Multiple mechanisms regulate NuMA dynamics at spindle poles

Abstract

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.

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

“…Scale bar, 5 mm. DOI: https://doi.org/10.7554/eLife.29328.005 pDsRed-N1, Clontech, gift from T. Schroer, Johns Hopkins University) (Quintyne and Schroer, 2002); mCherry-p50 (chicken p50 in mCherry-C1, Clontech, gift from M. Moffert and T. Schroer, Johns Hopkins University) (Shrum et al, 2009); GFP-NuMA (human NuMA in pEGFP-N1, Clontech, gift from D. Compton, Dartmouth Medical School) (Kisurina-Evgenieva et al, 2004); GFP-CAM-SAP1 (human CAMSAP1 in pEGFP-C1, Clontech, gift from A. Akhmanova, Utrecht University) .…”

Resistance to inhibitors of cholinesterase 8A catalyzes release of Gαi-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA/LGN/Gαi-GDP complexes

“…G␣i and NuMA bind LGN cooperatively in vivo, and stabilization of microtubules by Reactions rates (Kobs) were calculated from single exponential rate equations generated by using the ORIGIN 6.0 (Microcal Software) curve-fitting program. NuMA is precluded when NuMA is bound to LGN (13,14,20,21). Thus, dynamic release of NuMA from LGN at the sites of aster microtubule anchorage (the plasma membrane and͞or centrosomes) may be the mechanism to regulate aster microtubule forces during cell division.…”

“…LGN recruits NuMA to the plasma membrane and pericentriolar regions during mitosis (20,21). Binding of NuMA and G␣i-GDP to LGN is cooperative, and NuMA is unable to interact with microtubules when bound to LGN (20,21).…”

“…Binding of NuMA and G␣i-GDP to LGN is cooperative, and NuMA is unable to interact with microtubules when bound to LGN (20,21). Thus, the potential of NuMA to regulate aster microtubule pulling forces may be realized by dynamic binding and release of NuMA from LGN (13,21). This regulation could be achieved by Ric-8A-stimulated activation of G␣ and concomitant dissociation of GTP-G␣ and NuMA from LGN.…”

Automated Motile Cell Capture and Analysis with Optical Traps

“…FRAP has also found tremendous utility in the study of nuclear and chromosome dynamics, in part due to the stable nature of some protein scaVolds found near or on chromatin such as chromosomes and kinetochores (Gerlich et al, 2006;Howell et al, 2000;Shah et al, 2004), DNA damage machinery (Bekker-Jensen et al, 2005) and nucleoli (Chen and Huang, 2001), and other more transiently bound nuclear components (Misteli et al, 2000). Similarly, studies related to the cytoskeleton (Bulinski et al, 2001;Pearson et al, 2003) and cytoskeletal-associated structures (Khodjakov and Rieder, 1999;Kisurina-Evgenieva et al, 2004;von Wichert et al, 2003) use FRAP as a central methodology to investigate dynamics of protein association and turnover.…”