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Elmar Schiebel - One of the best experts on this subject based on the ideXlab platform.

  • duplication and nuclear envelope insertion of the yeast microtubule organizing centre the Spindle Pole Body
    Cells, 2018
    Co-Authors: Diana Rüthnick, Elmar Schiebel
    Abstract:

    The main microtubule organizing centre in the unicellular model organisms Saccharomyces cerevisiae and Schizosaccharomyces pompe is the Spindle Pole Body (SPB). The SPB is a multilayer structure, which duplicates exactly once per cell cycle. Unlike higher eukaryotic cells, both yeast model organisms undergo mitosis without breakdown of the nuclear envelope (NE), a so-called closed mitosis. Therefore, in order to simultaneously nucleate nuclear and cytoplasmic MTs, it is vital to embed the SPB into the NE at least during mitosis, similarly to the nuclear pore complex (NPC). This review aims to embrace the current knowledge of the SPB duplication cycle with special emphasis on the critical step of the insertion of the new SPB into the NE.

  • characterization of Spindle Pole Body duplication reveals a regulatory role for nuclear pore complexes
    Journal of Cell Biology, 2017
    Co-Authors: Diana Rüthnick, Annett Neuner, Michael Knop, Franziska Dietrich, Daniel Kirrmaier, Ulrike Engel, Elmar Schiebel
    Abstract:

    The Spindle Pole Body (SPB) of budding yeast duplicates once per cell cycle. In G1, the satellite, an SPB precursor, assembles next to the mother SPB (mSPB) on the cytoplasmic side of the nuclear envelope (NE). How the growing satellite subsequently inserts into the NE is an open question. To address this, we have uncoupled satellite growth from NE insertion. We show that the bridge structure that separates the mSPB from the satellite is a distance holder that prevents deleterious fusion of both structures. Binding of the γ-tubulin receptor Spc110 to the central plaque from within the nucleus is important for NE insertion of the new SPB. Moreover, we provide evidence that a nuclear pore complex associates with the duplicating SPB and helps to insert the SPB into the NE. After SPB insertion, membrane-associated proteins including the conserved Ndc1 encircle the SPB and retain it within the NE. Thus, uncoupling SPB growth from NE insertion unmasks functions of the duplication machinery.

  • Polo-like kinase Cdc5 regulates Spc72 recruitment to Spindle Pole Body in the methylotrophic yeast ogataea polymorpha
    eLife, 2017
    Co-Authors: Hiromi Maekawa, Diana Rüthnick, Gislene Pereira, Annett Neuner, Elmar Schiebel, Yoshinobu Kaneko
    Abstract:

    Before a cell divides, it needs to duplicate its genetic material to provide the new daughter cell with a full set of genetic information. To do so, the cell forms a complex of proteins called the Spindle apparatus, which is made up of string-like microtubules that divide the chromosomes evenly. In many organisms, the position of the Spindle determines where in the cell this separation happens. However, in baker’s yeast, the location where the cell will divide is determined well before the Spindle is formed. Unlike many other eukaryotic cells, these yeast cells divide asymmetrically and create buds that will form the new daughter cells. The position of this bud determines where the Spindle should be located and where the chromosomes separate. The Spindle itself is then organised by a structure called the Spindle Pole Body, which connects to microtubules inside the cell nucleus and microtubules in the cell plasma. Several proteins control where and how the Spindle forms, including a protein called the Spindle Pole component 72, or Spc72 for short, and an enzyme called Cdc5. However, until now it was unclear how Spindle formation is timed and controlled in other yeast species. Now, Maekawa et al. have used fluorescent markers and time lapse microscopy to examine how the Spindle forms in the yeast species Ogataea polymorpha, an important industrial yeast used to produce medicines and alcohol. The results show that in O. polymorpha, the positioning and orientation of the Spindle only occurred very late in the cell cycle and the microtubules in the cell plasma remained unstable until the chromosomes were about to separate. This was linked to changes in the level of Spc72, which increased at the Spindle Pole Body before the chromosomes separated and then dropped again. This was controlled by Cdc5. Understanding when and where microtubules are formed is an important step in understanding how cells divide. This is the first example of a budding yeast that creates new microtubules in the cell plasma every time the cell divides. Unravelling the molecular differences between yeast species could lead to new ways to optimise the use of industrial yeasts like O. polymorpha, or to combat disease-causing ones.

