβ-tubulin subunits, which are biochemical processes. Dynamic microtubules can also engage in mechanical processes, such as exerting forces by pushing or pulling against a load. Recent advances at the intersection of biochemistry and mechanics have revealed the existence of multiple conformations of αβ-tubulin subunits and their central role in dictating the mechanisms of microtubule dynamics and force generation. It has become apparent that microtubule-associated proteins (MAPs) selectively target specific tubulin conformations to regulate microtubule dynamics, and mechanical forces can also influence microtubule dynamics by altering the balance of tubulin conformations. Importantly, the conformational states of tubulin dimers are likely to be coupled throughout the lattice: the conformation of one dimer can influence the conformation of its nearest neighbours, and this effect can propagate over longer distances. This coupling provides a long-range mechanism by which MAPs and forces can modulate microtubule growth and shrinkage. These findings provide evidence that the interplay between biochemistry and mechanics is essential for the cellular functions of microtubules.
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Affiliation(s)
- Gary J Brouhard
- Department of Biology, McGill University, Montréal, Quebec, Canada.
| | - Luke M Rice
- Department of Biophysics, University of Texas Southwestern, Dallas, TX, USA.
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9
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Munguía B, Teixeira R, Veroli V, Melian E, Saldaña J, Minteguiaga M, Señorale M, Marín M, Domínguez L. Purification of native M. vogae and H. contortus tubulin by TOG affinity chromatography. Exp Parasitol 2017; 182:37-44. [DOI: 10.1016/j.exppara.2017.09.025] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2017] [Revised: 07/26/2017] [Accepted: 09/19/2017] [Indexed: 01/28/2023]
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10
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Amorim AF, Pinto D, Kuras L, Fernandes L. Absence of Gim proteins, but not GimC complex, alters stress-induced transcription. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2017; 1860:773-781. [PMID: 28457997 DOI: 10.1016/j.bbagrm.2017.04.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/04/2016] [Revised: 04/25/2017] [Accepted: 04/26/2017] [Indexed: 02/08/2023]
Abstract
Saccharomyces cerevisiae GimC (mammalian Prefoldin) is a hexameric (Gim1-6) cytoplasmic complex involved in the folding pathway of actin/tubulin. In contrast to a shared role in GimC complex, we show that absence of individual Gim proteins results in distinct stress responses. No concomitant alteration in F-actin integrity was observed. Transcription of stress responsive genes is altered in gim2Δ, gim3Δ and gim6Δ mutants: TRX2 gene is induced in these mutants but with a profile diverging from type cells, whereas CTT1 and HSP26 fail to be induced. Remaining gimΔ mutants display stress transcript abundance comparable to wild type cells. No alteration in the nuclear localization of the transcriptional activators for TRX2 (Yap1) and CTT1/HSP26 (Msn2) was observed in gim2Δ. In accordance with TRX2 induction, RNA polymerase II occupancy at TRX2 discriminates the wild type from gim2Δ and gim6Δ. In contrast, RNA polymerase II occupancy at CTT1 is similar in wild type and gim2Δ, but higher in gim6Δ. The absence of active RNA polymerase II at CTT1 in gim2Δ, but not in wild type and gim1Δ, explains the respective CTT1 transcript outputs. Altogether our results put forward the need of Gim2, Gim3 and Gim6 in oxidative and osmotic stress activated transcription; others Gim proteins are dispensable. Consequently, the participation of Gim proteins in activated-transcription is independent from the GimC complex.
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Affiliation(s)
- Ana Fátima Amorim
- Instituto Gulbenkian de Ciência, Oeiras, Portugal; Universidade de Lisboa, Faculdade de Ciências, Biosystems & Integrative Sciences Institute (BioISI), Lisboa, Portugal
| | - Dora Pinto
- Instituto Gulbenkian de Ciência, Oeiras, Portugal
| | - Laurent Kuras
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - Lisete Fernandes
- Instituto Gulbenkian de Ciência, Oeiras, Portugal; Universidade de Lisboa, Faculdade de Ciências, Biosystems & Integrative Sciences Institute (BioISI), Lisboa, Portugal; Instituto Politécnico de Lisboa, ESTeSL-Escola Superior de Tecnologia da Saúde de Lisboa, Lisboa, Portugal.
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11
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Sueth-Santiago V, Decote-Ricardo D, Morrot A, Freire-de-Lima CG, Lima MEF. Challenges in the chemotherapy of Chagas disease: Looking for possibilities related to the differences and similarities between the parasite and host. World J Biol Chem 2017; 8:57-80. [PMID: 28289519 PMCID: PMC5329715 DOI: 10.4331/wjbc.v8.i1.57] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2016] [Revised: 12/30/2016] [Accepted: 01/14/2017] [Indexed: 02/05/2023] Open
Abstract
Almost 110 years after the first studies by Dr. Carlos Chagas describing an infectious disease that was named for him, Chagas disease remains a neglected illness and a death sentence for infected people in poor countries. This short review highlights the enormous need for new studies aimed at the development of novel and more specific drugs to treat chagasic patients. The primary tool for facing this challenge is deep knowledge about the similarities and differences between the parasite and its human host.
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12
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Coordination of Cell Cycle Progression and Mitotic Spindle Assembly Involves Histone H3 Lysine 4 Methylation by Set1/COMPASS. Genetics 2016; 205:185-199. [PMID: 28049706 DOI: 10.1534/genetics.116.194852] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2016] [Accepted: 11/07/2016] [Indexed: 12/14/2022] Open
Abstract
Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chromatin, but it remains poorly understood how this post-translational modification contributes to the regulation of biological processes like the cell cycle. Here, we report a H3K4 methylation-dependent pathway in Saccharomyces cerevisiae that governs toxicity toward benomyl, a microtubule destabilizing drug. Benomyl-sensitive growth of wild-type cells required mono- and dimethylation of H3K4 and Pho23, a PHD-containing subunit of the Rpd3L complex. Δset1 and Δpho23 deletions suppressed defects associated with ipl1-2 aurora kinase mutant, an integral component of the spindle assembly checkpoint during mitosis. Benomyl resistance of Δset1 strains was accompanied by deregulation of all four tubulin genes and the phenotype was suppressed by tub2-423 and Δtub3 mutations, establishing a genetic link between H3K4 methylation and microtubule function. Most interestingly, sine wave fitting and clustering of transcript abundance time series in synchronized cells revealed a requirement for Set1 for proper cell-cycle-dependent gene expression and Δset1 cells displayed delayed entry into S phase. Disruption of G1/S regulation in Δmbp1 and Δswi4 transcription factor mutants duplicated both benomyl resistance and suppression of ipl1-2 as was observed with Δset1 Taken together our results support a role for H3K4 methylation in the coordination of cell-cycle progression and proper assembly of the mitotic spindle during mitosis.
