The nucleoporin MEL-28 promotes RanGTP-dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation

Abstract

The GTP-bound form of the Ran GTPase (RanGTP), produced around chromosomes, drives nuclear envelope and nuclear pore complex (NPC) re-assembly after mitosis. The nucleoporin MEL-28/ELYS binds chromatin in a RanGTP-regulated manner and acts to seed NPC assembly. Here we show that, upon mitotic NPC disassembly, MEL-28 dissociates from chromatin and re-localizes to spindle microtubules and kinetochores. MEL-28 directly binds microtubules in a RanGTP-regulated way via its C-terminal chromatin-binding domain. Using Xenopus egg extracts, we demonstrate that MEL-28 is essential for RanGTP-dependent microtubule nucleation and spindle assembly, independent of its function in NPC assembly. Specifically, MEL-28 interacts with the g-tubulin ring complex and recruits it to microtubule nucleation sites. Our data identify MEL-28 as a RanGTP target that functions throughout the cell cycle. Its cell cycle-dependent binding to chromatin or microtubules discriminates MEL-28 functions in interphase and mitosis, and ensures that spindle assembly occurs only after NPC breakdown.

Nuclear pore proteins and the control of genome functions

“…Consistently, Elys/Mel-28 was identified as a dual Nup/ kinetochore protein, and its depletion leads to severe defects in spindle assembly and chromosome segregation as well (Fernandez and Piano 2006;Galy et al 2006;Rasala et al 2006). At the molecular level, Nup107/Nup160 components and Elys were shown to recruit the microtubule nucleator g-tubulin ring complex (gTuRC) to unattached kinetochores to drive microtubule nucleation (Mishra et al 2010;Yokoyama et al 2014). All of the Nups of this complex could cooperate to promote proper spindle assembly, although it has also been proposed to be a predominant role for Elys (Yokoyama et al 2014).…”

“…At the molecular level, Nup107/Nup160 components and Elys were shown to recruit the microtubule nucleator g-tubulin ring complex (gTuRC) to unattached kinetochores to drive microtubule nucleation (Mishra et al 2010;Yokoyama et al 2014). All of the Nups of this complex could cooperate to promote proper spindle assembly, although it has also been proposed to be a predominant role for Elys (Yokoyama et al 2014). In addition, the Nup107/Nup160 complex might also regulate mitotic progression by controlling the spatial positioning of the chromosome passenger complex (CPC) (Platani et al 2009).…”

A Nucleoporin Docks Protein Phosphatase 1 to Direct Meiotic Chromosome Segregation and Nuclear Assembly

“…Recent work suggests that dynein on chromosomes may cause them to associate with microtubule minus ends (Muscat et al, 2015); another possibility, based on work in frog egg extracts (Yokoyama et al, 2014), is that MEL-28 itself links chromosomes to microtubules. More importantly, the mechanisms that elongate the spindle to separate homologous chromosomes remain unclear.…”

“…Using immunodepletions, extract experiments revealed a role for Y-complex proteins in spindle assembly (Mishra et al, 2010; Orjalo et al, 2006; Yokoyama et al, 2014), However, the spindles formed in these extracts do not perform robust chromosome segregation, which has limited analysis of kinetochore functions. RNA interference (RNAi) analysis in human cells, while complicated by potential indirect consequences of affecting nucleocytoplasmic transport and the extremely low turnover rate of core nuclear pore proteins (Toyama et al, 2013), has implicated Y-complex subunits in kinetochore composition, kinetochore-microtubule attachment strength, and chromosome segregation fidelity (Mishra et al, 2010; Platani et al, 2009; Ródenas et al, 2012; Zuccolo et al, 2007).…”

Nucleosome functions in spindle assembly and nuclear envelope formation

“…Nuclear pore complex proteins contribute to spindle function Several nucleoporins have secondary functions that connect NPCs with mitotic spindles. Both ELYS and the Nup107 complex can be detected in an interdependent manner at kinetochores and spindle poles [107][108][109][110], and seem to regulate both kinetochore and spindle function in a species-specific manner. During chromosome-induced spindle assembly in X. laevis egg extracts, immunodepletion of either the Nup107 complex or ELYS causes severely defective spindle assembly [110][111][112].…”