InteractiveFly: GeneBrief

Megator: Biological Overview | References

Gene name - Megator

Synonyms -

Cytological map position - 48C5-48C5

Function - signaling

Keywords - regulator of mitotic checkpoint, recruitments Mad2 and Mps1 to unattached kinetochores at entry into mitosis, regulation of cytoskeleton

Symbol - Mtor

FlyBase ID: FBgn0013756

Genetic map position - 2R:7,740,124..7,748,364 [+]

Classification - MLP2-like protein, chromosome segregation protein SMC

Cellular location - nuclear and mitotic spindle

NCBI link: EntrezGene
Mtor orthologs: Biolitmine

Recent literature
Liu, Y., Singh, S. R., Zeng, X., Zhao, J. and Hou, S. X. (2015). The nuclear matrix protein Megator regulates stem cell asymmetric division through the mitotic checkpoint complex in Drosophila testes. PLoS Genet 11: e1005750. PubMed ID: 26714316
In adult Drosophila testis, asymmetric division of germline stem cells (GSCs) is specified by an oriented spindle and cortically localized adenomatous coli tumor suppressor homolog 2 (Apc2). However, the molecular mechanism underlying these events remains unclear. This study identified Megator (Mtor), a nuclear matrix protein, which regulates GSC maintenance and asymmetric division through the spindle assembly checkpoint (SAC) complex. Loss of Mtor function results in Apc2 mis-localization, incorrect centrosome orientation, defective mitotic spindle formation, and abnormal chromosome segregation that lead to the eventual GSC loss. Expression of mitotic arrest-deficient-2 (Mad2) and monopolar spindle 1 (Mps1) of the SAC complex effectively rescues the GSC loss phenotype associated with loss of Mtor function. Collectively these results define a new role of the nuclear matrix-SAC axis in regulating stem cell maintenance and asymmetric division.
Aleman, J. R., Kuhn, T. M., Pascual-Garcia, P., Gospocic, J., Lan, Y., Bonasio, R., Little, S. C. and Capelson, M. (2021). Correct dosage of X chromosome transcription is controlled by a nuclear pore component. Cell Rep 35(11): 109236. PubMed ID: 34133927
Dosage compensation in Drosophila melanogaster involves a 2-fold transcriptional upregulation of the male X chromosome, which relies on the X-chromosome-binding males-specific lethal (MSL) complex (see msl-2). However, how such 2-fold precision is accomplished remains unclear. This study shows that a nuclear pore component, Mtor, is involved in setting the correct levels of transcription from the male X chromosome. Using larval tissues, this study demonstrated that the depletion of Mtor results in selective upregulation at MSL targets of the male X, beyond the required 2-fold. Mtor and MSL components interact genetically, and depletion of Mtor can rescue the male lethality phenotype of MSL components. Using RNA fluorescence in situ hybridization (FISH) analysis and nascent transcript sequencing, this study found that the effect of Mtor is not due to defects in mRNA export but occurs at the level of nascent transcription. These findings demonstrate a physiological role for Mtor in the process of dosage compensation, as a transcriptional attenuator of X chromosome gene expression.


A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. This report shows that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. It is proposed that Mtor/Tpr functions as a spatial regulator of the SAC ensuring the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible 'wait anaphase' signal to the vicinity of the spindle. It is also suggested that the regulatory role of Mtor upon Mad2 is indirect and may be catalyzed by Mps1 (Lince-Faria, 2009).

The mitotic spindle is composed of dynamic microtubules (MTs) and associated proteins that mediate chromosome segregation during mitosis. The requirement of an additional stationary or elastic structure forming a spindle matrix where molecular motors slide MTs has long been proposed to power chromosome motion and account for incompletely understood features of mitotic spindle dynamics. However, definitive evidence for its existence in living cells or on its biochemical nature and whether it plays a direct role during mitosis has been missing (Lince-Faria, 2009).