  • duplication of the yeast Spindle Pole Body once per cell cycle
    Molecular and Cellular Biology, 2016
    Co-Authors: Diana Rüthnick, Elmar Schiebel
    Abstract:

    : The yeast Spindle Pole Body (SPB) is the functional equivalent of the mammalian centrosome. Centrosomes and SPBs duplicate exactly once per cell cycle by mechanisms that use the mother structure as a platform for the assembly of the daughter. The conserved Sfi1 and centrin proteins are essential components of the SPB duplication process. Sfi1 is an elongated molecule that has, in its center, 20 to 23 binding sites for the Ca(2+)-binding protein centrin. In the yeastSaccharomyces cerevisiae, all Sfi1 N termini are in contact with the mother SPB whereas the free C termini are distal to it. During S phase and early mitosis, cyclin-dependent kinase 1 (Cdk1) phosphorylation of mainly serine residues in the Sfi1 C termini blocks the initiation of SPB duplication ("off" state). Upon anaphase onset, the phosphatase Cdc14 dephosphorylates Sfi1 ("on" state) to promote antiparallel and shifted incorporation of cytoplasmic Sfi1 molecules into the half-bridge layer, which thereby elongates into the bridge. The Sfi1 C termini of the two Sfi1 layers localize in the bridge center, whereas the N termini of the newly assembled Sfi1 molecules are distal to the mother SPB. These free Sfi1 N termini then assemble the new SPB in G1phase. Recruitment of Sfi1 molecules into the anaphase SPB and bridge formation were also observed inSchizosaccharomyces pombe, suggesting that the Sfi1 bridge cycle is conserved between the two organisms. Thus, restricting SPB duplication to one event per cell cycle requires only an oscillation between Cdk1 kinase and Cdc14 phosphatase activities. This clockwork regulates the "on"/"off" state of the Sfi1-centrin receiver.

  • Microtubule organization by the budding yeast Spindle Pole Body
    Biology of the Cell, 1999
    Co-Authors: Michael Knop, Gislene Pereira, Elmar Schiebel
    Abstract:

    In budding yeast microtubule organizing functions are provided by the Spindle Pole Body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the γ-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called γ-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of γ-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells.

Mark Winey - One of the best experts on this subject based on the ideXlab platform.

  • the aurora kinase ipl1 is necessary for Spindle Pole Body cohesion during budding yeast meiosis
    Journal of Cell Science, 2011
    Co-Authors: Katelan Shirk, Mark Winey, Thomas H Giddings, Hongguo Yu
    Abstract:

    In budding yeast, the microtubule-organizing center is called the Spindle Pole Body (SPB) and shares structural components with the centriole, the central core of the animal centrosome. During meiotic interphase I, the SPB is duplicated when DNA replication takes place. Duplicated SPBs are linked and then separate to form a bipolar Spindle required for homolog separation in meiosis I. During interphase II, SPBs are duplicated again, in the absence of DNA replication, to form four SPBs that establish two Spindles for sister-chromatid separation in meiosis II. Here, we report that the Aurora kinase Ipl1, which is necessary for sister-chromatid cohesion, is also required for maintenance of a tight association between duplicated SPBs during meiosis, which we term SPB cohesion. Premature loss of cohesion leads to SPB overduplication and the formation of multipolar Spindles. By contrast, the Polo-like kinase Cdc5 is necessary for SPB duplication and interacts antagonistically with Ipl1 at the meiotic SPB to ensure proper SPB separation. Our data suggest that Ipl1 coordinates SPB dynamics with the two chromosome segregation cycles during yeast meiosis.

  • structure function analysis of the c terminal domain of cnm67 a core component of the saccharomyces cerevisiae Spindle Pole Body
    Journal of Biological Chemistry, 2011
    Co-Authors: Vadim A Klenchin, Mark Winey, Jeremiah J Frye, Michelle Jones, Ivan Rayment
    Abstract:

    Abstract The Spindle Pole Body of the budding yeast Saccharomyces cerevisiae has served as a model system for understanding microtubule organizing centers, yet very little is known about the molecular structure of its components. We report here the structure of the C-terminal domain of the core component Cnm67 at 2.3 A resolution. The structure determination was aided by a novel approach to crystallization of proteins containing coiled-coils that utilizes globular domains to stabilize the coiled-coils. This enhances their solubility in Escherichia coli and improves their crystallization. The Cnm67 C-terminal domain (residues Asn-429—Lys-581) exhibits a previously unseen dimeric, interdigitated, all α-helical fold. In vivo studies demonstrate that this domain alone is able to localize to the Spindle Pole Body. In addition, the structure reveals a large functionally indispensable positively charged surface patch that is implicated in Spindle Pole Body localization. Finally, the C-terminal eight residues are disordered but are critical for protein folding and structural stability.

