Proteomic identification of NuMA Protein Interactions With the Kinesen Eg5

The nuclear mitotic apparatus protein (NuMA) plays a role in spindle fiber assembly, which in turn aids in the alignment of chromosomes during mitosis. During interphase, NuMA is localized in the nucleus but relocates to the spindle poles during early mitosis.¹ Experiments in Xenopus laevis have shown that NuMA protein functions to focus microtubules toward the mitotic spindle and tether microtubules to centrosomes.²

In their publication, Iwakiri et al.³ performed a proteomic analysis of mitotic cells to identify proteins that interact with NuMA and function together during mitosis. These results were validated by in vivo and in vitro binding studies.

Human NuMA protein was tagged with FLAG and expressed in Freestyle HEK-293F cells. After cells were arrested, FLAG-NuMA was precipitated and separated with SDS-PAGE. The stained gel was sliced, and proteins were digested with trypsin for LC-MS/MS on a Finnigan LTQ mass spectrometer (Thermo Scientific).

MS data revealed Eg5 as a potential novel NuMA-binding protein. Eg5 has been previously identified as a member of the kinesin-5 family of motor proteins.⁴ Eg5 is distributed along the spindles and functions to slide microtubules, which pushes centrosomes apart during mitosis.⁵

To further validate the interactions between NuMA and Eg5, NuMA protein and Eg5 were ectopically expressed in a coimmunoprecipitation assay and with HA-tagged LGN, which has been previously identified as a NuMA-binding protein.

Eg5 was successfully coprecipitated with anti-NuMA antibodies in synchronized HeLa cells but did not interact when cells were unsynchronized. Further immunoprecipitation assays led to the discovery that Eg5 binds to NuMA between the N-terminal motor domain and the C-terminal tail. Experiments to inhibit Eg5 revealed that it is required for accumulation of NuMA at the spindle poles, thereby leading proper spindle assembly and chromosome alignment. Further investigation is required to determine the exact mechanism behind the relocation of NuMA from the nucleus to the spindle poles and what role Eg5 and possibly additional proteins may play in that process.

References

1. Compton, D.A., and Cleveland, D.W. (1993) ‘NuMA is required for proper completion of mitosis‘, Journal of Cell Biology, 120 (4), (pp. 947-957)

2. Merdes, A., et al., (1996) ‘A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly‘, Cell, 87 (3), (pp. 447-458)

3. Iwakiri, Y., et al., (2013) ‘Interaction of NuMA protein with the kinesin Eg5: its possible role in bipolar spindle assembly and chromosome alignment‘, The Biochemical Journal, published online February 1, 2013. doi: 10.1042/BJ20121447

4. Ferenz, N.P., Gable, A., and Wadsworth, P. (2010) ‘Mitotic functions of kinesin-5‘, Seminars in Cell and Developmental Biology, 21 (3), (pp. 255-259)

5. Kapitein, L.C., et al., (2005) ‘The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks‘, Nature, 435 (7038), (pp. 114-118)