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13
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Frankl A, Mari M, Reggiori F. Electron microscopy for ultrastructural analysis and protein localization in Saccharomyces cerevisiae. MICROBIAL CELL 2015; 2:412-428. [PMID: 28357267 PMCID: PMC5349205 DOI: 10.15698/mic2015.11.237] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The yeast Saccharomyces cerevisiae is a key model system for studying of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. Ultrastructural examinations of this organism, though, are traditionally difficult because of the presence of a thick cell wall and the high density of cytoplasmic proteins. A series of recent methodological and technical developments, however, has revived interest in morphological analyses of yeast (e.g. 123). Here we present a review of established and new methods, from sample preparation to imaging, for the ultrastructural analysis of S. cerevisiae. We include information for the use of different fixation methods, embedding procedures, approaches for contrast enhancement, and sample visualization techniques, with references to successful examples. The goal of this review is to guide researchers that want to investigate a particular process at the ultrastructural level in yeast by aiding in the selection of the most appropriate approach to visualize a specific structure or subcellular compartment.
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Affiliation(s)
- Andri Frankl
- Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Muriel Mari
- Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Fulvio Reggiori
- Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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14
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Wang Q, Yang J, Dong Y, Zhang L. One-Step Fabrication of a Multifunctional Magnetic Nickel Ferrite/Multi-walled Carbon Nanotubes Nanohybrid-Modified Electrode for the Determination of Benomyl in Food. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2015; 63:4746-4753. [PMID: 25947038 DOI: 10.1021/acs.jafc.5b00973] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Abstract
Benomyl, as one kind of agricultural pesticide, has adverse impact on human health and the environment. It is urgent to develop effective and rapid methods for quantitative determination of benomyl. A simple and sensitive electroanalytical method for determination of benomyl using a magnetic nickel ferrite (NiFe2O4)/multi-walled carbon nanotubes (MWCNTs) nanohybrid-modified glassy carbon electrode (GCE) was presented. The electrocatalytic properties and electroanalysis of benomyl on the modified electrode were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). In the phosphate-buffered saline (PBS) of pH 6.0, this constructed biosensor exhibited two linear relationships with the benomyl concentration range from 1.00 × 10(-7) to 5.00 × 10(-7) mol/L and from 5.00 × 10(-7) to 1.00 × 10(-5) mol/L, respectively. The detection limit was 2.51 × 10(-8) mol/L (S/N = 3). Moreover, the proposed method was successfully applied to determine benomyl in real samples with satisfactory results. The NiFe2O4/MWCNTs/GCE showed good reproducibility and stability, excellent catalytic activity, and anti-interference.
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Affiliation(s)
- Qiong Wang
- College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, Liaoning 110036, People's Republic of China
| | - Jichun Yang
- College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, Liaoning 110036, People's Republic of China
| | - Yuanyuan Dong
- College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, Liaoning 110036, People's Republic of China
| | - Lei Zhang
- College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, Liaoning 110036, People's Republic of China
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15
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Abstract
Yeast prions, based on self-seeded highly ordered fibrous aggregates (amyloids), serve as a model for human amyloid diseases. Propagation of yeast prions depends on the balance between chaperones of the Hsp100 and Hsp70 families. The yeast prion [PSI(+)] can be eliminated by an excess of the chaperone Hsp104. This effect is reversed by an excess of the chaperone Hsp70-Ssa. Here we show that the actions of Hsp104 and Ssa on [PSI(+)] are modulated by the small glutamine-rich tetratricopeptide cochaperone Sgt2. Sgt2 is conserved from yeast to humans, has previously been implicated in the guided entry of tail-anchored proteins (GET) trafficking pathway, and is known to interact with Hsps, cytosolic Get proteins, and tail-anchored proteins. We demonstrate that Sgt2 increases the ability of excess Ssa to counteract [PSI(+)] curing by excess Hsp104. Deletion of SGT2 also restores trafficking of a tail-anchored protein in cells with a disrupted GET pathway. One region of Sgt2 interacts both with the prion domain of Sup35 and with tail-anchored proteins. Sgt2 levels are increased in response to the presence of a prion when major Hsps are not induced. Our data implicate Sgt2 as an amyloid "sensor" and a regulator of chaperone targeting to different types of aggregation-prone proteins.
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16
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Kathiravan MK, Salake AB, Chothe AS, Dudhe PB, Watode RP, Mukta MS, Gadhwe S. The biology and chemistry of antifungal agents: A review. Bioorg Med Chem 2012; 20:5678-98. [PMID: 22902032 DOI: 10.1016/j.bmc.2012.04.045] [Citation(s) in RCA: 427] [Impact Index Per Article: 32.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2012] [Revised: 04/21/2012] [Accepted: 04/21/2012] [Indexed: 01/16/2023]
Affiliation(s)
- Muthu K Kathiravan
- Sinhgad College of Pharmacy, Department of Pharmaceutical Chemistry, Vadgaon(Bk), Pune 410041, India.