  • the budding yeast Spindle Pole Body structure duplication and function
    Annual Review of Cell and Developmental Biology, 2004
    Co-Authors: Sue L Jaspersen, Mark Winey
    Abstract:

    ▪ Abstract Nucleation of microtubules by eukaryotic microtubule organizing centers (MTOCs) is required for a variety of functions, including chromosome segregation during mitosis and meiosis, cytokinesis, fertilization, cellular morphogenesis, cell motility, and intracellular trafficking. Analysis of MTOCs from different organisms shows that the structure of these organelles is widely varied even though they all share the function of microtubule nucleation. Despite their morphological diversity, many components and regulators of MTOCs, as well as principles in their assembly, seem to be conserved. This review focuses on one of the best-characterized MTOCs, the budding yeast Spindle Pole Body (SPB). We review what is known about its structure, protein composition, duplication, regulation, and functions. In addition, we discuss how studies of the yeast SPB have aided investigation of other MTOCs, most notably the centrosome of animal cells.

  • cdc28 cdk1 regulates Spindle Pole Body duplication through phosphorylation of spc42 and mps1
    Developmental Cell, 2004
    Co-Authors: Sue L Jaspersen, Thomas H Giddings, Brenda J Huneycutt, Katheryn A Resing, Mark Winey
    Abstract:

    Abstract Duplication of the Saccharomyces cerevisiae Spindle Pole Body (SPB) once per cell cycle is essential for bipolar Spindle formation and accurate chromosome segregation during mitosis. We have investigated the role that the major yeast cyclin-dependent kinase Cdc28/Cdk1 plays in assembly of a core SPB component, Spc42, to better understand how SPB duplication is coordinated with cell cycle progression. Cdc28 is required for SPB duplication and Spc42 assembly, and we found that Cdc28 directly phosphorylates Spc42 to promote its assembly into the SPB. The Mps1 kinase, previously shown to regulate Spc42 phosphorylation and assembly, is also a Cdc28 substrate, and Cdc28 phosphorylation of Mps1 is needed to maintain wild-type levels of Mps1 in cells. Analysis of nonphosphorylatable mutants in SPC42 and MPS1 indicates that direct Spc42 phosphorylation and indirect regulation of Spc42 through Mps1 are two overlapping pathways by which Cdc28 regulates Spc42 assembly and SPB duplication during the cell cycle.

  • the yeast protein kinase mps1p is required for assembly of the integral Spindle Pole Body component spc42p
    Journal of Cell Biology, 2002
    Co-Authors: Andrea R Castillo, Janet B Meehl, Garry Morgan, Amy Schutzgeschwender, Mark Winey
    Abstract:

    Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in Spindle Pole Body (SPB) duplication and the Spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1–8, that is defective in SPB duplication but not the Spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.

John V Kilmartin - One of the best experts on this subject based on the ideXlab platform.

  • lessons from yeast the Spindle Pole Body and the centrosome
    Philosophical Transactions of the Royal Society B, 2014
    Co-Authors: John V Kilmartin
    Abstract:

    The yeast Spindle Pole Body (SPB) is the functional equivalent of the centrosome. Most SPB components have been identified and their functions partly established. This involved a large variety of techniques which are described here, and the potential use of some of these in the centrosome field is highlighted. In particular, very useful structural information on the SPB was obtained from a reconstituted complex, the γ-tubulin complex, and also from a sub-particle, SPB cores, prepared by extraction of an enriched SPB preparation. The labelling of SPB proteins with GFP at the N or C termini, using GFP tags inserted into the genome, gave informative electron microscopy localization and fluorescence resonance energy transfer data. Examples are given of more precise functional data obtained by removing domains from one SPB protein, Spc110p, without affecting its essential function. Finally, a structural model for SPB duplication is described and the differences between SPB and centrosome duplication discussed.

