The deubiquitinating enzyme complex BRISC is required for proper mitotic spindle assembly in mammalian cells

Deubiquitinating enzymes (DUBs) negatively regulate protein ubiquitination and play an important role in diverse physiological processes, including mitotic division. The BRCC36 isopeptidase complex (BRISC) is a DUB that is specific for lysine 63–linked ubiquitin hydrolysis; however, its biological function remains largely undefined. Here, we identify a critical role for BRISC in the control of mitotic spindle assembly in cultured mammalian cells. BRISC is a microtubule (MT)-associated protein complex that predominantly localizes to the minus ends of K-fibers and spindle poles and directly binds to MTs; importantly, BRISC promotes the assembly of functional bipolar spindle by deubiquitinating the essential spindle assembly factor nuclear mitotic apparatus (NuMA). The deubiquitination of NuMA regulates its interaction with dynein and importin-β, which are required for its function in spindle assembly. Collectively, these results uncover BRISC as an important regulator of the mitotic spindle assembly and cell division, and have important implications for the development of anticancer drugs targeting BRISC.

Download Full-text

Abnormal mitotic spindle assembly and cytokinesis induced by D-Limonene in cultured mammalian cells

Mutagenesis ◽

10.1093/mutage/get040 ◽

2013 ◽

Vol 28 (6) ◽

pp. 631-635 ◽

Cited By ~ 10

Author(s):

M. Mauro ◽

I. Catanzaro ◽

F. Naselli ◽

G. Sciandrello ◽

F. Caradonna

Keyword(s):

Mitotic Spindle ◽

Mammalian Cells ◽

Spindle Assembly ◽

Mitotic Spindle Assembly

Download Full-text

A homologue of the human regulator of mitotic spindle assembly protein (RMSA-1) is present in crane fly and is associated with meiotic chromosomes

Journal of Cell Science ◽

10.1242/jcs.107.7.1845 ◽

1994 ◽

Vol 107 (7) ◽

pp. 1845-1851

Author(s):

J.P. Yeo ◽

A. Forer ◽

B.H. Toh

Keyword(s):

Sex Chromosomes ◽

Mitotic Spindle ◽

Mammalian Cells ◽

Animal Species ◽

Spindle Assembly ◽

Meiotic Spindle ◽

Chromosomal Protein ◽

Meiotic Chromosomes ◽

Mitotic Spindle Assembly ◽

Cdc 2

In a previous study, we have shown that a newly identified chromosomal protein, RMSA-1 (Regulator of Mitotic Spindle Assembly-1), identified and cloned using a human autoimmune, serum, is essential for mitotic spindle assembly; we proposed that RMSA-1 was a previously unknown physiological substrate for cdc 2 kinase. In the present study, we show that this protein is present in crane fly and is associated with the chromosomes of spermatocytes. A 31 kDa molecule in extracts from crane-fly nuclei, isolated from larvae, pupae and adults, reacts with affinity-purified anti-RMSA-1 autoantibody, shown by immunoblotting. The autoantibody reacts, as shown by immunofluorescence, with crane-fly spermatocyte chromosomes in prophase through anaphase of both meiosis-1 and meiosis-II but does not react with preprophase or telophase nuclei or with spermatid nuclei. In all meiotic stages, the crane-fly sex chromosomes stain more intensely than the autosomes. We conclude that, since RMSA-1 is present in insect and mammalian cells, it is conserved across a variety of animal species. Further, since RMSA-1 binds to chromosomes in meiotic cells, it also may be essential for assembly of the meiotic spindle.

Download Full-text

The nonmotor adaptor HMMR dampens Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregation

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0531 ◽

2018 ◽

Vol 29 (7) ◽

pp. 786-796 ◽

Cited By ~ 2

Author(s):

Helen Chen ◽

Marisa Connell ◽

Lin Mei ◽

Gregor S. D. Reid ◽

Christopher A. Maxwell

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly ◽

Adaptor Proteins ◽

Microtubule Organization ◽

Assembly Factor ◽

Mitotic Spindle Assembly ◽

Chemical Inhibition ◽

Kinesin Eg5 ◽

Interfering Rna

Mitotic spindle assembly and organization require forces generated by motor proteins. The activity of these motors is regulated by nonmotor adaptor proteins. However, there are limited studies reporting the functional importance of adaptors on the balance of motor forces and the promotion of faithful and timely cell division. Here we show that genomic deletion or small interfering RNA silencing of the nonmotor adaptor Hmmr/HMMR disturbs spindle microtubule organization and bipolar chromosome–kinetochore attachments with a consequent elevated occurrence of aneuploidy. Rescue experiments show a conserved motif in HMMR is required to generate interkinetochore tension and promote anaphase entry. This motif bears high homology with the kinesin Kif15 and is known to interact with TPX2, a spindle assembly factor. We find that HMMR is required to dampen kinesin Eg5-mediated forces through localizing TPX2 and promoting the formation of inhibitory TPX2-Eg5 complexes. In HMMR-silenced cells, K-fiber stability is reduced while the frequency of unattached chromosomes and the time needed for chromosome segregation are both increased. These defects can be alleviated in HMMR-silenced cells with chemical inhibition of Eg5 but not through the silencing of Kif15. Together, our findings indicate that HMMR balances Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregation.

Download Full-text

Regulation of mitotic spindle assembly factor NuMA by Importin-β

The Journal of Cell Biology ◽

10.1083/jcb.201705168 ◽

2017 ◽

Vol 216 (11) ◽

pp. 3453-3462 ◽

Cited By ~ 18

Author(s):

Chih-Chia Chang ◽

Tzu-Lun Huang ◽

Yuta Shimamoto ◽

Su-Yi Tsai ◽

Kuo-Chiang Hsia

Keyword(s):

Mitotic Spindle ◽

Molecular Mechanisms ◽

Spindle Assembly ◽

Mitotic Apparatus ◽

Microtubule Binding ◽

Binding Function ◽

C Terminus ◽

Importin Α ◽

Mitotic Spindle Assembly ◽

Guanosine Triphosphatase

Ran–guanosine triphosphatase orchestrates mitotic spindle assembly by modulation of the interaction between Importin-α/-β and spindle assembly factors (SAFs). The inhibition of SAFs performed by importins needs to be done without much sequestration from abundant nuclear localization signal (NLS) –containing proteins. However, the molecular mechanisms that determine NLS-binding selectivity and that inhibit activity of Importin-β–regulated SAFs (e.g., nuclear mitotic apparatus protein [NuMA]) remain undefined. Here, we present a crystal structure of the Importin-α–NuMA C terminus complex showing a novel binding pattern that accounts for selective NLS recognition. We demonstrate that, in the presence of Importin-α, Importin-β inhibits the microtubule-binding function of NuMA. Further, we have identified a high-affinity microtubule-binding region that lies carboxyl-terminal to the NLS, which is sterically masked by Importin-β on being bound by Importin-α. Our study provides mechanistic evidence of how Importin-α/-β regulates the NuMA functioning required for assembly of higher-order microtubule structures, further illuminating how Ran-governed transport factors regulate diverse SAFs and accommodate various cell demands.

Download Full-text