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17
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Widlund PO, Podolski M, Reber S, Alper J, Storch M, Hyman AA, Howard J, Drechsel DN. One-step purification of assembly-competent tubulin from diverse eukaryotic sources. Mol Biol Cell 2012; 23:4393-401. [PMID: 22993214 PMCID: PMC3496613 DOI: 10.1091/mbc.e12-06-0444] [Citation(s) in RCA: 111] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
A method is presented that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. It eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research. We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.
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Affiliation(s)
- Per O Widlund
- Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
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18
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Lama R, Sandhu R, Zhong B, Li B, Su B. Identification of selective tubulin inhibitors as potential anti-trypanosomal agents. Bioorg Med Chem Lett 2012; 22:5508-16. [PMID: 22850214 DOI: 10.1016/j.bmcl.2012.07.023] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2012] [Revised: 07/05/2012] [Accepted: 07/06/2012] [Indexed: 11/16/2022]
Abstract
The potency of a series of sulfonamide tubulin inhibitors against the growth of Trypanosoma brucei (T. brucei), as well as human cancer and primary fibroblast cells were evaluated with the aim of determining whether compounds that selectively inhibit parasite proliferation could be identified. Several compounds showed excellent selectivity against T. brucei growth, and have the potential to be used for the treatment of Human African trypanosomiasis. A T. brucei tubulin protein homology model was built based on the crystal structure of the bovine tubulin. The colchicine-binding domain, which is also the binding site of the tested sulfonamide tubulin inhibitors, showed clear differences between the tubulin structures and presumably explained the selectivity of the compounds.
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Affiliation(s)
- Rati Lama
- Department of Chemistry, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Ave., Cleveland, OH 44115, USA
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19
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Erlemann S, Neuner A, Gombos L, Gibeaux R, Antony C, Schiebel E. An extended γ-tubulin ring functions as a stable platform in microtubule nucleation. J Cell Biol 2012; 197:59-74. [PMID: 22472440 PMCID: PMC3317808 DOI: 10.1083/jcb.201111123] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2011] [Accepted: 03/05/2012] [Indexed: 11/28/2022] Open
Abstract
γ-Tubulin complexes are essential for microtubule (MT) nucleation. The γ-tubulin small complex (γ-TuSC) consists of two molecules of γ-tubulin and one molecule each of Spc97 and Spc98. In vitro, γ-TuSCs oligomerize into spirals of 13 γ-tubulin molecules per turn. However, the properties and numbers of γ-TuSCs at MT nucleation sites in vivo are unclear. In this paper, we show by fluorescence recovery after photobleaching analysis that γ-tubulin was stably integrated into MT nucleation sites and was further stabilized by tubulin binding. Importantly, tubulin showed a stronger interaction with the nucleation site than with the MT plus end, which probably provides the basis for MT nucleation. Quantitative analysis of γ-TuSCs on single MT minus ends argued for nucleation sites consisting of approximately seven γ-TuSCs with approximately three additional γ-tubulin molecules. Nucleation and anchoring of MTs required the same number of γ-tubulin molecules. We suggest that a spiral of seven γ-TuSCs with a slight surplus of γ-tubulin nucleates MTs in vivo.
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Affiliation(s)
- Sarah Erlemann
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ–ZMBH Allianz, 69120 Heidelberg, Germany
| | - Annett Neuner
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ–ZMBH Allianz, 69120 Heidelberg, Germany
| | - Linda Gombos
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ–ZMBH Allianz, 69120 Heidelberg, Germany
| | - Romain Gibeaux
- Cell Biology and Biophysics, European Molecular Biology Laboratory Heidelberg, 69117 Heidelberg, Germany
| | - Claude Antony
- Cell Biology and Biophysics, European Molecular Biology Laboratory Heidelberg, 69117 Heidelberg, Germany
| | - Elmar Schiebel
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ–ZMBH Allianz, 69120 Heidelberg, Germany
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20
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Drummond DR, Kain S, Newcombe A, Hoey C, Katsuki M, Cross RA. Purification of tubulin from the fission yeast Schizosaccharomyces pombe. Methods Mol Biol 2011; 777:29-55. [PMID: 21773919 DOI: 10.1007/978-1-61779-252-6_3] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
The fission yeast Schizosaccharomyces pombe is an attractive source of tubulin for biochemical experiments as it contains few tubulin isoforms and is amenable to genetic manipulation. We describe the preparation of milligram quantities of highly purified native tubulin from S. pombe suitable for use in microtubule dynamics assays as well as structural and other biochemical studies. S. pombe cells are grown in bulk in a fermenter and then lysed using a bead mill. The soluble protein fraction is bound to anion-exchange chromatography resin by batch binding, packed in a -chromatography column and eluted by a salt gradient. The tubulin-containing fraction is ammonium sulphate precipitated to further concentrate and purify the protein. A round of high-resolution anion-exchange chromatography is carried out before a cycle of polymerisation and depolymerisation to select functional tubulin. Gel filtration is used to remove residual contaminants before a final desalting step. The purified tubulin is concentrated, and then frozen and stored in liquid nitrogen.
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Affiliation(s)
- Douglas R Drummond
- Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK.
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21
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Abstract
Tubulin is a highly conserved, negatively charged protein that is found in essentially all eukaryotic cells. These properties ensure that isolation protocols successful in one system will likely work, with a few modifications, in most systems. Tubulin has been isolated most frequently from mammalian brain, and the main difference encountered in other systems versus brain is that tubulin is much less abundant in nearly all other sources than it is in brain. This means that attempting to purify tubulin by direct polymerization from a homogenate will often fail or be quite inefficient. However, the conservation of negative charge on tubulin means that an initial ion exchange step can be used to both purify and concentrate the protein from most systems. Polymerization-competent tubulin can usually be obtained by inducing polymerization in the salt eluate from the ion exchange step. We describe protocols for this procedure and describe its application to a number of vertebrate, fungal, protozoal, and plant sources.
