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First published online 23 November 2004
doi: 10.1242/jcs.01568
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Research Article |
1 Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA
2 Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA
3 Center for Cell Signaling, Box 800977, HSC, University of Virginia, Charlottesville, VA 22908, USA
Author for correspondence (e-mail: duane.a.compton{at}dartmouth.edu)
Accepted 29 September 2004
The large coiled-coil protein NuMA plays an essential role in organizing microtubule minus ends at spindle poles in vertebrate cells. Here, we use both in vivo and in vitro methods to examine NuMA dynamics at mitotic spindle poles. Using fluorescence recovery after photobleaching, we show that an exogenously expressed green-fluorescent-protein/NuMA fusion undergoes continuous exchange between soluble and spindle-associated pools in living cells. These dynamics require cellular energy and display an average half-time for fluorescence recovery of 
3 minutes. To explore how NuMA dynamics at spindle poles is regulated, we exploited the association of NuMA with microtubule asters formed in mammalian mitotic extracts. Using a monoclonal antibody specific for human NuMA, we followed the fate of human NuMA associated with microtubule asters upon dilution with a hamster mitotic extract. Consistent with in vivo data, this assay shows that NuMA can be displaced from the core of pre-assembled asters into the soluble pool. The half-time of NuMA displacement from asters under these conditions is 5 minutes. Using this assay, we show that protein kinase activity and the NuMA-binding protein LGN regulate the dynamic exchange of NuMA on microtubule asters. Thus, the dynamic properties of NuMA are regulated by multiple mechanisms including protein phosphorylation and binding to the LGN protein, and the rate of exchange between soluble and microtubule-associated pools suggests that NuMA associates with an insoluble matrix at spindle poles.
Key words: NuMA, Mitotic Spindle, Kinetochore, Microtubule, Centrosome
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