Analysis of Regulation of Major Mitotic Assembly Events In Vitro by Nuclear Import and Export Receptors

This dissertation is an analysis of regulation of major mitotic assembly events by nuclear transport receptors, or karyopherins. Karyopherins, composed of both importins and exportins, are a large family of proteins that transport specific proteins and RNAs between the cytoplasm and nucleus of the eukaryotic cell. Each karyopherin has a distinct set of cargoes that it binds and transports through the nuclear pore complex. A high concentration of RanGTP inside the nucleus, created by the chromatin-bound enzyme RCC1, is harnessed by karyopherins to achieve transport directionality. During mitosis, upon nuclear breakdown, RCC1 continues to produce a high concentration of RanGTP around the mitotic chromosomes. Away from the chromosomes, where RanGTP is low, Importins α and β inhibit assembly factors for spindle, nuclear membrane, and nuclear pore assembly. However, near the chromosomes, where RanGTP levels are high, Importins α and β release assembly factors with the result that mitotic structures only assemble around the chromosomes. Thus, evolution has co-opted the RanGTP/karyopherin system to spatially regulate the assembly of multiple critical structures in mitosis. Much of this has been determined in Xenopus egg extracts, which are unique in that one can reconstitute cellular events in cell cycle-specific states. Chapter 2 extends the role of RanGTP/karyopherin spatial regulation by dissecting the mechanism of action by which the import receptor Transportin regulates mitotic assembly events. Using a variety of molecular tools and Xenopus egg extracts, we show that Transportin inhibits the assembly of major mitotic structures, not by titrating RanGTP, but by directly blocking proteins needed for assembly everywhere except in areas near mitotic chromosomes.Chapter 3 presents an extensive published review of karyopherins as global regulators of mitotic assembly events, both in vivo and in vitro, including protein interactions, checkpoints, and critical proteins required for proper assembly.Chapter 4 dissects whether export receptors, or exportins, play a role in mitotic assembly regulation, focusing on Crm1/Exportin-1, Exportin-tRNA, and Exportin-5. Using Xenopus egg extracts, we find that exportins are potent inhibitors of spindle assembly, nuclear membrane fusion, and nuclear pore formation, and their inhibition can be, to an extent, counteracted by RanGTP.