scholarly journals Distinct Dgrip84 Isoforms Correlate with Distinct γ-Tubulins in Drosophila

2008 ◽  
Vol 19 (1) ◽  
pp. 368-377 ◽  
Author(s):  
Christiane Wiese
Keyword(s):  
Cell Cycle ◽  
Spindle Pole ◽  
Multiprotein Complex ◽  
Microtubule Organization ◽  
Developmentally Regulated ◽  
Tubulin Genes ◽  
Tubulin Isotype ◽  
Ring Complex ◽  
Cycle Dependent ◽  
Drosophila Embryos

γ-Tubulin is an indispensable component of the animal centrosome and is required for proper microtubule organization. Within the cell, γ-tubulin exists in a multiprotein complex containing between two (some yeasts) and six or more (metazoa) additional highly conserved proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). γ-Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named the γ-tubulin ring complex (γTuRC). Curiously, many organisms (including humans) have two distinct γ-tubulin genes. In Drosophila, where the two γ-tubulin isotypes have been studied most extensively, the γ-tubulin genes are developmentally regulated: the “maternal” γ-tubulin isotype (named γTub37CD according to its location on the genetic map) is expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the meiotic and early embryonic cleavages. The second γ-tubulin isotype (γTub23C) is ubiquitously expressed and persists in most of the cells of the adult fly. In those rare cases where both γ-tubulins coexist in the same cell, they show distinct subcellular distributions and cell-cycle-dependent changes: γTub37CD mainly localizes to the centrosome, where its levels vary only slightly with the cell cycle. In contrast, the level of γTub23C at the centrosome increases at the beginning of mitosis, and γTub23C also associates with spindle pole microtubules. Here, we show that γTub23C forms discrete complexes that closely resemble the complexes formed by γTub37CD. Surprisingly, however, γTub23C associates with a distinct, longer splice variant of Dgrip84. This may reflect a role for Dgrip84 in regulating the activity and/or the location of the γ-tubulin complexes formed with γTub37CD and γTub23C.

scholarly journals CP60: a microtubule-associated protein that is localized to the centrosome in a cell cycle-specific manner.

1995 ◽  
Vol 6 (12) ◽  
pp. 1673-1684 ◽  
Author(s):  
D R Kellogg ◽  
K Oegema ◽  
J Raff ◽  
K Schneider ◽  
B M Alberts
Keyword(s):  
Cell Cycle ◽  
Dependent Manner ◽  
Multiprotein Complex ◽  
Cyclin Dependent Kinases ◽  
A Cell ◽  
Late Telophase ◽  
Cycle Dependent ◽  
Centrosomal Proteins ◽  
Drosophila Embryos

DMAP190 is a microtubule-associated protein from Drosophila that is localized to the centrosome. In a previous study, we used affinity chromatography to identify proteins that interact with DMAP190, and identified a 60-kDa protein that we named DMAP60 (Kellogg and Alberts, 1992). Like DMAP190, DMAP60 interacts with microtubules and is localized to the centrosome, and the two proteins associate as part of a multiprotein complex. We now report the cloning and sequencing of the cDNA encoding DMAP60. The amino acid sequence of DMAP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. As judged by in situ hybridization, the gene for DMAP60 maps to chromosomal region 46A. In agreement with others working on Drosophila centrosomal proteins, we have changed the names for DMAP190 and DMAP60 to CP190 and CP60, respectively, to give these proteins a consistent nomenclature. Antibodies that recognize CP60 reveal that it is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically during late telophase or early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated, and dephosphorylated CP60 is a good substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. We find that bacterially expressed CP60 binds to purified microtubules, and this binding is blocked by CP60 phosphorylation.

scholarly journals Microtubule Organization Requires Cell Cycle-dependent Nucleation at Dispersed Cytoplasmic Sites: Polar and Perinuclear Microtubule Organizing Centers in the Plant Pathogen Ustilago maydis