A functional spindle matrix would be expected to (a) form a fusiform structure coalescent with spindle MTs, (b) persist in the absence of MTs, (c) be resilient in response to changes of spindle shape and length, and (d) affect spindle assembly and/or function if one or more of its components are perturbed. In Drosophila, a complex of at least four nuclear proteins, Skeletor, Megator (Mtor), Chromator, and EAST (enhanced adult sensory threshold), form a putative spindle matrix that persists in the absence of MTs in fixed preparations (Johansen, 2007). From this complex, Mtor is the only protein that shows clear sequence conservation with proteins in other organisms, such as the nuclear pore complex (NPC) protein translocated promoter region (Tpr) in mammals (Cordes, 1997; Zimowska, 1997), its respective counterparts Mlp1 and Mlp2 in yeast (Strambio-de-Castillia, 1999), and nuclear pore anchor in plants (Xu, 2007). NPC proteins, including Mtor/Tpr orthologues in yeast, have been shown to functionally interact with spindle assembly checkpoint (SAC) components (Iouk, 2002; Scott, 2005). The SAC ensures correct chromosome segregation by providing time for proper kinetochore (KT) attachments to spindle MTs while inhibiting the activity of the anaphase-promoting complex/cyclosome (Lince-Faria, 2009).

Assuming that any critical function by the spindle matrix is widely conserved, this study focused on understanding the mitotic role of Mtor in living Drosophila somatic cells. The results provide a new conceptual view of a spindle matrix not as a rigid structural scaffold but as a spatial determinant of key mitotic regulators (Lince-Faria, 2009).

Mtor localizes to a dynamic nuclear derived spindle matrix in living cells. To investigate the localization of Mtor in living cells, a Drosophila S2 cell line was generated stably coexpressing Mtor-mCherry and GFP-α-tubulin. Mtor-mCherry is nuclear in interphase and at nuclear envelope breakdown (NEB) reorganizes into a fusiform structure coalescent with spindle MTs. Mtor-mCherry shows a highly adaptable morphology in response to changes in spindle shape and dynamics throughout mitosis, which is inconsistent with a static structure. Similar to endogenous Mtor, Mtor-mCherry retracts and loses the fusiform shape upon MT depolymerization but is retained in a conspicuous milieu around chromosomes, suggesting that MTs exert a pushing force on the Mtor-defined matrix (Lince-Faria, 2009).

Previous electron microscopy analysis revealed the existence of a membranous network surrounding the spindle from prophase to metaphase in S2 cells (Maiato, 2006). This study used immunofluorescence to show that lamin B is not fully disintegrated at this stage. Similar results have recently been reported in living Drosophila embryos and neuroblasts, where a spindle envelope was proposed to limit the diffusion of nuclear derived Nup107 before anaphase (Katsani, 2008). To test whether this membranous network works as a diffusion barrier around the spindle, the dynamic behavior of Mtor-mCherry relative was compared to GFP-α-tubulin and a known MT-associated protein, Jupiter, upon colchicine addition. GFP-α-tubulin or Jupiter-GFP fluorescence is gradually lost from the spindle region with an equivalent gain in the cytoplasm. In contrast, Mtor-mCherry remains confined to the spindle region with no detectable fluorescence gain in the cytoplasm. These results argue against the existence of a diffusion barrier around the metaphase spindle in Drosophila S2 cells and suggest that Mtor is being selectively retained in this region (Lince-Faria, 2009).

FRAP was used to shed light on the dynamic properties of Mtor. In interphase nuclei, there is ~50% recovery of fluorescence in the bleached region with an equivalent loss from a similar unbleached region and undetectable cytoplasmic exchange, suggesting that Mtor in the nucleoplasm is mobile. In mitosis, FRAP of Mtor-mCherry in one half-spindle is mirrored by an equivalent loss of fluorescence from the unbleached half-spindle as if Mtor exchanges between half-spindles. However, this recovery was slower than in interphase nuclei and had a minor contribution from a cytoplasmic pool. In both interphase and mitosis, the recovery curves of Mtor-mCherry fitted a single exponential, suggesting affinity to a yet unidentified substrate, whereas GFP-α-tubulin in the spindle displayed biphasic recovery kinetics and best fit the sum of two exponentials as result of a rapid diffusion phase followed by a slower recovery phase associated with MT turnover. Finally, in S2 cells that sporadically form two spindles in the same cytoplasm, fluorescence exchange was found within the same spindle and from surrounding cytoplasm with no apparent loss from the neighboring unbleached spindle, supporting that Mtor is unable to exchange between two spindles located <10 µm apart. Collectively, these data indicate that Mtor is part of a dynamic, nuclear derived spindle matrix surrounded by a fenestrated membranous system containing lamin B and shows mobility properties that are distinct from MTs and associated proteins (Lince-Faria, 2009).

