Dataset Information

Regulation of mitotic spindle assembly factor NuMA by Importin-?.

ABSTRACT: Ran-guanosine triphosphatase orchestrates mitotic spindle assembly by modulation of the interaction between Importin-?/-? 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-?-regulated SAFs (e.g., nuclear mitotic apparatus protein [NuMA]) remain undefined. Here, we present a crystal structure of the Importin-?-NuMA C terminus complex showing a novel binding pattern that accounts for selective NLS recognition. We demonstrate that, in the presence of Importin-?, Importin-? 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-? on being bound by Importin-?. Our study provides mechanistic evidence of how Importin-?/-? 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.

SUBMITTER: Chang CC

PROVIDER: S-EPMC5674899 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Regulation of mitotic spindle assembly factor NuMA by Importin-β.

Chang Chih-Chia CC Huang Tzu-Lun TL Shimamoto Yuta Y Tsai Su-Yi SY Hsia Kuo-Chiang KC

The Journal of cell biology 20170922 11

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]) rema ...[more]

PMID: 28939615

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Project description:BackgroundThe Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains.ResultsHere we show that RhoA is a substrate for ARHGEF10. In both G1/S and M phases, ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa cells. Furthermore, RNA interference-based knockdown of ARHGEF10 resulted in multipolar spindle formation in M phase. Each spindle pole seems to contain a centrosome consisting of two centrioles and the pericentriolar material. Downregulation of RhoA elicited similar phenotypes, and aberrant mitotic spindle formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively activated RhoA. Multinucleated cells were not increased upon ARHGEF10 knockdown in contrast to treatment with Y-27632, a specific pharmacological inhibitor for the RhoA effector kinase ROCK, which induced not only multipolar spindle formation, but also multinucleation. Therefore, unregulated centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10 knockdown-dependent multipolar spindle formation. We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10. Knockdown of KIF3B again caused multipolar spindle phenotypes. The supernumerary centrosome phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of ARHGEF10, RhoA or KIF3B was abrogated by RNA interference.ConclusionCollectively, our results suggest that a novel RhoA-dependent signaling pathway under the control of ARHGEF10 has a pivotal role in the regulation of the cell division cycle. This pathway is not involved in the regulation of cytokinesis, but instead may regulate centrosome duplication. The kinesin-like motor protein KIF3B may modulate the ARHGEF10-RhoA pathway through the binding to ARHGEF10.

Dataset Information

Regulation of mitotic spindle assembly factor NuMA by Importin-?.

Publications

Regulation of mitotic spindle assembly factor NuMA by Importin-β.

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