2003 ◽  
Vol 14 (2) ◽  
pp. 642-657 ◽  
Author(s):  
Anne Straube ◽  
Marianne Brill ◽  
Berl R. Oakley ◽  
Tetsuya Horio ◽  
Gero Steinberg
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Organization ◽  
Microtubule Organizing Centers ◽  
Nucleation Sites ◽  
Cycle Dependent ◽  
Green Fluorescent

Growth of most eukaryotic cells requires directed transport along microtubules (MTs) that are nucleated at nuclear-associated microtubule organizing centers (MTOCs), such as the centrosome and the fungal spindle pole body (SPB). Herein, we show that the pathogenic fungusUstilago maydis uses different MT nucleation sites to rearrange MTs during the cell cycle. In vivo observation of green fluorescent protein-MTs and MT plus-ends, tagged by a fluorescent EB1 homologue, provided evidence for antipolar MT orientation and dispersed cytoplasmic MT nucleating centers in unbudded cells. On budding γ-tubulin containing MTOCs formed at the bud neck, and MTs reorganized with >85% of all minus-ends being focused toward the growth region. Experimentally induced lateral budding resulted in MTs that curved out of the bud, again supporting the notion that polar growth requires polar MT nucleation. Depletion or overexpression of Tub2, the γ-tubulin from U. maydis, affected MT number in interphase cells. The SPB was inactive in G2 phase but continuously recruited γ-tubulin until it started to nucleate mitotic MTs. Taken together, our data suggest that MT reorganization in U. maydis depends on cell cycle-specific nucleation at dispersed cytoplasmic sites, at a polar MTOC and the SPB.

An abundant high-mobility-group-like protein is targeted to micronuclei in a cell cycle-dependent and developmentally regulated fashion in Tetrahymena thermophila

1993 ◽  
Vol 13 (1) ◽  
pp. 163-173
Author(s):  
T Wang ◽  
C D Allis
Keyword(s):  
Cell Cycle ◽  
Tetrahymena Thermophila ◽  
Polyclonal Antibodies ◽  
Dna Recombination ◽  
High Mobility ◽  
High Mobility Group ◽  
Sexual Cycle ◽  
Developmentally Regulated ◽  
Early Stages ◽  
Cycle Dependent

In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei. Biochemical data document the fact that HMG B is extremely labile in micronuclei. Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei. Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication. This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair. Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin. However, micronuclei are transcriptionally active during early stages of conjugation. Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented. Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration. We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.

scholarly journals Cdk1 promotes cytokinesis in fission yeast through activation of the septation initiation network

2014 ◽  
Vol 25 (15) ◽  
pp. 2250-2259 ◽  
Author(s):  
Nicole Rachfall ◽  
Alyssa E. Johnson ◽  
Sapna Mehta ◽  
Jun-Song Chen ◽  
Kathleen L. Gould
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Complete Removal ◽  
Spindle Pole ◽  
Cyclin Dependent Kinase ◽  
Gtpase Activating Protein ◽  
Pole Body ◽  
Kinase Cascade ◽  
Septation Initiation Network ◽  
Cycle Dependent

In Schizosaccharomyces pombe, late mitotic events are coordinated with cytokinesis by the septation initiation network (SIN), an essential spindle pole body (SPB)–associated kinase cascade, which controls the formation, maintenance, and constriction of the cytokinetic ring. It is not fully understood how SIN initiation is temporally regulated, but it depends on the activation of the GTPase Spg1, which is inhibited during interphase by the essential bipartite GTPase-activating protein Byr4-Cdc16. Cells are particularly sensitive to the modulation of Byr4, which undergoes cell cycle–dependent phosphorylation presumed to regulate its function. Polo-like kinase, which promotes SIN activation, is partially responsible for Byr4 phosphorylation. Here we show that Byr4 is also controlled by cyclin-dependent kinase (Cdk1)–mediated phosphorylation. A Cdk1 nonphosphorylatable Byr4 phosphomutant displays severe cell division defects, including the formation of elongated, multinucleate cells, failure to maintain the cytokinetic ring, and compromised SPB association of the SIN kinase Cdc7. Our analyses show that Cdk1-mediated phosphoregulation of Byr4 facilitates complete removal of Byr4 from metaphase SPBs in concert with Plo1, revealing an unexpected role for Cdk1 in promoting cytokinesis through activation of the SIN pathway.