  • sfi1p has conserved centrin binding sites and an essential function in budding yeast Spindle Pole Body duplication
    Journal of Cell Biology, 2003
    Co-Authors: John V Kilmartin
    Abstract:

    Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the Spindle Pole Body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.

  • Spindle Pole Body duplication a model for centrosome duplication
    Trends in Cell Biology, 2000
    Co-Authors: Ian R Adams, John V Kilmartin
    Abstract:

    The yeast Spindle Pole Body (SPB) is the functional equivalent of the centrosome and forms the two Poles of the mitotic Spindle. Before mitosis, both SPBs and centrosomes are present as single copies and must be duplicated to form the bipolar Spindle. SPB components have been identified using a combination of biochemistry and genetics, and their role during SPB duplication has been analysed using temperature-sensitive mutants. In this article, we describe structural aspects of SPB duplication and their possible relationship to centrosome duplication.

  • localization of core Spindle Pole Body spb components during spb duplication in saccharomyces cerevisiae
    Journal of Cell Biology, 1999
    Co-Authors: Ian R Adams, John V Kilmartin
    Abstract:

    We have examined the process of Spindle Pole Body (SPB) duplication in Saccharomyces cerevisiae by electron microscopy and found several stages. These include the assembly, probably from the satellite, of a large plaque-like structure, the duplication plaque, on the cytoplasmic face of the half-bridge and its insertion into the nuclear envelope. We analyzed the role of the main SPB components in the formation of these structures by identifying them from an SPB core fraction by mass spectrometry. Temperature-sensitive mutants for two of the components, Spc29p and Nud1p, were prepared to partly define their function. The composition of two of the intermediates in SPB duplication, the satellite and the duplication plaque, was examined by immunoelectron microscopy. Both contain cytoplasmic SPB components showing that duplication has already been partly achieved by the end of the preceding cell cycle when the satellite is formed. We show that by overexpression of SPB components the structure of the satellite can be changed and SPB duplication inhibited by disrupting the attachment of the plaque-like intermediate to the half-bridge. We present a model for SPB duplication where binding of SPB components to either end of the bridge structure ensures two separate SPBs.

  • saccharomyces cerevisiae cells with defective Spindle Pole Body outer plaques accomplish nuclear migration via half bridge organized microtubules
    Molecular Biology of the Cell, 1998
    Co-Authors: Arndt Brachat, John V Kilmartin, Achim Wach, Peter Philippsen
    Abstract:

    Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the Spindle Pole Body (SPB). Deletion of CNM67 (YNL225c) frequently results in Spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for Spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

Trisha N Davis - One of the best experts on this subject based on the ideXlab platform.

  • The Spindle Pole Body of Saccharomyces cerevisiae: architecture and assembly of the core components.
    Current Topics in Developmental Biology, 2020
    Co-Authors: Susan E. Francis, Trisha N Davis
    Abstract:

    Publisher Summary The budding yeast Spindle Pole Body (SPB) is a multilayered cylindrical organelle that is embedded in the nuclear envelope throughout the cell cycle. This chapter focuses on the assembly, organization, and regulation of the core of the SPB. The SPB functions as the microtubule organizing center (MTOC) in the budding yeast Saccharomyces cerevisiae, in a manner analogous to the centrosome of animal cells. It is the only MTOC in the yeast cell, serving to initiate nuclear microtubules that form the mitotic Spindle as well as cytoplasmic microtubules that position the nucleus and align the Spindle during cell division. Monoclonal antibodies prepared against enriched SPB fraction led to the identification of three abundant and essential SPB constituents: Spc110p, Spc42p, and Spc98p. The critical part of the SPB is the γ-tubulin complex consisting of Spc97p, Spc98p, and Tub4p and found in both the inner and outer plaques. A composite picture of the structure of the SPB core can be drawn from immunolocalization, biochemical, and genetic data. Once the yeast genome is sequenced, the highly enriched preparations of SPBs are characterized by mass spectrometry to identify 23 Spindle Pole components.

