scholarly journals Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

2013 ◽  
Vol 24 (18) ◽  
pp. 2894-2906 ◽  
Author(s):  
Hirohisa Masuda ◽  
Risa Mori ◽  
Masashi Yukawa ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Unique Role ◽  
Pole Body ◽  
Organizing Center ◽  
Core Components

γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1–6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1–3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not. Recently a novel conserved component, MOZART1, was identified in humans and plants, but its precise functions remain to be determined. In this paper, we characterize the fission yeast homologue Mzt1, showing that it is essential for cell viability. Mzt1 is present in approximately equal stoichiometry with Alp4/GCP2 and localizes to all the MTOCs, including the SPB and interphase and equatorial MTOCs. Temperature-sensitive mzt1 mutants display varying degrees of compromised microtubule organization, exhibiting multiple defects during both interphase and mitosis. Mzt1 is required for γ-TuC recruitment, but not sufficient to localize to the SPB, which depends on γ-TuC integrity. Intriguingly, the core γ-TuC assembles in the absence of Mzt1. Mzt1 therefore plays a unique role within the γ-TuC components in attachment of this complex to the major MTOC site.

Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 407-422 ◽  
Author(s):  
E A Vallen ◽  
W Ho ◽  
M Winey ◽  
M D Rose
Keyword(s):  
Copy Number ◽  
Gene Dosage ◽  
Spindle Pole Body ◽  
Direct Interaction ◽  
Spindle Pole ◽  
High Copy Number ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Recessive Mutations ◽  
Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

scholarly journals 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

2017 ◽  
Vol 28 (25) ◽  
pp. 3647-3659 ◽  
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.

scholarly journals A Fourth Component of the Fission Yeast γ-Tubulin Complex, Alp16, Is Required for Cytoplasmic Microtubule Integrity and Becomes Indispensable When γ-Tubulin Function Is Compromised

2002 ◽  
Vol 13 (7) ◽  
pp. 2360-2373 ◽  
Author(s):  
Akiko Fujita ◽  
Leah Vardy ◽  
Miguel Angel Garcia ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Multicopy Suppressor ◽  
Large Complex ◽  
Synthetically Lethal ◽  
And Function

γ-Tubulin functions as a multiprotein complex, called the γ-tubulin complex (γ-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved γ-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16 + , as a multicopy suppressor of temperature-sensitive alp6-719mutants. alp16 + encodes a 759-amino-acid protein with two conserved regions found in all other members of γ-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16 + is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225,alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with γ-tubulin and cosediments with the γ-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the γ-TuC between yeast and higher eukaryotes.

scholarly journals Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle

2011 ◽  
Vol 195 (3) ◽  
pp. 467-484 ◽  
Author(s):  
Tiina Tamm ◽  
Agnes Grallert ◽  
Emily P.S. Grossman ◽  
Isabel Alvarez-Tabares ◽  
Frances E. Stevens ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Microtubule Organizing Center ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Organizing Center ◽  
Nuclear Component ◽  
Anaphase B

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.

scholarly journals Two spatially distinct Kinesin-14 Pkl1 and Klp2 generate collaborative inward forces against Kinesin-5 Cut7 inS. pombe

2017 ◽  
Author(s):  
Masashi Yukawa ◽  
Yusuke Yamada ◽  
Tomoaki Yamauchi ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Force Balance ◽  
Temperature Sensitive ◽  
Spindle Microtubule ◽  
Pole Body ◽  
Summary Statement ◽  
Proper Force ◽  
Individual Motor