To address the mitotic role of Mtor, RNAi was used in Drosophila S2 cells stably coexpressing GFP-α-tubulin and the KT marker mCherry-centromere identifier (CID). Mtor-depleted cells show no major spindle defects but typically form a poorly defined metaphase plate as the result of progressing ~15% faster through mitosis when compared with controls. Such problems in completing chromosome congression are corrected if anaphase onset is delayed by treating cells with the proteasome inhibitor MG132. As in Mtor RNAi, S2 cells depleted of the SAC protein Mad2 undergo a faster mitosis. Moreover, Mtor-depleted cells show a lower mitotic index as well as a weakened response to MT depolymerization, suggesting that Mtor is required for proper SAC response (Lince-Faria, 2009).

Quantitative analysis of anaphase revealed a significant attenuation in the velocity of chromosome separation in Mtor-depleted cells by affecting spindle elongation. These results could be accounted for if Mtor is part of a structural scaffold where motor proteins assemble to generate force. However, an alternative hypothesis is that Mtor may function to provide the necessary time for proper maturation of a competent spindle. To test this, anaphase onset was delayed by treating Mtor-depleted cells with MG132, and half-spindle elongation velocity was measured after drug washout. No difference was found in half-spindle elongation velocity between Mtor RNAi and control cells treated with MG132. Additionally, half-spindle elongation in Mad2-depleted cells, which progresses faster through mitosis, was similar to Mtor-depleted cells, supporting the spindle maturation hypothesis (Lince-Faria, 2009).

To shed light on the role of Mtor in SAC response, the recruitment of Mad2 and BubR1 to unattached KTs after Mtor depletion was analyzed. It was found that although BubR1 was unaltered after Mtor depletion, Mad2 KT accumulation was significantly reduced. Decreased Mad2 levels at KTs explain why Mtor-depleted cells enter anaphase prematurely, presumably because it requires binding of fewer MTs to remove all Mad2 from KTs and satisfy the SAC, whereas residual Mad2 at KTs may be sufficient to produce a weakened response to colchicine (Lince-Faria, 2009).

Next, how Mtor regulates the recruitment of Mad2 to KTs in living S2 cells stably coexpressing GFP-α-tubulin and monomeric RFP (mRFP)-Mad2 was investigated. In interphase, mRFP-Mad2 is nuclear, accumulating at unattached KTs and spindles as cells transit into mitosis. The spindle accumulation of Mad2 is thought to result from dynein-dependent poleward transport as MTs attach to KTs. Interestingly, however, it was found that a distinct pool of mRFP-Mad2 localizes to a nuclear derived spindle matrix even when MTs have just started invading the nuclear space. A similar behavior has been observed in vertebrate cells, where GFP-Mad2 accumulates as an ill-defined nuclear derived matrix during early prometaphase after its initial recruitment to unattached KTs. Like Mtor, the retention of Mad2 in the spindle matrix is resistant to MT depolymerization, suggesting that spindle-associated Mad2 is not freely diffusible. mRFP-Mad2 remains associated with the spindle matrix in the absence of Mtor, but it is unable to accumulate at KTs even after MT depolymerization with colchicine. Stable expression of Mtor-mCherry (which is RNAi insensitive) rescues normal Mad2 localization at KTs after Mtor RNAi, indicating that the observed phenotype is specific and supporting that Mtor-mCherry is a functional protein. Lastly, Mtor depletion does not affect normal Mad2 expression levels and vice versa, which rules out unspecific effects of Mtor over Mad2 mRNA transport to the cytoplasm (Lince-Faria, 2009).