  • novel phosphorylation states of the yeast Spindle Pole Body
    Biology Open, 2018
    Co-Authors: Kimberly K Fong, Alex Zelter, Beth Graczyk, Jill M Hoyt, Michael Riffle, Richard S Johnson, Michael J Maccoss, Trisha N Davis
    Abstract:

    ABSTRACT Phosphorylation regulates yeast Spindle Pole Body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of enriched Saccharomyces cerevisiae SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a cdc4-1 temperature-sensitive mutation, with particular focus on phosphorylation events on the γ-tubulin small complex (γ-TuSC). The cdc4-1 arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of Spindle length. Additionally, in vivo analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites that influence microtubule formation.

  • novel phosphorylation states of the yeast Spindle Pole Body
    bioRxiv, 2018
    Co-Authors: Kimberly K Fong, Alex Zelter, Beth Graczyk, Jill M Hoyt, Michael Riffle, Richard S Johnson, Michael J Maccoss, Trisha N Davis
    Abstract:

    Phosphorylation regulates yeast Spindle Pole Body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purified Saccharomyces cerevisiae SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a cdc4-1 temperature sensitive mutation, with particular interest in phosphorylation events on the γ-tubulin small complex (γ-TuSC). The cdc4-1 arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of Spindle length. Additionally, in vivo analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites influencing microtubule regulation.

  • identification of saccharomyces cerevisiae Spindle Pole Body remodeling factors
    PLOS ONE, 2010
    Co-Authors: Kristen B Greenland, Huiming Ding, Michael Costanzo, Charles Boone, Trisha N Davis
    Abstract:

    The Saccharomyces cerevisiae centrosome or Spindle Pole Body (SPB) is a dynamic structure that is remodeled in a cell cycle dependent manner. The SPB increases in size late in the cell cycle and during most cell cycle arrests and exchanges components during G1/S. We identified proteins involved in the remodeling process using a strain in which SPB remodeling is conditionally induced. This strain was engineered to express a modified SPB component, Spc110, which can be cleaved upon the induction of a protease. Using a synthetic genetic array analysis, we screened for genes required only when Spc110 cleavage is induced. Candidate SPB remodeling factors fell into several functional categories: mitotic regulators, microtubule motors, protein modification enzymes, and nuclear pore proteins. The involvement of candidate genes in SPB assembly was assessed in three ways: by identifying the presence of a synthetic growth defect when combined with an Spc110 assembly defective mutant, quantifying growth of SPBs during metaphase arrest, and comparing distribution of SPB size during asynchronous growth. These secondary screens identified four genes required for SPB remodeling: NUP60, POM152, and NCS2 are required for SPB growth during a mitotic cell cycle arrest, and UBC4 is required to maintain SPB size during the cell cycle. These findings implicate the nuclear pore, urmylation, and ubiquitination in SPB remodeling and represent novel functions for these genes.

  • the organization of the core proteins of the yeast Spindle Pole Body
    Molecular Biology of the Cell, 2005
    Co-Authors: Eric G D Muller, Brian E Snydsman, Isabella Novik, Dale W Hailey, Daniel R Gestaut, Christine A Niemann, Eileen T Otoole, Tom H Giddings, Bryan A Sundin, Trisha N Davis
    Abstract:

    The Spindle Pole Body (SPB) is the microtubule organizing center of Saccharomyces cerevisiae. Its core includes the proteins Spc42, Spc110 (kendrin/pericentrin ortholog), calmodulin (Cmd1), Spc29, and Cnm67. Each was tagged with CFP and YFP and their proximity to each other was determined by fluorescence resonance energy transfer (FRET). FRET was measured by a new metric that accurately reflected the relative extent of energy transfer. The FRET values established the topology of the core proteins within the architecture of SPB. The N-termini of Spc42 and Spc29, and the C-termini of all the core proteins face the gap between the IL2 layer and the central plaque. Spc110 traverses the central plaque and Cnm67 spans the IL2 layer. Spc42 is a central component of the central plaque where its N-terminus is closely associated with the C-termini of Spc29, Cmd1, and Spc110. When the donor-acceptor pairs were ordered into five broad categories of increasing FRET, the ranking of the pairs specified a unique geometry for the positions of the core proteins, as shown by a mathematical proof. The geometry was integrated with prior cryoelectron tomography to create a model of the interwoven network of proteins within the central plaque. One prediction of the model, the dimerization of the calmodulin-binding domains of Spc110, was confirmed by in vitro analysis.

