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A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization

Nature Cell Biology volume 3, pages 1069–1075 (2001)Cite this article

Abstract

Asymmetric cell division requires the orientation of mitotic spindles along the cell-polarity axis. In Drosophila neuroblasts, this involves the interaction of the proteins Inscuteable (Insc) and Partner of inscuteable (Pins). We report here that a human Pins-related protein, called LGN, is instead essential for the assembly and organization of the mitotic spindle. LGN is cytoplasmic in interphase cells, but associates with the spindle poles during mitosis. Ectopic expression of LGN disrupts spindle-pole organization and chromosome segregation. Silencing of LGN expression by RNA interference also disrupts spindle-pole organization and prevents normal chromosome segregation. We found that LGN binds the nuclear mitotic apparatus protein NuMA, which tethers spindles at the poles, and that this interaction is required for the LGN phenotype. Anti-LGN antibodies and the LGN-binding domain of NuMA both trigger microtubule aster formation in mitotic Xenopusegg extracts, and the NuMA-binding domain of LGN blocks aster assembly in egg extracts treated with taxol. Thus, we have identified a mammalian Pins homologue as a key regulator of spindle organization during mitosis.

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Figure 1: Expression and subcellular localization of LGN.
Figure 2: Effects of LGN overexpression on microtubule organization in mitotic MDCK cells.
Figure 3: LGN interacts directly with NuMA.
Figure 4: LGN and NuMA interact in vivo.
Figure 5: Effects of LGN on NuMA localization in MDCK cells.
Figure 6: Knockdown of endogenous LGN in HeLa cells by RNAi causes mitotic defects.
Figure 7: LGN negatively regulates aster formation in Xenopus mitotic egg extracts.

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Acknowledgements

We thank G. Post (University of Kentucky) for the human LGN cDNA, J. Casanova (University of Virginia) for the MDCK T23 cell line, Duane Compton (Dartmouth Medical School) for anti-NuMA antibodies and NuMA cDNA, and members of the Macara group for reagents and helpful discussions. We also thank T. Tuschl for information on designing and using siRNA duplexes. This work was supported by grant CA40042 from the NIH, DHHS.

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Authors and Affiliations

  1. Centre for Cell Signalling and Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, 22908, Virginia, USA

    Quansheng Du & Ian G. Macara

  2. Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, 22908, Virginia, USA

    P. Todd Stukenberg

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Correspondence to Quansheng Du.

Supplementary information

Supplementary Figures

Figure S1 Effects of LGN overexpression on microtubule organization in mitotic HeLa cells. (PDF 397 kb)

Figure S2 Ectopically expressed Myc–LGN-N(1′340) co-localizes with NuMA in interphase cell nuclei and disrupts the normal distribution of NuMA.

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Du, Q., Stukenberg, P. & Macara, I. A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization. Nat Cell Biol 3, 1069–1075 (2001). https://doi.org/10.1038/ncb1201-1069

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