Multiple mechanisms regulate NuMA dynamics at spindle poles.
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 approximately 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 approximately 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.
Kisurina-Evgenieva
Olga
O
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
Mack
Gary
G
Du
Quansheng
Q
Macara
Ian
I
Khodjakov
Alexey
A
Compton
Duane A
DA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D034961:Antigens, Nuclear; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism; D006224:Cricetinae; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D018386:Kinetochores / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D013997:Time Factors
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C102754:GPSM2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins