scholarly journals Saccharomyces cerevisiae Duo1p and Dam1p, Novel Proteins Involved in Mitotic Spindle Function

1998 ◽  
Vol 143 (4) ◽  
pp. 1029-1040 ◽  
Author(s):  
Christian Hofmann ◽  
Iain M. Cheeseman ◽  
Bruce L. Goode ◽  
Kent L. McDonald ◽  
Georjana Barnes ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Spindle Pole Bodies ◽  
Intranuclear Microtubules ◽  
Spindle Microtubules ◽  
Green Fluorescent

In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP–Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

scholarly journals Implication of a novel multiprotein Dam1p complex in outer kinetochore function

2001 ◽  
Vol 155 (7) ◽  
pp. 1137-1146 ◽  
Author(s):  
Iain M. Cheeseman ◽  
Christine Brew ◽  
Michael Wolyniak ◽  
Arshad Desai ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Phosphorylation Event ◽  
Spindle Microtubules ◽  
Green Fluorescent ◽  
Kinetochore Function

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

scholarly journals Aspergillus nidulans Dis1/XMAP215 Protein AlpA Localizes to Spindle Pole Bodies and Microtubule Plus Ends and Contributes to Growth Directionality

2007 ◽  
Vol 6 (3) ◽  
pp. 555-562 ◽  
Author(s):  
Cathrin Enke ◽  
Nadine Zekert ◽  
Daniel Veith ◽  
Carolin Schaaf ◽  
Sven Konzack ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Spindle Pole Bodies ◽  
Family Protein ◽  
Associated Proteins ◽  
Hyphal Morphology ◽  
Conventional Kinesin ◽  
Green Fluorescent

ABSTRACTThe dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. InSchizosaccharomyces pombeand in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA fromAspergillus nidulansand show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA.alpAdeletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics.

scholarly journals Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

1997 ◽  
Vol 17 (9) ◽  
pp. 5001-5015 ◽  
Author(s):  
N I Zanchin ◽  
P Roberts ◽  
A DeSilva ◽  
F Sherman ◽  
D S Goldfarb
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Acid Protein ◽  
Conserved Gene ◽  
Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

scholarly journals Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2

1999 ◽  
Vol 145 (5) ◽  
pp. 979-991 ◽  
Author(s):  
Roberta Fraschini ◽  
Elisa Formenti ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Cycle Progression ◽  
Spindle Pole Bodies

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

scholarly journals Mitosis in the fission yeast Schizosaccharomyces pombe as revealed by freeze-substitution electron microscopy

1986 ◽  
Vol 80 (1) ◽  
pp. 253-268
Author(s):  
K. Tanaka ◽  
T. Kanbe
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mitotic Spindle ◽  
Nuclear Division ◽  
Freeze Substitution ◽  
Spindle Pole ◽  
Nuclear Space ◽  
Spindle Pole Bodies ◽  
Transmission Electron ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules

Nuclear division in Schizosaccharomyces pombe has been studied in transmission electron micrographs of sections of cells fixed by a method of freeze-substitution. We have found cytoplasmic microtubules in the vicinity of the spindle pole bodies and two kinds of microtubules, short discontinuous ones and long, parallel ones in the intranuclear mitotic spindle. For most of the time taken by nuclear division the spindle pole bodies face each other squarely across the nuclear space but early in mitosis they briefly appear twisted out of alignment with each other, thereby imparting a sigmoidal shape to the bundle of spindle microtubules extending between them. This configuration is interpreted as indicating active participation of the spindle in the initial elongation of the dividing nucleus. It is proposed that mitosis is accompanied by the shortening of chromosomal microtubules simultaneously with the elongation of the central pole-to-pole bundle of microtubules of the intranuclear spindle. Daughter nuclei are separated by the sliding apart of interdigitating microtubules of the spindle at telophase. Some of the latter bear dense knobs at their ends.

scholarly journals Marker Fusion Tagging, a New Method for Production of Chromosomally Encoded Fusion Proteins

2010 ◽  
Vol 9 (5) ◽  
pp. 827-830 ◽  
Author(s):  
Julian Lai ◽  
Seng Kah Ng ◽  
Fang Fang Liu ◽  
Rajesh Narhari Patkar ◽  
Yanfen Lu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Spindle Pole ◽  
Hygromycin B ◽  
Content Type ◽  
Spindle Pole Bodies ◽  
New Gene ◽  
Green Fluorescent

ABSTRACT A new gene-tagging method (marker fusion tagging [MFT]) is demonstrated for Neurospora crassa and Magnaporthe oryzae. Translational fusions between the hygromycin B resistance gene and various markers are inserted into genes of interest by homologous recombination to produce chromosomally encoded fusion proteins. This method can produce tags at any position and create deletion alleles that maintain N- and C-terminal sequences. We show the utility of MFT by producing enhanced green fluorescent protein (EGFP) tags in proteins localized to nuclei, spindle pole bodies, septal pore plugs, Woronin bodies, developing septa, and the endoplasmic reticulum.