The colocalization of Mtor and Mad2 in the spindle matrix suggests that these proteins may interact. Indeed, Mad2 was found to coimmunoprecipitate with Mtor in lysates obtained from Drosophila embryos harvested between 0-3 h after egg laying. Given that Mtor does not specifically accumulate at KTs, this interaction might represent an important regulatory step for the subsequent recruitment of Mad2 to unattached KTs. Several proteins such as Mad1, Rod, Ndc80, or Mps1 are involved in recruiting Mad2 to unattached KTs (Musacchio, 2007). Although Mad1, Rod, and Ndc80 are effectively targeted to unattached KTs after Mtor depletion, Mps1 accumulation is significantly reduced. Mps1 kinase activity has been shown to be required to specifically target Mad2 but not Mad1 to unattached KTs in human cells (Tighe, 2008). To investigate whether the same regulatory role upon Mad2 is true in Drosophila, an mps1 kinase-dead (mps1KD) allele was generated by homologous recombination in flies. In agreement with the results in human cells, neuroblasts from mps1KD third instar larvae show reduced or undetectable Mad2 accumulation at KTs upon colchicine treatment. Collectively, these results support that the regulatory role of Mtor upon Mad2 is indirect and may be catalyzed by Mps1 (Lince-Faria, 2009).

Like Tpr, Mad1, Mad2, and Mps1 localize at the NPC during interphase in human cells. During mitosis, Tpr remains associated with the nuclear envelope until prometaphase. Moreover, a fraction of Tpr is associated with the mitotic spindle from late prometaphase until anaphase and is recruited to the reforming nuclear envelope during telophase. This confirms the previous identification of Tpr in isolated human mitotic spindles (Sauer, 2005), no enriched fraction of Tpr was dected that resists MT depolymerization with nocodazole, including KTs. To see whether Tpr has a conserved regulatory role in the recruitment of Mad2 to unattached KTs in human cells, RNAi was used to deplete Tpr in HeLa cells. Like in S2 cells, Tpr RNAi leads to reduced accumulation of Mad2 but not Mad1 to unattached KTs accompanied by a decrease in the normal mitotic index and a weakened SAC response in the presence of nocodazole. Tpr knockdown does not enrich for cells in G2 and slightly increases the number of cells in G1 (Loiodice, 2004), supporting that the lower mitotic index is not caused by the inability of cells to enter mitosis but rather reflects a faster exit. Moreover, Tpr, Mad1, Mad2, and Mps1 coimmunoprecipitate in mitotic enriched HeLa cell extracts prepared in the presence of nocodazole, extending the results obtained in Drosophila and reinforcing that this complex forms independently of MTs and an intact nuclear envelope. While this paper was under revision, Tpr was independently found to interact with Mad1 and Mad2 in human cells (Lee, 2008). In agreement with the current results, the authors propose that Tpr is important for controlling the SAC but reject the possibility that Tpr is playing a role in mitotic timing. However, quantification of the NEB to anaphase duration in Tpr-depleted cells does show a 25% acceleration of mitosis during this period (Lee, 2008). Finally, the results are not consistent with a model in which KT-associated Tpr serves as a docking place for Mad1 because Tpr (or Mtor) were not detected at KTs, including those that were positive for Mad1, and no impairment was found in Mad1 KT recruitment in Tpr- or Mtor-depleted cells (Lince-Faria, 2009).

Overall, the results support a model in which Mtor/Tpr acts as a spatial regulator of SAC, ensuring a timely and effective recruitment of Mad2 and Mps1 to unattached KTs as cells enter mitosis. In budding yeast, Mps1 phosphorylates Mad1, which is continuously recycled to KTs from Mlps at NPCs, but N-terminal deletion mutants of Mad1 lacking the Mlp-binding domain have a functional SAC. In humans and Drosophila, Mps1 regulates Mad2 but not Mad1 accumulation at KTs. Because Mad1 localization at KTs does not depend on Mlps/Mtor/Tpr and Mps1 kinase activity, the residual Mad2 at KTs after Mtor/Tpr RNAi possibly corresponds to the Mad1-bound fraction. One possibility is that Mps1 phosphorylation of Mad1 regulates the recruitment of a fast-exchanging pool of Mad2 to KTs. Parallelly, Mtor/Tpr may spatially regulate Mps1 autophosphorylation, which is important for its normal KT accumulation, together with Mad2. The presence of Mad2 in the complex may work as a positive feedback mechanism to ensure continuous Mps1 kinase activity upon SAC activation (Lince-Faria, 2009).