scholarly journals Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

1998 ◽  
Vol 9 (4) ◽  
pp. 775-793 ◽  
Author(s):  
Gislene Pereira ◽  
Michael Knop ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Nuclear Localization Sequence ◽  
Checkpoint Control ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pole Body ◽  
Cycle Dependent ◽  
Cytoplasmic Side

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.

scholarly journals Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast.

1993 ◽  
Vol 121 (5) ◽  
pp. 961-976 ◽  
Author(s):  
H Funabiki ◽  
I Hagan ◽  
S Uzawa ◽  
M Yanagida
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Topoisomerase Ii ◽  
Nuclear Periphery ◽  
Spindle Pole Body ◽  
Critical Length ◽  
Spindle Pole ◽  
Dna Topoisomerase ◽  
Motor Cells ◽  
Cycle Dependent

Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin of a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase II, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm1 mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.

scholarly journals Cell Cycle-regulated, Microtubule-independent Organelle Division in Cyanidioschyzon merolae

2005 ◽  
Vol 16 (5) ◽  
pp. 2493-2502 ◽  
Author(s):  
Keiji Nishida ◽  
Fumi Yagisawa ◽  
Haruko Kuroiwa ◽  
Toshiyuki Nagata ◽  
Tsuneyoshi Kuroiwa
Keyword(s):  
Cell Cycle ◽  
Spindle Pole ◽  
Chloroplast Division ◽  
Mitochondrial Morphology ◽  
Cyanidioschyzon Merolae ◽  
Microtubule Organization ◽  
Mitochondrial Division ◽  
Protein Levels ◽  
Division Site ◽  
Organelle Division

Mitochondrial and chloroplast division controls the number and morphology of organelles, but how cells regulate organelle division remains to be clarified. Here, we show that each step of mitochondrial and chloroplast division is closely associated with the cell cycle in Cyanidioschyzon merolae. Electron microscopy revealed direct associations between the spindle pole bodies and mitochondria, suggesting that mitochondrial distribution is physically coupled with mitosis. Interconnected organelles were fractionated under microtubule-stabilizing condition. Immunoblotting analysis revealed that the protein levels required for organelle division increased before microtubule changes upon cell division, indicating that regulation of protein expression for organelle division is distinct from that of cytokinesis. At the mitochondrial division site, dynamin stuck to one of the divided mitochondria and was spatially associated with the tip of a microtubule stretching from the other one. Inhibition of microtubule organization, proteasome activity or DNA synthesis, respectively, induced arrested cells with divided but shrunk mitochondria, with divided and segregated mitochondria, or with incomplete mitochondrial division restrained at the final severance, and repetitive chloroplast division. The results indicated that mitochondrial morphology and segregation but not division depend on microtubules and implied that the division processes of the two organelles are regulated at distinct checkpoints.

scholarly journals An abundant high-mobility-group-like protein is targeted to micronuclei in a cell cycle-dependent and developmentally regulated fashion in Tetrahymena thermophila.

1993 ◽  
Vol 13 (1) ◽  
pp. 163-173 ◽  
Author(s):  
T Wang ◽  
C D Allis
Keyword(s):  
Cell Cycle ◽  
Tetrahymena Thermophila ◽  
Polyclonal Antibodies ◽  
Dna Recombination ◽  
High Mobility ◽  
High Mobility Group ◽  
Sexual Cycle ◽  
Developmentally Regulated ◽  
Early Stages ◽  
Cycle Dependent

In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei. Biochemical data document the fact that HMG B is extremely labile in micronuclei. Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei. Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication. This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair. Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin. However, micronuclei are transcriptionally active during early stages of conjugation. Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented. Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration. We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.

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