In Vivo Dynamics ofDrosophilaNuclear Envelope Components

Abstract

Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells. INTRODUCTIONIn eukaryotes, the nuclear envelope (NE) defines the limits between the nucleus and the cytoplasm. The outer NE membrane is considered to be structurally and functionally part of the endoplasmic reticulum network, whereas the inner membrane, with its distinct protein composition, provides anchoring points for the chromatin and nuclear lamina. Nuclear pore complexes (NPCs) are embedded at the points of fusion between the inner and outer NE membrane and represent the sole channels of transport across the NE. NPCs are composed of multiple copies of ϳ30 different proteins termed nucleoporins (Nups), most of which are organized into subcomplexes that associate with each other to build up the mature NPCs (for reviews, see Hetzer et al., 2005;Schwartz, 2005;Lim and Fahrenkrog, 2006;Tran and Wente, 2006). During cell division, the NE and NPCs are subjected to major rearrangements. However, the extent to which the NE and NPCs disassemble at mitotic entry varies among organisms (for reviews, see Margalit et al., 2005;Prunuske and Ullman, 2006). Unlike in most yeast and fungi, characterized by a "closed mitosis," NE disassembly is required in animal cells to allow spindle microtubule access to chromosomes. In vertebrates, cell division leads to complete NE breakdown at the prophase-prometaphase transition. During this "open mitosis," integral membrane proteins of the NE and the soluble subcomplexes of the NPCs redistribute throughout the endoplasmic reticulum and the mitotic cytoplasm (for reviews, see Hetzer et al., 2005;Margalit et al., 2005;Prunuske and Ullman, 2006). In Drosophila and Caenorhabditis elegans embryos, however, the NE only partially disassembles near spindle poles in early mitosis. NPCs disassemble during prometapha...

A mitotic role for Mad1 beyond the spindle checkpoint

“…All were fully functional, rescuing in a single copy the lethality, the rough eyes and the mitotic defects of mad1 1 homozygotes described below (supplementary material Table S1 and data not shown). The fluorescently tagged mad1 transgene recapitulated the distribution of Mad1 previously described using antibodies in Drosophila (Katsani et al, 2008;Lince-Faria et al, 2009) and has a similar distribution to that seen in mammalian cells. Mad1 associates with nuclear envelope and nucleoplasm during interphase, but localizes to kinetochores at mitotic entry.…”

“…Western blots of mad1 1 mutant and transgenic mad1 variants were performed as previously described (Buffin et al, 2007) using mouse anti-Mad1 (Katsani et al, 2008). In brief, protein extracts from 50 to 60 brains of wild type, mad1-KAPA and GFP-mad1 (each expressed in the mad1 1 mutant background), and mad1 1 homozygous or mad1 1 /Df(2R)45-30n third instar larvae were loaded onto 10% SDSacrylamide gels and electroblotted to nitrocellulose membrane.…”

The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis

“…Antibodies were used at the following dilutions: mouse anti-Orb 6H4, 1:100 (purified IgG, Developmental Studies Hybridoma Bank, University of Iowa, IA, USA); mAb414, 1:400 (Covance); mouse anti-GFP, 1:200 (Roche, Basel, Switzerland); mouse anti-Mtor, 1:100 (Qi et al, 2004); rabbit anti-dmNup153, 1:200 (Dimaano et al, 2001); and rabbit anti-dmNup107, 1:1000 (Katsani et al, 2008). Secondary antibodies were anti-rabbit and anti-mouse conjugated to Alexa Fluor 594 and Alexa Fluor 488 (Molecular Probes-Invitrogen) and used at 1:1000.…”

“…Moreover, recent evidence indicates that in HeLa cells and Xenopus egg extracts, the Nup107-160 complex mediates microtubule nucleation at kinetochores via its interaction with the -TuRC complex (Mishra et al, 2010). Unlike in other metazoans, in Drosophila Nup107 fails to localize to kinetochores at mitosis but is found concentrated in the spindle region (Katsani et al, 2008). In summary, the Nup107-160 complex is multifunctional, with roles in both nucleocytoplasmic transport and cell cycle regulation.…”

A novel function of Rab5 in mitosis

“…2 Notably, in the fruit fly, the nuclear membrane never disassembles completely but it fenestrates allowing spindle microtubules access to chromosomes in what is defined a "semi-open" mitosis. 11 Similarly, also in Caenorabditis elegans nuclear envelope breakdown occurs very late, compared with vertebrates, fully disassembling only during mid-late anaphase.…”