Michael Knop - One of the best experts on this subject based on the ideXlab platform.

  • characterization of Spindle Pole Body duplication reveals a regulatory role for nuclear pore complexes
    Journal of Cell Biology, 2017
    Co-Authors: Diana Rüthnick, Annett Neuner, Michael Knop, Franziska Dietrich, Daniel Kirrmaier, Ulrike Engel, Elmar Schiebel
    Abstract:

    The Spindle Pole Body (SPB) of budding yeast duplicates once per cell cycle. In G1, the satellite, an SPB precursor, assembles next to the mother SPB (mSPB) on the cytoplasmic side of the nuclear envelope (NE). How the growing satellite subsequently inserts into the NE is an open question. To address this, we have uncoupled satellite growth from NE insertion. We show that the bridge structure that separates the mSPB from the satellite is a distance holder that prevents deleterious fusion of both structures. Binding of the γ-tubulin receptor Spc110 to the central plaque from within the nucleus is important for NE insertion of the new SPB. Moreover, we provide evidence that a nuclear pore complex associates with the duplicating SPB and helps to insert the SPB into the NE. After SPB insertion, membrane-associated proteins including the conserved Ndc1 encircle the SPB and retain it within the NE. Thus, uncoupling SPB growth from NE insertion unmasks functions of the duplication machinery.

  • nud1p the yeast homolog of centriolin regulates Spindle Pole Body inheritance in meiosis
    The EMBO Journal, 2006
    Co-Authors: Oren Gordon, Christof Taxis, Philipp J Keller, Aleksander Benjak, Ernst H K Stelzer, Giora Simchen, Michael Knop
    Abstract:

    Nud1p, a protein homologous to the mammalian centrosome and midBody component Centriolin, is a component of the budding yeast Spindle Pole Body (SPB), with roles in anchorage of microtubules and regulation of the mitotic exit network during vegetative growth. Here we analyze the function of Nud1p during yeast meiosis. We find that a nud1-2 temperature-sensitive mutant has two meiosis-related defects that reflect genetically distinct functions of Nud1p. First, the mutation affects spore formation due to its late function during spore maturation. Second, and most important, the mutant loses its ability to distinguish between the ages of the four Spindle Pole bodies, which normally determine which SPB would be preferentially included in the mature spores. This affects the regulation of genome inheritance in starved meiotic cells and leads to the formation of random dyads instead of non-sister dyads under these conditions. Both functions of Nud1p are connected to the ability of Spc72p to bind to the outer plaque and half-bridge (via Kar1p) of the SPB.

  • Microtubule organization by the budding yeast Spindle Pole Body
    Biology of the Cell, 1999
    Co-Authors: Michael Knop, Gislene Pereira, Elmar Schiebel
    Abstract:

    In budding yeast microtubule organizing functions are provided by the Spindle Pole Body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the γ-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called γ-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of γ-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells.

  • spc29p is a component of the spc110p subcomplex and is essential for Spindle Pole Body duplication
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Sarah Elliott, Michael Knop, Gabriel Schlenstedt, Elmar Schiebel
    Abstract:

    In yeast, microtubules are organized by the Spindle Pole Body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the γ-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p influences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p/Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication.

  • spc98p and spc97p of the yeast γ tubulin complex mediate binding to the Spindle Pole Body via their interaction with spc110p
    The EMBO Journal, 1997
    Co-Authors: Michael Knop, Elmar Schiebel
    Abstract:

    Previously, we have shown that the yeast γ-tubulin, Tub4p, forms a 6S complex with the Spindle Pole Body components Spc98p and Spc97p. In this paper we report the purification of the Tub4p complex. It contained one molecule of Spc98p and Spc97p, and two or more molecules of Tub4p, but no other protein. We addressed how the Tub4p complex binds to the yeast microtubule organizing center, the Spindle Pole Body (SPB). Genetic and biochemical data indicate that Spc98p and Spc97p of the Tub4p complex bind to the N-terminal domain of the SPB component Spc110p. Finally, we isolated a complex containing Spc110p, Spc42p, calmodulin and a 35 kDa protein, suggesting that these four proteins interact in the SPB. We discuss in a model, how the N-terminus of Spc110p anchors the Tub4p complex to the SPB and how Spc110p itself is embedded in the SPB.