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Affiliation(s)
- Dan L Sackett
- Laboratory of Integrative and Medical Biophysics, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
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22
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Miller LM, Xiao H, Burd B, Horwitz SB, Angeletti RH, Verdier-Pinard P. Methods in tubulin proteomics. Methods Cell Biol 2010; 95:105-26. [PMID: 20466132 DOI: 10.1016/s0091-679x(10)95007-3] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
New analytical methods are needed for the successful outcome of experiments aimed at characterizing mechanisms of microtubule dynamics and at understanding the effects of drugs on microtubules. The identification of tubulin isotypes and of regions of the microtubule involved in drug interactions has been advanced by proteomic methodologies. The diversity of tubulin sequences and posttranslational modifications (PTMs) can generate a complex mixture of heterodimers with unique molecular dynamics driving specific functions. Mass spectrometry (MS)-based approaches have been developed, and in combination with chromatographic and/or electrophoretic separation of tubulin polypeptides or peptides, they have contributed to our understanding of tubulin proteomics. We present protocols that we have used for the analysis of tubulin isotypes and PTMs present in tubulin isolated from cells in culture or tissues and for the identification of tubulin regions altered by microtubule-stabilizing agents. Tubulin proteomics complements structural and computer modeling information for a high-resolution view of microtubule dynamics and its alteration by drugs. These methodologies will help in providing insights into tubulin isotype-specific functions and in the design of drugs targeting either all tubulin heterodimers indiscriminately or only those containing specific isotypes.
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Affiliation(s)
- Leah M Miller
- Laboratory of Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, New York 10461, USA
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23
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Abstract
Tubulin and other flagellar and ciliary proteins are the substrates for a host of posttranslational modifications (PTMs), many of which have been highly conserved over evolutionary time. In addition to the binding of MAPs (microtubule-associated proteins) that provide a specific functionality, or the use of different tubulin isotypes to convey a specific function, most cells rely on an array of PTMs. These include phosphorylation, acetylation, glycylation, glutamylation, and methylation. The first and the last of this list are not unique to the tubulin in cilia and flagella, while the others are. This chapter will review briefly these varying modifications and will conclude with detailed methods for their detection and localization at the limit of resolution provided by electron microscopy.
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Affiliation(s)
- Roger D Sloboda
- Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, USA
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24
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Koo BS, Park H, Kalme S, Park HY, Han JW, Yeo YS, Yoon SH, Kim SJ, Lee CM, Yoon MY. α- and β-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening. Appl Microbiol Biotechnol 2009; 82:513-24. [DOI: 10.1007/s00253-008-1821-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2008] [Revised: 11/24/2008] [Accepted: 12/08/2008] [Indexed: 10/21/2022]
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25
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Clément MJ, Rathinasamy K, Adjadj E, Toma F, Curmi PA, Panda D. Benomyl and colchicine synergistically inhibit cell proliferation and mitosis: evidence of distinct binding sites for these agents in tubulin. Biochemistry 2008; 47:13016-25. [PMID: 19049291 DOI: 10.1021/bi801136q] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Benomyl, a tubulin-targeted antimitotic antifungal agent, belongs to the benzimidazole group of compounds, which are known to inhibit the binding of colchicine to tubulin. Therefore, benomyl was thought to bind at or near the colchicine-binding site on tubulin. However, recent mutational studies in yeast and fluorescence studies involving competitive binding of benomyl and colchicine on goat brain tubulin suggested that benomyl may bind to tubulin at a site distinct from the colchicine-binding site. We set out to examine whether colchicine and benomyl bind to tubulin at distinct sites using a human cervical cancer (HeLa) cell line with the thinking that these agents should exert either additive or synergistic activity on cell proliferation if their binding sites on tubulin are different. We found that benomyl and colchicine synergistically inhibited the proliferation of HeLa cells and blocked their cell cycle progression at mitosis. The synergistic activity of benomyl and colchicine was also apparent from their strong depolymerizing effects on both the spindle and interphase microtubules when used in combinations, providing further evidence that these agents bind to tubulin at different sites. Using NMR spectroscopy, we finally demonstrated that benomyl and colchicine bind to tubulin at different sites and that the binding of colchicine seems to positively influence the binding of benomyl to tubulin and vice versa. Further, an analysis of the saturation transfer difference NMR data yielded an interesting insight into the colchicine-tubulin interaction. The data presented in this study provided a mechanistic understanding of the synergistic effects of benomyl and colchicine on HeLa cell proliferation.
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Affiliation(s)
- Marie-Jeanne Clément
- Laboratoire Structure et Activite des Biomolecules Normales et Pathologiques, INSERM U829, Universite Evry-Val d'Essonne, EA3637, Evry, F-91025 France
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26
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Rathinasamy K, Panda D. Kinetic stabilization of microtubule dynamic instability by benomyl increases the nuclear transport of p53. Biochem Pharmacol 2008; 76:1669-80. [DOI: 10.1016/j.bcp.2008.09.001] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2008] [Revised: 08/28/2008] [Accepted: 09/02/2008] [Indexed: 01/26/2023]
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27
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Almeida B, Silva A, Mesquita A, Sampaio-Marques B, Rodrigues F, Ludovico P. Drug-induced apoptosis in yeast. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2008; 1783:1436-48. [PMID: 18252203 DOI: 10.1016/j.bbamcr.2008.01.005] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/31/2007] [Revised: 12/21/2007] [Accepted: 01/07/2008] [Indexed: 01/04/2023]
Abstract
In order to alter the impact of diseases on human society, drug development has been one of the most invested research fields. Nowadays, cancer and infectious diseases are leading targets for the design of effective drugs, in which the primary mechanism of action relies on the modulation of programmed cell death (PCD). Due to the high degree of conservation of basic cellular processes between yeast and higher eukaryotes, and to the existence of an ancestral PCD machinery in yeast, yeasts are an attractive tool for the study of affected pathways that give insights into the mode of action of both antitumour and antifungal drugs. Therefore, we covered some of the leading reports on drug-induced apoptosis in yeast, revealing that in common with mammalian cells, antitumour drugs induce apoptosis through reactive oxygen species (ROS) generation and altered mitochondrial functions. The evidence presented suggests that yeasts may be a powerful model for the screening/development of PCD-directed drugs, overcoming the problem of cellular specificity in the design of antitumour drugs, but also enabling the design of efficient antifungal drugs, targeted to fungal-specific apoptotic regulators that do not have major consequences for human cells.