SAC proteins evolved from systems with a closed mitosis like budding yeast, where the spindle assembles inside an intact nuclear envelope into more complex systems like animals and plants, where the nuclear envelope is thought to fully or partially disintegrate during spindle formation, justifying the requirement of a nuclear derived spindle matrix for an effective SAC response. What retains matrix components around the spindle in systems where mitosis is thought to be open remains an intriguing question. In this regard, lamin B was proposed to tether several factors that mediate spindle assembly in Xenopus laevis egg extracts and possibly in human cells. Additionally, a continuous endoplasmic reticulum surrounding the mitotic spindle is thought to be recycled from the nuclear envelope after its disassembly and has been observed in many systems undergoing an open mitosis, including humans. Although such fenestrated membranous systems cannot work as diffusion barriers, it is possible that they indirectly help to generate local gradients or concentrate matrix-affine substrates. The enrichment of Mad2 in the spindle matrix provides an explanation for an unsolved SAC paradigm in which the 'wait anaphase' signal emanating from unattached KTs must be diffusible to prevent premature anaphase onset of already bioriented chromosomes but at the same time is known to be restricted to the vicinity of the spindle (Lince-Faria, 2009).

The proposed role of Mtor/Tpr further supports the necessity of spindle maturation for proper KT-MT attachments and anaphase spindle elongation in which the spindle matrix may help extend the duration of mitosis for the assembly of a competent chromosome segregation machinery. Mtor/Tpr-depleted cells have a weakened SAC response that, as opposed to complete checkpoint loss, may be compatible with cell viability and lead to cancer. The involvement of Tpr in the activation of several oncogenes may translate into an unfavorable combination that facilitates transformation and tumorigenesis in humans (Lince-Faria, 2009).

Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator

Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila Titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In Western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which was identified by mass spectrometry analysis as being titin. The spindle matrix proteins Skeletor, Megator and Chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein (Fabian, 2007).

Although no functional data is available on the role of spindle D-titin, several aspects of spindle physiology are congruent with known roles of D-titin. For one, a putative spindle matrix might provide flexibility and elasticity as an underlying component of spindles, and the matrix might even be involved in force production. The term 'putative' was used because there has been no definitive molecular or morphological description of a matrix, except that the Drosophila proteins Skeletor, Megator and Chromator would seem to be components of (or at least markers of) such a matrix. Skeletor, Megator and Chromator staining reveals the expected morphology, and Skeletor and Megator distribution maintain a spindle shape even after spindle microtubules have been depolymerized. Since titin has a major role in the elasticity of skeletal and cardiac muscle, and since it is closely associated with skeletor in the spindle, it is reasonable to think that Skeletor and D-titin function physiologically as part of a spindle matrix. Spindle actin and myosin are closely associated with D-titin and Skeletor, so they, too, might be part of such a matrix. The data suggest that the interactions among these proteins (D-titin, Skeletor, myosin and actin) and between these proteins and the spindle microtubules change during the course of cell division. Skeletor, for example, is present along the spindle fibers and in between them (the 'matrix') throughout the course of division, but it accumulates progressively in the kinetochore microtubule bundles as division proceeds towards anaphase. D-titin in chromosomes is present in high concentration in prophase but by metaphase and anaphase, the staining of the chromosomes is somewhat reduced, as determined by comparing their staining intensities with that of muscle fibers in the same preparations. In prophase, D-titin is present in the cytoplasmic asters. After nuclear membrane breakdown, as the development of the spindle proceeds towards anaphase, D-titin becomes increasingly concentrated at the poles and along kinetochore fibers with the staining along the kinetochore fibers eventually being as high as 80-85% the levels of staining observed in muscle fibers. Thus, it can be speculated that the interactions among these proteins develop and continue throughout prometaphase to culminate in a 'mature' matrix by metaphase (Fabian, 2007).

Another potential role for D-titin in spindles may be as part of the elastic 'tethers' extending between arms of separating half-bivalents in crane-fly spermatocytes. Evidence for these tethers comes from experiments showing that laser-severed chromosome arms or entire chromosomes move backwards across the equator. D-titin extends between the arms of separating half-bivalents, so it is reasonable to suggest that the tethers contain D-titin, and that D-titin is responsible for tether elasticity, at least in part (Fabian, 2007).

Another possible role is suggested by recent experiments in which titin modulates the velocity of actin filaments that slide along an HMM-coated surface, titin-actin interactions acting as a `viscous bumper mechanism' to slow the movements. One could envisage a similar effect on the velocity of chromosome movement because titin can interact with microtubules in addition to muscle proteins (Fabian, 2007).

