scholarly journals Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

2020 ◽  
Author(s):  
Takumi Chinen ◽  
Kaho Yamazaki ◽  
Kaho Hashimoto ◽  
Ken Fujii ◽  
Koki Watanabe ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Spindle Pole ◽  
Human Cells ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
The Individual ◽  
Chemical Perturbation

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. While centrosomes facilitate bipolar spindle formation, the individual functions of the centriole and PCM in mitosis remain elusive. Herein, we show the redundant roles of the centriole and PCM in bipolar spindle formation in human cells. Upon depletion of the PCM scaffold components, pericentrin and CDK5RAP2, centrioles remained able to recruit CEP192 onto their walls, which was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the chemical perturbation of polo-like kinase 1, a critical kinase for PCM assembly, efficiently suppressed the proliferation of various cancer cell lines from which centrioles were removed. Overall, these data suggest that the centriole and PCM cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation in human cells.

scholarly journals Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Takumi Chinen ◽  
Kaho Yamazaki ◽  
Kaho Hashimoto ◽  
Ken Fujii ◽  
Koki Watanabe ◽  
...  
Keyword(s):  
A549 Cells ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Scaffold Proteins ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Mitotic Cells

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

scholarly journals Aster-free spindle poles in insect spermatocytes: evidence for chromosome-induced spindle formation?

1986 ◽  
Vol 102 (5) ◽  
pp. 1679-1687 ◽  
Author(s):  
W Steffen ◽  
H Fuge ◽  
R Dietz ◽  
M Bastmeyer ◽  
G Müller
Keyword(s):  
Spindle Pole ◽  
Serial Sections ◽  
Spindle Formation ◽  
Microtubule Polymerization ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Lysis Buffer ◽  
Bipolar Spindles

Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.

scholarly journals Centriole-independent mitotic spindle assembly relies on the PCNT–CDK5RAP2 pericentriolar matrix

2020 ◽  
Vol 219 (12) ◽  
Author(s):  
Sadanori Watanabe ◽  
Franz Meitinger ◽  
Andrew K. Shiau ◽  
Karen Oegema ◽  
Arshad Desai
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Hela Cells ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Cell Type ◽  
Matrix Assembly ◽  
Spindle Formation ◽  
Pericentriolar Material ◽  
Mitotic Spindle Assembly

Centrosomes, composed of centrioles that recruit a pericentriolar material (PCM) matrix assembled from PCNT and CDK5RAP2, catalyze mitotic spindle assembly. Here, we inhibit centriole formation and/or remove PCNT–CDK5RAP2 in RPE1 cells to address their relative contributions to spindle formation. While CDK5RAP2 and PCNT are normally dispensable for spindle formation, they become essential when centrioles are absent. Acentriolar spindle assembly is accompanied by the formation of foci containing PCNT and CDK5RAP2 via a microtubule and Polo-like kinase 1–dependent process. Foci formation and spindle assembly require PCNT-CDK5RAP2–dependent matrix assembly and the ability of CDK5RAP2 to recruit γ-tubulin complexes. Thus, the PCM matrix can self-organize independently of centrioles to generate microtubules for spindle assembly; conversely, an alternative centriole-anchored mechanism supports spindle assembly when the PCM matrix is absent. Extension to three cancer cell lines revealed similar results in HeLa cells, whereas DLD1 and U2OS cells could assemble spindles in the absence of centrioles and PCNT-CDK5RAP2, suggesting cell type variation in spindle assembly mechanisms.

scholarly journals Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation

2015 ◽  
Vol 35 (15) ◽  
pp. 2626-2640 ◽  
Author(s):  
Lingjun Meng ◽  
Jung-Eun Park ◽  
Tae-Sung Kim ◽  
Eun Hye Lee ◽  
Suk-Youl Park ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Spindle Assembly ◽  
Human Cells ◽  
Mitotic Progression ◽  
Spindle Formation ◽  
Content Type ◽  
Bipolar Spindle ◽  
Egg Extracts ◽  
Cultured Human Cells ◽  
Bipolar Spindles

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study withXenopus laevisegg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif ofXenopusCep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous toXenopusp-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

scholarly journals TRIM37 prevents formation of condensate-organized ectopic spindle poles to ensure mitotic fidelity

2021 ◽  
Vol 220 (7) ◽  
Author(s):  
Franz Meitinger ◽  
Dong Kong ◽  
Midori Ohta ◽  
Arshad Desai ◽  
Karen Oegema ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Ubiquitin Ligase ◽  
Spindle Pole ◽  
Mitotic Entry ◽  
Centriole Duplication ◽  
Mulibrey Nanism ◽  
Spindle Poles ◽  
Pathological Mechanism ◽  
Pericentriolar Material ◽  
Centrosomal Proteins