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Affiliation(s)
- B Almeida
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, Braga, Portugal
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28
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Singh P, Rathinasamy K, Mohan R, Panda D. Microtubule assembly dynamics: An attractive target for anticancer drugs. IUBMB Life 2008; 60:368-75. [DOI: 10.1002/iub.42] [Citation(s) in RCA: 115] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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29
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Sweet TJ, Boyer B, Hu W, Baker KE, Coller J. Microtubule disruption stimulates P-body formation. RNA (NEW YORK, N.Y.) 2007; 13:493-502. [PMID: 17307817 PMCID: PMC1831866 DOI: 10.1261/rna.355807] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/10/2023]
Abstract
Processing bodies (P-bodies) are subcellular ribonucleoprotein (RNP) granules that have been hypothesized to be sites of mRNA degradation, mRNA translational control, and/or mRNA storage. Importantly, P-bodies are conserved from yeast to mammals and contain a common set of evolutionarily conserved protein constituents. P-bodies are dynamic structures and their formation appears to fluctuate in correlation with alterations in mRNA metabolism. Despite these observations, little is understood about how P-body structures are formed within the cell. In this study, we demonstrate a relationship between P-bodies and microtubules in the budding yeast, Saccharomyces cerevisiae. First, we demonstrate that disruption of microtubules by treatment with the drug benomyl leads to aggregation of P-body components. Consistent with this finding, we also demonstrate that disruption of microtubules by a temperature-sensitive allele of the major alpha tubulin, TUB1 (tub1-724) stimulates P-body formation. Second, we find that the alpha-tubulin protein Tub1 colocalizes with P-bodies upon microtubule destabilization. Third, we determine that a putative tubulin tyrosine ligase, encoded by YBR094W, is a protein component of P-bodies, providing additional evidence for a physical connection between P-bodies and microtubules. Finally, we establish that P-bodies formed by microtubule destabilization fail to correlate with global changes in the stability of mRNA or in general mRNA translation. These findings demonstrate that the aggregation of P-body components is linked to the intracellular microtubule network, and, further, that P-bodies formed by disruption of microtubules aggregate independent of broad alterations in either mRNA decay or mRNA translation.
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Affiliation(s)
- Thomas J Sweet
- Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106, USA
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30
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Zou G, Ying SH, Shen ZC, Feng MG. Multi-sited mutations of beta-tubulin are involved in benzimidazole resistance and thermotolerance of fungal biocontrol agent Beauveria bassiana. Environ Microbiol 2007; 8:2096-105. [PMID: 17107551 DOI: 10.1111/j.1462-2920.2006.01086.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Fungicide resistance and thermotolerance of biocontrol agents in mitosporic fungi are of merits for enhancing fungal formulations against insect pests in the field. Among 20 wild strains of Beauveria bassiana (a well-known fungal biocontrol agent) tested in this study, 19 were sensitive or highly sensitive to carbendazim (methyl 2-benzimidazole carbamate), a typical benzimidazole fungicide, despite low resistance found in one strain. Sequential mutagenesis of a carbendazim-sensitive wild strain [minimal inhibitory concentration (MIC) = 1.32 microg ml(-1)] under artificial selection pressure generated 11 mutants sharing a common MIC of > 1000 microg ml(-1) without visible variation in colony growth and conidiation capacity. This represents at least 758-fold enhancement of the resistance among the mutants. However, accompanied with the enhanced resistance, all the mutants became less thermotolerable. Stressed at 48 degrees C, conidial LT(50)s of the mutants varied from 1.8 to 9.6 min and were lower than the parental LT(50) (36 min). Moreover, the contents of hydrophobin-like proteins in conidial walls declined significantly among the mutants compared with that of the wild parent. Mutations commonly relating to benzimidazole resistance in fungi were located at Q134, F167 and/or E198 around the taxol-binding site of beta-tubulin by sequencing the beta-tubulin of the mutants. Also, mutations of other 37 amino acid residues in the sequences (each having one to five residues mutated) were found for the first time and they were diverse in spatial structure. All mutations restricted to the half of beta-tubulin close to alpha-tubulin were likely involved in variation in each of the traits concerned but their interactions were complicated.
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Affiliation(s)
- Gen Zou
- Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou 310058, People's Republic of China
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31
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Rathinasamy K, Panda D. Suppression of microtubule dynamics by benomyl decreases tension across kinetochore pairs and induces apoptosis in cancer cells. FEBS J 2006; 273:4114-28. [PMID: 16903866 DOI: 10.1111/j.1742-4658.2006.05413.x] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
We found that benomyl, a benzimidazole fungicide, strongly suppressed the reassembly of cold-depolymerized spindle microtubules in HeLa cells. Benomyl perturbed microtubule-kinetochore attachment and chromosome alignment at the metaphase plate. Benomyl also significantly decreased the distance between the sister kinetochore pairs in metaphase cells and increased the level of the checkpoint protein BubR1 at the kinetochore region, indicating that benomyl caused loss of tension across the kinetochores. In addition, benomyl decreased the intercentrosomal distance in mitotic HeLa cells and blocked the cells at mitosis. Further, we analyzed the effects of benomyl on the signal transduction pathways in relation to mitotic block, bcl2 phosphorylation and induction of apoptosis. The results suggest that benomyl causes loss of tension across the kinetochores, blocks the cell cycle progression at mitosis and subsequently, induces apoptosis through the bcl2-bax pathway in a manner qualitatively similar to the powerful microtubule targeted anticancer drugs like the vinca alkaloids and paclitaxel. Considering the very high toxicity of the potent anticancer drugs and the low toxicity of benomyl in humans, we suggest that benomyl could be useful as an adjuvant in combination with the powerful anticancer drugs in cancer therapy.
