Megator, an Essential Coiled-Coil Protein that Localizes to the Putative Spindle Matrix during Mitosis inDrosophila

Abstract

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH 2 -terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH 2 -terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

“…This seems to be an evolutionarily conserved interaction, because small interfering RNA-induced depletion of Tpr (the vertebrate orthologue of yeast Mlp1/2-Drosophila Mtor) in HeLa cells significantly reduces Mad2 labeling at the NE (our unpublished data). Our in vivo study further revealed that in syncytial embryos, a fraction of GFP-Mad2 is found within the spindle area in metaphase and subsequently localizes between the two reforming nuclei during telophase ( Figure 7), localizations similar to that of Mtor (Qi et al, 2004). It is thus conceivable that an interaction between Mad1-Mad2 and Mtor may also occur at this stage of mitosis.…”

“…Drosophila NPC disassembly was completed by metaphase and preceded lamina depolymerization, a property shared by C. elegans embryos (Lee et al, 2000). Compared with Nup107, ketel GFP (which, as discussed above, likely interacts with peripheral FG-Nups), as well as Nup153 and Mtor (the Drosophila orthologue of Tpr; Qi et al, 2004), two asymmetrically localized nuclear basket Figure 8. Association of Mad2 with the NPCs at the end of mitosis is a two-step process.…”

“…In budding and fission yeasts, the NE localization of Mad2 relies on Mad1 (Iouk et al, 2002;Ikui et al, 2002) and in Saccharomyces cerevisiae, the basket nucleoporins Mlp1/2 were further demonstrated to be major binding sites for Mad1 at NPCs (Scott et al, 2005). This prompted us to analyze the behavior of Drosophila Mad1 (CG2072, listed in Flybase as TXBP181-like) and Mtor (Megator, the Drosophila orthologue of S. cerevisiae Mlp1/2 and vertebrate Tpr; Qi et al, 2004) in fixed mRFP-Nup107 Drosophila em-bryos. As observed for GFP-Mad2, and unlike mRFPNup107 or Mtor, Mad1 was mainly nuclear in early embryos, and its NE localization only became apparent in late preblastoderm or cellularized embryos ( Figure 9A, compare top and bottom panels, and Figure 9B).…”

“…In prometaphase, a fraction of Nup107 was still detectable at the NE, whereas Mad1 and Mtor were localized within the spindle area ( Figure 9B, bottom, brackets). In metaphase, Mtor displayed its typical localization at the spindle matrix (Qi et al, 2004), and a fraction of Mad1 also persisted within the spindle area, whereas mRFP-Nup107 had a more diffuse localization throughout the cell ( Figure 9B, top, brackets). On NE reassembly, Mad1 was first excluded from the reforming nuclei, even from those already ringed with an apparently continuous mRFP-Nup107 signal (arrowheads in Figure 9B).…”

Comparative interactomics provides evidence for functional specialization of the nuclear pore complex

“…5 Additionally, they have retained similar functions to those in other eukarya, by creating heterochromatin free zones around nuclear pores that are evident by electron microscopy in all eukaryotic nuclei, as well as recapitulating interactions with the spindle organizer. 4,38,[79][80][81] Thus, in conjunction with divergent lamin-like proteins, 35,82 and unconventional kinetochores, 83 it seems that the trypanosome nucleus uses unique protein complexes in parallel with conserved core elements such as the spindle microtubules and outer kinetochore components, Ndc80/Nuf2 to facilitate trypanosome biology. 84,85 Did trypanosomes retain an ancient symmetric assembly or reconfigure their molecular biology through the loss of introns and individual gene promoters (Fig.…”