ABSTRACTKinesin motors play central roles in bipolar spindle assembly. In many eukaryotes, spindle pole separation is driven by Kinesin-5 that generates outward force. This outward force is balanced by antagonistic inward force elicited by Kinesin-14 and/or Dynein. In fission yeast, two Kinesin-14s, Pkl1 and Klp2, play an opposing role against Kinesin-5/Cut7. However, how these two Kinesin-14s coordinate individual activities remains elusive. Here we show that while deletion of eitherpkl1orklp2rescues temperature sensitivecut7mutants, onlypkl1deletion can bypass the lethality caused bycut7deletion. Pkl1 is tethered to the spindle pole body, while Klp2 is localized along the spindle microtubule. Forced targeting of Klp2 to the spindle pole body, however, compensates for Pkl1 functions, indicating that cellular localizations, rather than individual motor specificities, differentiate between the two Kinesin-14s. Interestingly, human Kinesin-14/HSET can replace either Pkl1 or Klp2. Moreover, overproducing HSET induces monopolar spindles, reminiscent of the phenotype of Cut7 inactivation. Taken together, this study has uncovered the biological mechanism of how two different Kinesin-14s exert their antagonistic roles against Kinesin-5 in a spatially distinct manner.SUMMARY STATEMENTProper force-balance generated by Kinesin-5 and Kinesin-14 is crucial for spindle bipolarity. Two fission yeast Kinesin-14s localize to different structures, thereby collaboratively producing inward forces against Kinesin-5-mediated outward force.Abbreviations usedGBPGFP-binding proteinMWP complexMsd1-Wdr8-Pkl1 complexSPBspindle pole bodytstemperature sensitiveγ-TuCthe γ-tubulin complex

scholarly journals Generation of temperature sensitive mutations with error-prone PCR in a gene encoding a component of the spindle pole body in fission yeast

2019 ◽  
Vol 83 (9) ◽  
pp. 1717-1720 ◽  
Author(s):  
Ngang Heok Tang ◽  
Chii Shyang Fong ◽  
Hirohisa Masuda ◽  
Isabelle Jourdain ◽  
Masashi Yukawa ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Pole Body

scholarly journals Generation of Temperature Sensitive Mutations with Error-Prone PCR in a Gene Encoding a Component of the Spindle Pole Body in Fission Yeast

2019 ◽  
Author(s):  
Ngang Heok Tang ◽  
Chii Shyang Fong ◽  
Hirohisa Masuda ◽  
Isabelle Jourdain ◽  
Masashi Yukawa ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Simple Procedure ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Essential Genes ◽  
Temperature Sensitive ◽  
Cellular Functions ◽  
Gene Encoding ◽  
Pole Body ◽  
Ts Mutations

AbstractTemperature-sensitive (ts) mutants provide powerful tools, thereby investigating cellular functions of essential genes. We report here a simple procedure to generate ts mutations using error-prone PCR in pcp1 that encodes a spindle pole body (SPB) component in Schizosaccharomyces pombe. This manipulation is not restricted to analysis of Pcp1, and can be suited to any essential genes involved in other processes.

scholarly journals gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation.

1996 ◽  
Vol 134 (2) ◽  
pp. 429-441 ◽  
Author(s):  
A Spang ◽  
S Geissler ◽  
K Grein ◽  
E Schiebel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Checkpoint Control ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Spindle Formation ◽  
Pole Body ◽  
Gamma Tubulin

Tub4p is a novel tubulin in Saccharomyces cerevisiae that most closely resembles gamma-tubulin. We report in this manuscript that the essential Tub4p is associated with the inner and outer plaques of the yeast microtubule organizing center, the spindle pole body (SPB). These SPB substructures are involved in the attachment of the nuclear and cytoplasmic microtubules, respectively (Byers, B., and L. Goetsch. 1975. J. Bacteriol. 124:511-523). Study of a temperature sensitive tub4-1 allele revealed that TUB4 has essential functions in microtubule organization. Remarkably, SPB duplication and separation are not impaired in tub4-1 cells incubated at the nonpermissive temperature. However, SPBs from such cells contain less or misdirected nuclear microtubules. Further analysis revealed that tub4-1 cells are able to assemble a short bipolar spindle, suggesting that the defect in microtubule organization occurs after spindle formation. A role of Tub4p in microtubule organization is further suggested by an increase in chromosome loss in tub4-1 cells. In addition, cell cycle arrest and survival of tub4-1 cells is dependent on the mitotic checkpoint control gene BUB2 (Hoyt, M.A., L. Totis, B.T. Roberts. 1991. Cell. 66:507-517), one of the cell's monitors of spindle integrity.

scholarly journals Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function.

1996 ◽  
Vol 134 (2) ◽  
pp. 443-454 ◽  
Author(s):  
L G Marschall ◽  
R L Jeng ◽  
J Mulholland ◽  
T Stearns
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Microtubule Organizing Center ◽  
Pole Body ◽  
Monopolar Spindle ◽  
Organizing Center ◽  
Gamma Tubulin

gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.

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