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Affiliation(s)
- K Rathinasamy
- School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India
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32
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Akagi T, Mitani S, Ito K, Shigehara I, Komyoji T, Matsuo N. Structure-activity relationships of pyridylcarbamates active against both benzimidazole-sensitive and -resistant isolates ofBotrytis cinerea. ACTA ACUST UNITED AC 2006. [DOI: 10.1002/ps.2780440107] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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33
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Foland TB, Dentler WL, Suprenant KA, Gupta ML, Himes RH. Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae. Yeast 2005; 22:971-8. [PMID: 16134117 PMCID: PMC3008355 DOI: 10.1002/yea.1284] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Wild-type Saccharomyces cerevisiae tubulin does not bind the anti-mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for beta-tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule-stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated beta-tubulin gene into a S. cerevisiae strain with diminished transporter activity and developed the first paclitaxel-sensitive budding yeast strain. In the presence of paclitaxel, cytoplasmic microtubules were stable to cold depolymerization. Paclitaxel-treated cells showed evidence of a mitotic block, with an increase in large-budded cells and cells with a 2N DNA content and DNA fragmentation, identified by FACS analysis and the TUNEL assay. In the presence of paclitaxel, the number of dead cells in cultures increased three-fold and cells containing reactive oxygen species were present. We conclude that paclitaxel blocks mitosis in this strain, leading to an apoptotic-like cell death. This strain will also be useful in further studies of the effect of microtubule dynamics on various cellular processes in S. cerevisiae.
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Affiliation(s)
| | | | | | | | - Richard H. Himes
- Correspondence to: Richard H. Himes, Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045-7534, USA.
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34
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Dorn JF, Jaqaman K, Rines DR, Jelson GS, Sorger PK, Danuser G. Yeast kinetochore microtubule dynamics analyzed by high-resolution three-dimensional microscopy. Biophys J 2005; 89:2835-54. [PMID: 16192284 PMCID: PMC1366782 DOI: 10.1529/biophysj.104.058461] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.
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Affiliation(s)
- J F Dorn
- Laboratory for Computational Cell Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
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35
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Werbovetz KA, Sackett DL, Delfín D, Bhattacharya G, Salem M, Obrzut T, Rattendi D, Bacchi C. Selective antimicrotubule activity of N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-5) against kinetoplastid parasites. Mol Pharmacol 2003; 64:1325-33. [PMID: 14645662 DOI: 10.1124/mol.64.6.1325] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Analogs of the antimitotic herbicide oryzalin (3,5-dinitro-N4,N4-di-n-propylsulfanilamide) were recently prepared that were more potent in vitro than the parent compound against the kinetoplastid parasite Leishmania donovani (Bioorg Med Chem Lett 12:2395-2398, 2002). In the present work, we show that the most active molecule in the group, N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-5), is a potent, selective antimitotic agent against kinetoplastid parasites. GB-II-5 possesses IC50 values of 0.41 and 0.73 microM in vitro against two strains of the related parasite Trypanosoma brucei but is much less toxic to J774 murine macrophages and PC3 prostate cancer cells, exhibiting IC50 values of 29 and 35 microM against these lines, respectively. Selectivity is also observed for GB-II-5 with purified leishmanial and mammalian tubulin. The assembly of 15 microM leishmanial tubulin is completely inhibited by 10 microM GB-II-5, whereas 40 microM GB-II-5 inhibits the assembly of 15 microM porcine brain tubulin by only 17%. In cultured L. donovani and T. brucei, treatment with 5 and 0.5 microM GB-II-5, respectively, causes a striking increase in the fraction of G2M cells compared with control. Given the potency and selectivity of this agent against kinetoplastid tubulin, GB-II-5 emerges as an exciting new antitrypanosomal and antileishmanial lead compound.
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Affiliation(s)
- Karl A Werbovetz
- Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA.
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36
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Lila T, Renau TE, Wilson L, Philips J, Natsoulis G, Cope MJ, Watkins WJ, Buysse J. Molecular basis for fungal selectivity of novel antimitotic compounds. Antimicrob Agents Chemother 2003; 47:2273-82. [PMID: 12821479 PMCID: PMC161869 DOI: 10.1128/aac.47.7.2273-2282.2003] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Compounds that selectively disrupt fungal mitosis have proven to be effective in controlling agricultural pests, but no specific mitotic inhibitor is available for the treatment of systemic mycoses in mammalian hosts. In an effort to identify novel mitotic inhibitors, we used a cell-based screening strategy that exploited the hypersensitivity of a yeast alpha-tubulin mutant strain to growth inhibition by antimitotic agents. The compounds identified inhibited yeast nuclear division and included one structural class of compounds shown to be fungus specific. MC-305904 and structural analogs inhibited fungal cell mitosis and inhibited the in vitro polymerization of fungal tubulin but did not block mammalian cell microtubule function or mammalian tubulin polymerization. Extensive analysis of yeast mutations that specifically alter sensitivity to MC-305904 structural analogs suggested that compounds in the series bind to a site on fungal beta-tubulin near amino acid 198. Features of the proposed binding site explain the observed fungal tubulin specificity of the series and are consistent with structure-activity relationships among a library of related compounds.
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Affiliation(s)
- Thomas Lila
- Essential Therapeutics, Mountain View, California 94043, USA.
