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MBC in Press, published online ahead of print February 20, 2008
Mol. Biol. Cell 10.1091/mbc.E07-10-1050

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Submitted on October 19, 2007
Revised on February 1, 2008
Accepted on February 8, 2008

Localized RanGTP Accumulation Promotes Microtubule Nucleation at Kinetochores in Somatic Mammalian Cells

Liliana Torosantucci, Maria De Luca, Giulia Guarguaglini, Patrizia Lavia, and Francesca Degrassi

IBPM Institute of Molecular Biology and Pathology, CNR National Research Council, c/o University of Rome "La Sapienza", 00185 Rome, Italy

Monitoring Editor: Stephen Doxsey

Centrosomes are the major sites for microtubule nucleation in mammalian cells, although both chromatin- and kinetochore-mediated microtubule nucleation have been observed during spindle assembly. As yet, it is still unclear whether these pathways are coregulated and the molecular requirements for microtubule nucleation at kinetochore are not fully understood. This work demonstrates that kinetochores are initial sites for microtubule nucleation during spindle reassembly after nocodazole. This process requires local RanGTP accumulation concomitant with delocalization from kinetochores of the hydrolysis factor RanGAP1. Kinetochore-driven microtubule nucleation is also activated after cold-induced microtubule disassembly when centrosome nucleation is impaired, e.g., after Polo-like kinase 1 depletion, indicating that dominant centrosome activity normally masks the kinetochore-driven pathway. In cells with unperturbed centrosome nucleation, defective RanGAP1 recruitment at kinetochores after treatment with the Crm1 inhibitor leptomycin B activates kinetochore microtubule nucleation after cold. Finally, nascent microtubules associate with the RanGTP regulated microtubule-stabilizing protein HURP in both cold- and nocodazole-treated cells. These data support a model for spindle assembly in which RanGTP-dependent abundance of nucleation/stabilization factors at centrosomes and kinetochores orchestrates the contribution of the two spindle assembly pathways in mammalian cells. The complex between RanGTP, the export receptor Crm1 and NES-bearing proteins regulates microtubule nucleation at kinetochores.

Address correspondence to: Francesca Degrassi (f.degrassi{at}caspur.it)






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