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

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

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

Bioscience Biotechnology and Biochemistry ◽

10.1080/09168451.2019.1611414 ◽

2019 ◽

Vol 83 (9) ◽

pp. 1717-1720 ◽

Cited By ~ 2

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

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

Molecular Biology of the Cell ◽

10.1091/mbc.e13-05-0235 ◽

2013 ◽

Vol 24 (18) ◽

pp. 2894-2906 ◽

Cited By ~ 41

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.

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

10.1101/595447 ◽

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.

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Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation

Journal of Cell Science ◽

10.1242/jcs.110.15.1805 ◽

1997 ◽

Vol 110 (15) ◽

pp. 1805-1812 ◽

Cited By ~ 8

Author(s):

M.J. Moser ◽

M.R. Flory ◽

T.N. Davis

Keyword(s):

Cell Growth ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Chromosome Segregation ◽

Fluorescent Protein ◽

Budding Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Temperature Sensitive ◽

Pole Body

The essential calmodulin genes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe were precisely replaced with genes encoding fusions between calmodulin and the green fluorescent protein (GFP). In living budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) localized simultaneously to sites of cell growth and to the spindle pole body (SPB), the yeast analog of the centrosome. Having demonstrated proper localization of GFP-calmodulin in budding yeast, we examined the localization of a fusion between GFP and calmodulin (GFP-Camlp) in fission yeast, where calmodulin had not been localized by any method. We find GFP-Camlp also localizes both to sites of polarized cell growth and to the fission yeast SPB. The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence. Antiserum to S. pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum. This pattern was identical to that seen using antiserum to Sad1p, a known SPB component. We then characterized the defects in a temperature-sensitive S. pombe calmodulin mutant. Mutant cam1-E14 cells synchronized in S phase completed DNA synthesis, but lost viability during transit of mitosis. Severe defects in chromosome segregation, including hypercondensation, fragmentation, and unequal allocation of chromosomal material were observed. Immunofluorescence analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells. Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects suggest that calmodulin performs an essential role during mitosis at the fission yeast SPB.

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The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability.

The Journal of Cell Biology ◽

10.1083/jcb.129.4.1033 ◽

1995 ◽

Vol 129 (4) ◽

pp. 1033-1047 ◽

Cited By ~ 265

Author(s):

I Hagan ◽

M Yanagida

Keyword(s):

Fission Yeast ◽

Nuclear Membrane ◽

Essential Gene ◽

Nuclear Periphery ◽

Spindle Pole Body ◽

Spindle Pole ◽

Temperature Sensitive ◽

Spindle Formation ◽

Isolation And Characterization ◽

Pole Body

Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected both spindle formation and function. The sad1+ gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.

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Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body

10.7287/peerj.preprints.3377v1 ◽

2017 ◽

Author(s):

Ngang Heok Tang ◽

Chii Shyang Fong ◽

Hirohisa Masuda ◽

Isabelle Jourdain ◽

Masashi Yukawa ◽

...

Keyword(s):

Fission Yeast ◽

Genetic Manipulation ◽

Spindle Pole Body ◽

Spindle Pole ◽

Temperature Sensitive ◽

Cellular Processes ◽

Temperature Sensitive Mutants ◽

Pole Body ◽

Genes Encoding ◽

Mitosis And Meiosis

The spindle pole body (SPB) in fungi is the equivalent of the animal centrosome. A number of previous studies have identified many, if not all, components of the SPB. The SPB is the structural platform for microtubule nucleation and plays important roles, both in mitosis and meiosis. The SPB is absolutely essential for cell survival and its abnormalities give rise to aberrant cell division and morphogenesis. Therefore, it is crucial to understand how the SPB organises itself and how the functions of individual SPB components are regulated. We report here a procedure to generate temperature sensitive mutants in the fission yeast, Schizosaccharomyces pombe. The approach has proved useful to characterise functions of individual SPB components. This original genetic manipulation is however not restricted to analysis of SPB functions, and can be suited to investigate other cellular processes in S. pombe.

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