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Woyke T, Roberson RW, Pettit GR, Winkelmann G, Pettit RK. Effect of auristatin PHE on microtubule integrity and nuclear localization in Cryptococcus neoformans. Antimicrob Agents Chemother 2002; 46:3802-8. [PMID: 12435680 PMCID: PMC132747 DOI: 10.1128/aac.46.12.3802-3808.2002] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2002] [Revised: 06/12/2002] [Accepted: 08/19/2002] [Indexed: 11/20/2022] Open
Abstract
The mechanism of action of the fungicidal peptide auristatin PHE was investigated in Cryptococcus neoformans. Since auristatin PHE causes budding arrest in C. neoformans (T. Woyke, G. R. Pettit, G. Winkelmann, and R. K. Pettit, Antimicrob. Agents Chemother. 45:3580-3584, 2001), microtubule integrity and nuclear localization in auristatin PHE-treated cells were examined. Iterative deconvolution in conjunction with an optimized C. neoformans microtubule immunolabeling procedure enabled detailed visualization of the microtubule cytoskeleton in auristatin PHE-treated C. neoformans. The effect of auristatin PHE on C. neoformans microtubule organization was compared with that of the tubulin-binding agent nocodazole. Both drugs produced complete disruption first of cytoplasmic and then of spindle microtubules in a time- and concentration-dependent manner. Sub-MICs of auristatin PHE caused complete microtubule disruption within 4.5 h, while 1.5 times the nocodazole MIC was required for the same effect. For both drugs, disruption of microtubules was accompanied by blockage of nuclear migration and of nuclear and cellular division, resulting in cells arrested in a uninucleate, large-budded stage. Nocodazole and the linear peptide auristatin PHE are remarkably different in structure and spectrum of activity, yet on the cellular level, they have similar effects.
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Affiliation(s)
- Tanja Woyke
- Cancer Research Institute, Arizona State University, Tempe 85287, USA
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Ochola DOK, Prichard RK, Lubega GW. Classical ligands bind tubulin of trypanosomes and inhibit their growth in vitro. J Parasitol 2002; 88:600-4. [PMID: 12099434 DOI: 10.1645/0022-3395(2002)088[0600:clbtot]2.0.co;2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Tubulin ligands known to be toxic to certain organisms or cells were tested for their ability to inhibit proliferation of trypanosomes in culture. Tubulin was purified from Trypanosoma brucei brucei or rat brain by poly-L-lysine affinity chromatography and used in binding studies in order to compare the binding of [3H]mebendazole to trypanosome and mammalian tubulin. All the compounds tested in culture inhibited trypanosome proliferation in a concentration-dependent manner. The concentration required to inhibit trypanosome proliferation by 50 or 90% (IC50 or IC90) in 24 hr was determined for each compound. There were no significant differences (P > 0.05) among the benzimidazoles (BZs), but colchicine and vinblastine caused significantly greater inhibitions than the BZs (P < 0.02 and P < 0.005, respectively). Increasing the incubation time to 72 hr caused a 2- to 4-fold lowering of the IC50 and IC90 values for all the drugs. In the binding assays, there was higher total binding of [3H]mebendazole to trypanosome than rat brain tubulin. The results suggest that the inhibition of trypanosome growth was caused by the specific interaction of these ligands with trypanosome tubulin. Trypanosome tubulin is, therefore, a reasonable target against which novel drugs can be developed to control trypanosomiasis.
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Affiliation(s)
- Donasian O K Ochola
- Molecular Biology Laboratory, Faculty of Veterinary Medicine, Makerere University, Kampala, Uganda
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Caron JM, Vega LR, Fleming J, Bishop R, Solomon F. Single site alpha-tubulin mutation affects astral microtubules and nuclear positioning during anaphase in Saccharomyces cerevisiae: possible role for palmitoylation of alpha-tubulin. Mol Biol Cell 2001; 12:2672-87. [PMID: 11553707 PMCID: PMC59703 DOI: 10.1091/mbc.12.9.2672] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
We generated a strain of Saccharomyces cerevisiae in which the sole source of alpha-tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S alpha-tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15 degrees C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in alpha-tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated alpha-tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated alpha-tubulin in C377S tub1 cells. Our results suggest that cys-377 of alpha-tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle.
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Affiliation(s)
- J M Caron
- Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030, USA.
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Affiliation(s)
- J Vogel
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA
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42
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Wigge PA, Kilmartin JV. The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation. J Cell Biol 2001; 152:349-60. [PMID: 11266451 PMCID: PMC2199619 DOI: 10.1083/jcb.152.2.349] [Citation(s) in RCA: 273] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.
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Affiliation(s)
- Philip A. Wigge
- Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom
| | - John V. Kilmartin
- Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom
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Howlett NG, Schiestl RH. Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay. Mutat Res 2000; 454:53-62. [PMID: 11035159 DOI: 10.1016/s0027-5107(00)00097-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV. The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy. In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events. Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event. Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate. Actinomycin D and benomyl strongly induced aneuploidy. EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction. In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid. Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae.
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Affiliation(s)
- N G Howlett
- Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA
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Bentley KS, Kirkland D, Murphy M, Marshall R. Evaluation of thresholds for benomyl- and carbendazim-induced aneuploidy in cultured human lymphocytes using fluorescence in situ hybridization. Mutat Res 2000; 464:41-51. [PMID: 10633176 DOI: 10.1016/s1383-5718(99)00165-5] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Threshold mechanisms of activity for mutagenic agents have been debated for some time, especially for those substances which induce aneuploidy by inhibiting mitotic spindle function. No observed effect levels (NOELs) or "practical thresholds" have been demonstrated for several aneugens both in vitro and in vivo generally by either counting chromosomes in metaphase preparations or by observing micronuclei. Recently, fluorescence in situ hybridization (FISH) has proven to be a sensitive and useful technique for the assessment of aneuploidy at low concentrations. Using binucleate human lymphocytes coupled with FISH, we have been able to characterize a threshold mechanism of action for two spindle inhibitors, benomyl and its active metabolite, carbendazim. Test chemicals were added 24 h following culture initiation. After a further 20 h, cytochalasin B was added, and cells were harvested 28 h later (72 h post initiation). The distribution of chromosomes between the nuclei of binucleate cells was evaluated by fluorescence microscopy for the simultaneous detection of centromeres labeled with FITC (green) or Cy-3 (red). Six human chromosomes were investigated in pairs (1 and 8, 11 and 18, and X and 17). Abnormalities were classified as chromosome loss (including centromeric positive micronuclei), chromosome gain, non-disjunction, or polyploidy. Dose-response data were generated over a range of closely spaced concentrations at 100 ng/ml intervals. The threshold, defined as the lowest "effect" concentration using statistical methods, was determined for each chromosome. Non-disjunction proved to be the most sensitive endpoint for the detection of aneuploidy occurring at higher frequencies and lower concentrations. Results for the six chromosomes demonstrated similar dose-response data which included a series of concentrations with no statistically significant increase above background, followed by a second range of higher concentrations with a statistically significant, concentration-dependent increase. Nearly equimolar threshold concentrations were determined for benomyl- and carbendazim-induced non-disjunction.