Centrosomes are composed of a centriolar core surrounded by pericentriolar material that nucleates microtubules. The ubiquitin ligase TRIM37 localizes to centrosomes, but its centrosomal roles are not yet defined. We show that TRIM37 does not control centriole duplication, structure, or the ability of centrioles to form cilia but instead prevents assembly of an ectopic centrobin-scaffolded structured condensate that forms by budding off of centrosomes. In ∼25% of TRIM37-deficient cells, the condensate organizes an ectopic spindle pole, recruiting other centrosomal proteins and acquiring microtubule nucleation capacity during mitotic entry. Ectopic spindle pole–associated transient multipolarity and multipolar segregation in TRIM37-deficient cells are suppressed by removing centrobin, which interacts with and is ubiquitinated by TRIM37. Thus, TRIM37 ensures accurate chromosome segregation by preventing the formation of centrobin-scaffolded condensates that organize ectopic spindle poles. Mutations in TRIM37 cause the disorder mulibrey nanism, and patient-derived cells harbor centrobin condensate-organized ectopic poles, leading us to propose that chromosome missegregation is a pathological mechanism in this disorder.

scholarly journals Kinetochore-mediated outward force promotes spindle pole separation in fission yeast

2019 ◽  
Vol 30 (22) ◽  
pp. 2802-2813 ◽  
Author(s):  
Yutaka Shirasugi ◽  
Masamitsu Sato
Keyword(s):  
Fission Yeast ◽  
Motor Proteins ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Double Knockout ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Bipolar Spindles ◽  
Meiosis I

Bipolar spindles are organized by motor proteins that generate microtubule-­dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

scholarly journals The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

2020 ◽  
Vol 134 (1) ◽  
pp. jcs251025
Author(s):  
Zoë Geraghty ◽  
Christina Barnard ◽  
Pelin Uluocak ◽  
Ulrike Gruneberg
Keyword(s):  
Molecular Mechanisms ◽  
Metaphase Plate ◽  
Direct Interaction ◽  
Mitotic Chromosomes ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

scholarly journals Interdependency of Fission Yeast Alp14/TOG and Coiled Coil Protein Alp7 in Microtubule Localization and Bipolar Spindle Formation

2004 ◽  
Vol 15 (4) ◽  
pp. 1609-1622 ◽  
Author(s):  
Masamitsu Sato ◽  
Leah Vardy ◽  
Miguel Angel Garcia ◽  
Nirada Koonrugsa ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Microtubule Organization ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
C Terminus ◽  
Step Model

The Dis1/TOG family plays a pivotal role in microtubule organization. In fission yeast, Alp14 and Dis1 share an essential function in bipolar spindle formation. Here, we characterize Alp7, a novel coiled-coil protein that is required for organization of bipolar spindles. Both Alp7 and Alp14 colocalize to the spindle pole body (SPB) and mitotic spindles. Alp14 localization to these sites is fully dependent upon Alp7. Conversely, in the absence of Alp14, Alp7 localizes to the SPBs, but not mitotic spindles. Alp7 forms a complex with Alp14, where the C-terminal region of Alp14 interacts with the coiled-coil domain of Alp7. Intriguingly, this Alp14 C terminus is necessary and sufficient for mitotic spindle localization. Overproduction of either full-length or coiled-coil region of Alp7 results in abnormal V-shaped spindles and stabilization of interphase microtubules, which is induced independent of Alp14. Alp7 may be a functional homologue of animal TACC. Our results shed light on an interdependent relationship between Alp14/TOG and Alp7. We propose a two-step model that accounts for the recruitment of Alp7 and Alp14 to the SPB and microtubules.

scholarly journals Centriole number and the reproductive capacity of spindle poles.

1985 ◽  
Vol 100 (3) ◽  
pp. 887-896 ◽  
Author(s):  
G Sluder ◽  
C L Rieder
Keyword(s):  
Spindle Pole ◽  
Reproductive Capacity ◽  
Centriole Duplication ◽  
Voltage Electron ◽  
Spindle Poles ◽  
Mother And Daughter ◽  
Pericentriolar Material ◽  
History Of ◽  
Bipolar Spindles

The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.

scholarly journals In vitro antiproliferative activity of some fluoro-substituted benzimidazole-based scaffolds

2021 ◽  
Vol 4 (1) ◽  
pp. 10-17
Author(s):  
Ronak Haj Ersan ◽  
Nizami Duran
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Mtt Assay ◽  
Antiproliferative Activity ◽  
Cancer Cell Lines ◽  
Positive Control ◽  
Human Cells ◽  
Benzimidazole Derivatives ◽  
Antiproliferative Agents

In the present work, a series of fluoro-substituted benzimidazole derivatives were designed and synthesized as antiproliferative agents. The antiproliferative activity of these compounds was investigated using MTT assay. Fluoro-substituted benzimidazole derivatives showed significant antiproliferative activity against all the tested cancer cell lines. All the derivatives were found to be less toxic as compared to methotrexate (positive control) in human cells, indicating selective and efficient antiproliferative activity of these benzimidazole derivatives. These findings suggest that compounds ORT14 and ORT15 among this series are most effective and have potential for detailed investigations.

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