In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis

2001 ◽  
Vol 114 (14) ◽  
pp. 2627-2640 ◽  
Author(s):  
Anabelle Decottignies ◽  
Patrick Zarzov ◽  
Paul Nurse
Keyword(s):  
Fission Yeast ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Periphery ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cyclin Dependent Kinase ◽  
Spindle Pole Bodies ◽  
Mitosis And Meiosis

We investigated the in vivo localisation of fission yeast cyclin-dependent kinase cdc2p during mitosis and meiosis. Fusion to yellow fluorescent protein (YFP) revealed that cdc2-YFP is present in the cytoplasm at all stages of the cell cycle. Nuclear cdc2-YFP fluorescence oscillates with that of cdc13-YFP cyclin. At G1/S, at least one of cdc13p, cig1p or cig2p B-type cyclins is required for the accumulation of cdc2-YFP into the nucleus. Cdc2-YFP and cdc13-YFP are highly enriched on the spindle pole body of cells in late G2 or arrested at S phase. Both accumulate on the spindle pole bodies and the spindle in prophase and metaphase independently of the microtubule-associated protein dis1p. In anaphase, the cdc2p/cdc13p complex leaves the spindle prior to sister chromatid separation, and cdc13-YFP is enriched at the nuclear periphery before fluorescence disappears. If cdc13p cannot be recognized by the anaphase-promoting complex, cdc2-YFP and cdc13-YFP remain associated with the spindle. In mating cells, cdc2-YFP enters the nucleus as soon as the cells undergo fusion. During karyogamy and meiotic prophase, cdc2-YFP is highly enriched on the centromeres. In meiosis I, association of cdc2-YFP with the spindle and the spindle pole bodies shows differences to mitotic cells, suggesting different mechanisms of spindle formation. This study suggests that changes in cdc2p localisation are important for both mitosis and meiosis regulation.

Fine structure of an organelle associated with the nucleus and cytoplasmic microtubules in the cellular slime mould Polysphondylium violaceum

1975 ◽  
Vol 18 (2) ◽  
pp. 315-326
Author(s):  
U.P. Roos
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Interphase Nuclei ◽  
Spindle Pole Bodies ◽  
Nucleation Sites ◽  
Cellular Slime ◽  
Granular Matrix ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
Opaque Body

Polysphondylium violaceum was grown in association with Escherichia coli. Vegetative amoebae and pseudoplasmodia were fixed under different conditions and processed for electron microscopy. An electron-opaque body (nucleus-associated body, NAB) lies in the cytoplasm near the tapered end of interphase nuclei. The NAB consists of a disk-shaped, multilayered core, approximately 200 nm in diameter and 150 nm thick, embedded in a granular matrix from which electron-opaque nodules protrude. The nodules are termination points of microtubules radiating from the NAB into the cytoplasm or running along the nucleus. On the average there are 16 nodules per NAB. One or two microtubules terminate in each nodule. Spindle pole bodies, arising by duplication of the NAB at the beginning of mitosis, are unstructured foci for spindle microtubules in mitotic cells. It is suggested that cytoplasmic microtubules do not determine cell shape, but they probably cause the tapering deformation of the nucleus. They may, furthermore, represent a storage form of subunits for utilization during the formation of the mitotic spindle. The nodules of the NAB are potential nucleation sites of cytoplasmic microtubules during interphase. Spindle pole bodies presumably acquire a microtubule organizing capability by integration of the decondensed nodules.

scholarly journals Nuclear Import of Histone H2a and H2b Is Mediated by a Network of Karyopherins

2001 ◽  
Vol 153 (2) ◽  
pp. 251-262 ◽  
Author(s):  
Nima Mosammaparast ◽  
Kelley R. Jackson ◽  
Yurong Guo ◽  
Cynthia J. Brame ◽  
Jeffrey Shabanowitz ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Fluorescent Protein ◽  
Nuclear Localization Sequence ◽  
Temperature Sensitive ◽  
Histone H2a ◽  
Amino Terminal ◽  
Core Histones ◽  
Localization Sequence ◽  
Green Fluorescent

The first step in the assembly of new chromatin is the cell cycle–regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

scholarly journals Novel Phosphorylation States of the Yeast Spindle Pole Body

2018 ◽  
Author(s):  
Kimberly K. Fong ◽  
Alex Zelter ◽  
Beth Graczyk ◽  
Jill M. Hoyt ◽  
Michael Riffle ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Phosphorylation Sites ◽  
Microtubule Nucleation ◽  
Data Set ◽  
Pole Body

ABSTRACTPhosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purifiedSaccharomyces cerevisiaeSPBs 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 acdc4-1temperature sensitive mutation, with particular interest in phosphorylation events on the γ-tubulin small complex (γ-TuSC). Thecdc4-1arrest 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 analyzedin vivoby fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally,in vivoanalysis 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.

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