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Affiliation(s)
- K S Bentley
- DuPont Agricultural Products, Wilmington, DE 19880-0038, USA.
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Simple Flavones Possessing Complex Biological Activity. ACTA ACUST UNITED AC 2000. [DOI: 10.1016/s1572-5995(00)80033-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/07/2023]
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Ploubidou A, Robinson DR, Docherty RC, Ogbadoyi EO, Gull K. Evidence for novel cell cycle checkpoints in trypanosomes: kinetoplast segregation and cytokinesis in the absence of mitosis. J Cell Sci 1999; 112 ( Pt 24):4641-50. [PMID: 10574712 DOI: 10.1242/jcs.112.24.4641] [Citation(s) in RCA: 145] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Trypanosoma brucei has a single nucleus and a single kinetoplast (the mitochondrial genome). Each of these organelles has a distinct S phase, which is followed by a segregation period, prior to cell division. The segregation of the two genomes takes place in a specific temporal order by interaction with microtubule-based structures, the spindle for nuclear DNA and the flagellum basal bodies for the kinetoplast DNA. We used rhizoxin, the anti-microtubule agent and polymerisation inhibitor, or the nuclear DNA synthesis inhibitor aphidicolin, to interfere with cell cycle events in order to study how such events are co-ordinated. We show that T. brucei cytokinesis is not dependent upon either mitosis or nuclear DNA synthesis, suggesting that there are novel cell cycle checkpoints in this organism. Moreover, use of monoclonal antibodies to reveal cytoplasmic events such as basal body duplication shows that some aphidicolin treated cells appear to be in G(1) phase (1K1N) but have activated some cytoplasmic events characteristic of G(2) phase (basal body segregation). We discuss a possible dominant role in trypanosomes for kinetoplast/basal body segregation in control of later cell cycle events such as cytokinesis
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Affiliation(s)
- A Ploubidou
- University of Manchester, School of Biological Sciences, Stopford Building 2.205, Oxford Road, Manchester, M13 9PT, UK
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Abstract
Species of the trypanosomatid parasite genera Trypanosoma and Leishmania exhibit a particular range of cell shapes that are defined by their internal cytoskeletons. The cytoskeleton is characterized by a subpellicular corset of microtubules that are cross-linked to each other and to the plasma membrane. Trypanosomatid cells possess an extremely precise organization of microtubules and filaments, with some of their organelles, such as the mitochondria, kinetoplasts, basal bodies, and flagella, present as single copies in each cell. The duplication of these structures and changes in their position during life cycle differentiations provide markers and insight into events involved in determining cell form and division. We have a rapidly increasing catalog of these structures, their molecular cytology, and their ontogeny. The current sophistication of available molecular genetic techniques for use in these organisms has allowed a new functional analysis of the cytoskeleton, including functions that are intrinsic to the proliferation and pathogenicity of these parasites.
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Affiliation(s)
- K Gull
- School of Biological Sciences, University of Manchester, United Kingdom.
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Ekwall K, Cranston G, Allshire RC. Fission yeast mutants that alleviate transcriptional silencing in centromeric flanking repeats and disrupt chromosome segregation. Genetics 1999; 153:1153-69. [PMID: 10545449 PMCID: PMC1460827 DOI: 10.1093/genetics/153.3.1153] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
In the fission yeast Schizosaccharomyces pombe genes are transcriptionally silenced when placed within centromeres, within or close to the silent mating-type loci or adjacent to telomeres. Factors required to maintain mating-type silencing also affect centromeric silencing and chromosome segregation. We isolated mutations that alleviate repression of marker genes in the inverted repeats flanking the central core of centromere I. Mutations csp1 to 13 (centromere: suppressor of position effect) defined 12 loci. Ten of the csp mutants have no effect on mat2/3 or telomere silencing. All csp mutants allow some expression of genes in the centromeric flanking repeat, but expression in the central core is undetectable. Consistent with defective centromere structure and function, chromosome loss rates are elevated in all csp mutants. Mutants csp1 to 6 are temperature-sensitive lethal and csp3 and csp6 cells are defective in mitosis at 36 degrees. csp7 to 13 display a high incidence of lagging chromosomes on late anaphase spindles. Thus, by screening for mutations that disrupt silencing in the flanking region of a fission yeast centromere a novel collection of mutants affecting centromere architecture and chromosome segregation has been isolated.
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Affiliation(s)
- K Ekwall
- Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland
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Abstract
This review traces the principal advances in the study of mitosis in filamentous fungi from its beginnings near the end of the 19(th) century to the present day. Meiosis and mitosis had been accurately described and illustrated by the second decade of the present century and were known to closely resemble nuclear divisions in higher eukaryotes. This information was effectively lost in the mid-1950s, and the essential features of mitosis were then rediscovered from about the mid-1960s to the mid-1970s. Interest in the forces that separate chromatids and spindle poles during fungal mitosis followed closely on the heels of detailed descriptions of the mitotic apparatus in vivo and ultrastructurally during this and the following decade. About the same time, fundamental studies of the structure of fungal chromatin and biochemical characterization of fungal tubulin were being carried out. These cytological and biochemical studies set the stage for a surge of renewed interest in fungal mitosis that was issued in by the age of molecular biology. Filamentous fungi have provided model studies of the cytology and genetics of mitosis, including important advances in the study of mitotic forces, microtubule-associated motor proteins, and mitotic regulatory mechanisms.
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Affiliation(s)
- J R Aist
- Department of Plant Pathology, College of Agriculture and Life Sciences, Ithaca, New York 14853, USA
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