Microtubule Nucleation at Non-Spindle Pole Body Microtubule-Organizing Centers Requires Fission Yeast Centrosomin-Related Protein mod20p

Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the gamma-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.

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In vitro approaches for the study of microtubule nucleation at the fission yeast spindle pole body

Methods in Cell Biology - Centrosomes and Spindle Pole Bodies ◽

10.1016/s0091-679x(01)67012-2 ◽

2001 ◽

pp. 167-177

Author(s):

Hirohisa Masuda ◽

Saeko Takada ◽

Takehiko Shibata ◽

W. Zacheus Cande ◽

Yasushi Hiraoka

Keyword(s):

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Microtubule Nucleation ◽

Pole Body

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Faculty Opinions recommendation of Fission yeast Myo51 is a meiotic spindle pole body component with discrete roles during cell fusion and spore formation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2137023.1737135 ◽

2010 ◽

Author(s):

Christopher McInerny

Keyword(s):

Fission Yeast ◽

Cell Fusion ◽

Spindle Pole Body ◽

Spindle Pole ◽

Spore Formation ◽

Meiotic Spindle ◽

Pole Body ◽

Body Component

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Distinct nuclear and spindle pole body populations of cyclin–cdc2 in fission yeast

Nature ◽

10.1038/347680a0 ◽

1990 ◽

Vol 347 (6294) ◽

pp. 680-682 ◽

Cited By ~ 110

Author(s):

Caroline E. Alfa ◽

Bernard Ducommun ◽

David Beach ◽

Jeremy S. Hyams

Keyword(s):

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body

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The Fission Yeast Homolog of the Human Transcription Factor EAP30 Blocks Meiotic Spindle Pole Body Amplification

Developmental Cell ◽

10.1016/j.devcel.2005.04.016 ◽

2005 ◽

Vol 9 (1) ◽

pp. 63-73 ◽

Cited By ~ 20

Author(s):

Ye Jin ◽

Joel J. Mancuso ◽

Satoru Uzawa ◽

Daniela Cronembold ◽

W. Zacheus Cande

Keyword(s):

Transcription Factor ◽

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Meiotic Spindle ◽

Pole Body ◽

Yeast Homolog ◽

Human Transcription Factor

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A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0497 ◽

2017 ◽

Vol 28 (25) ◽

pp. 3647-3659 ◽

Cited By ~ 23

Author(s):

Masashi Yukawa ◽

Tomoki Kawakami ◽

Masaki Okazaki ◽

Kazunori Kume ◽

Ngang Heok Tang ◽

...

Keyword(s):

Fission Yeast ◽

Chromosome Segregation ◽

Spindle Pole Body ◽

Spindle Assembly ◽

Spindle Pole ◽

Temperature Sensitive ◽

Bipolar Spindle ◽

Pole Body ◽

Cross Linker ◽

Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

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The Mammalian γ-Tubulin Complex Contains Homologues of the Yeast Spindle Pole Body Components Spc97p and Spc98p

The Journal of Cell Biology ◽

10.1083/jcb.141.3.663 ◽

1998 ◽

Vol 141 (3) ◽

pp. 663-674 ◽

Cited By ~ 138

Author(s):

Steven M. Murphy ◽

Lenore Urbani ◽

Tim Stearns

Keyword(s):

Mammalian Cells ◽

Spindle Pole Body ◽

Spindle Pole ◽

Microtubule Organizing Centers ◽

Molecular Weights ◽

Microtubule Polymerization ◽

Pole Body ◽

Human Proteins ◽

Body Components ◽

Cytoplasmic Complex

γ-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. γ-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic γ-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human γ-tubulin were made. The epitope-tagged γ-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic γ-tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100- and 101-kD components of the γ-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with γ-tubulin at the centrosome, cosediment with γ-tubulin in sucrose gradients, and coimmunoprecipitate with γ-tubulin, indicating that they are part of the γ-tubulin complex. The conservation of a complex involving γ-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation.

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Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast

Molecular Genetics and Genomics ◽

10.1007/s00438-003-0938-8 ◽

2003 ◽

Vol 270 (6) ◽

pp. 449-461 ◽

Cited By ~ 74

Author(s):

F. Miki ◽

A. Kurabayashi ◽

Y. Tange ◽

K. Okazaki ◽

M. Shimanuki ◽

...

Keyword(s):

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Hybrid Search ◽

Pole Body ◽

Membrane Bound ◽

Two Hybrid

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The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope

The Journal of Cell Biology ◽

10.1083/jcb.200601062 ◽

2006 ◽

Vol 174 (5) ◽

pp. 665-675 ◽

Cited By ~ 105

Author(s):

Sue L. Jaspersen ◽

Adriana E. Martin ◽

Galina Glazko ◽

Thomas H. Giddings ◽

Garry Morgan ◽

...

Keyword(s):

Nuclear Envelope ◽

Spindle Pole Body ◽

Spindle Pole ◽

Integral Membrane Proteins ◽

Microtubule Nucleation ◽

The Core ◽

Pole Body ◽

C Terminus ◽

Bridge Structures ◽

Sun Domain

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication.

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