The identification of D-titin in spindles, together with other muscle proteins, actin, myosin and zyxin, suggests that these proteins indeed function during mitosis, perhaps in a spindle matrix and in producing forces for chromosome movement. If the arrangements (and polarities) of these proteins in the spindle were known, their function might be better understood. With respect to D-titin, following several markers could, in theory, give information about how the molecules are arranged; since D-titin is so large, double staining with different antibodies could result in resolvable separation for epitopes reasonably far apart on the molecule. Separate dots were indeed found after staining with the three different antibodies. Because the staining is punctate, and the molecules are arranged in three dimensions and not just the two observed in the image, certainty about the arrangement of the individual D-titin molecules is not possible. Nonetheless, if it is true that two D-titin epitopes are 2 microm apart along the length of the kinetochore microtubules, but only 0.2 microm apart at an oblique angle, as seen in double stained images, this would suggest that the individual D-titin molecules may be arranged obliquely to the kinetochore microtubules rather than along them (Fabian, 2007).

The presence of D-titin in prophase nuclear membranes and in nascent membranes formed around the daughter nuclei in telophase, is consistent with the finding that D-titin is associates with and binds to C. elegans lamins (Fabian, 2007).

An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region

The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. This paper describes evidence for a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, this study demonstrates that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous 'spindle envelope' confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function (Schweizer, 2015).

The cytoplasm is more crowded than the nucleoplasm, meaning that there is more “free” space in the nucleus for nano-sized particles. When proteins like Megator, soluble tubulin, or Mad2, which are restricted to the nucleus or the cytoplasm during interphase, diffusively equilibrate in all available spaces within the cell after NEB, this consequently leads to an enrichment of these proteins in the less crowded spindle region. While other roles cannot be excluded, the spindle envelope might act as a selective barrier to impede the invasion of the spindle region by large organelles, while retaining Megator complexes that might show gel-like properties. Importantly, cyclin B/Cdk1 and many other mitotic regulators, such as Polo/Plk1, Mad2, and Fizzy/Cdc20, as well as soluble tubulin and chromosomes, accumulate in the spindle region during mitosis. This might facilitate interactions between chromosomes and MTs during spindle assembly, not only by providing a physical constraint for MT growth and chromosome distribution, but also by creating a biochemical milieu that favors spindle assembly. Importantly, a MT-independent organelle-exclusion system is conserved in human cells and might therefore represent a common strategy to control mitosis in space and time (Schweizer, 2015).

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila

Immunocytochemistry and cross-immunoprecipitation analysis was used 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 NH2-terminal coiled-coil region remains in the cytoplasm; however, it is capable of assembling into spherical structures. On the basis of these findings it is proposed that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex (Qi, 2004).

This study shows that the Bx34 antigen in addition to the previously reported localization to the extrachromosomal space and nuclear rim at interphase (Zimowska, 1997) also interacts with the putative spindle matrix proteins, Skeletor and Chromator, during mitosis. The organization of the Bx34 antigen with a large NH2-terminal coiled-coil domain and a shorter acidic COOH-terminal domain is similar to the structure of the mammalian Tpr (translocated promoter region) protein (Mitchell, 1992) and like Tpr the Bx34 antigen is found at the nuclear rim, likely in association with nuclear pore complexes (Zimowska, 1997). However, comparison of Tpr and the Bx34 antigen sequences show a very low level of identity on the amino acid level (Zimowska, 1997) and although the Bx34 antigen is abundant in the nuclear interior, mammalian Tpr is restricted to the nuclear periphery (Frosst, 2002). Furthermore, mammalian Tpr has not been observed to localize to the spindle at metaphase. Thus, although structurally similar, there is likely to be significant functional differences between the Bx34 antigen and mammalian Tpr, wherefore the Bx34 antigen in Drosophila has been named Megator (Qi, 2004).

The presence of a large coiled-coil domain in Megator raises the intriguing possibility that it could comprise the structural element of a potential spindle matrix. Because both Chromator and Skeletor localize to chromosomes as well as to the spindle-like structure, it was not clear whether the physical interactions observed in co-ip and pull-down experiments between these molecules reflected interactions in chromosomal complexes or interactions on the spindle-like structure or both (Rath, 2004). However, because Megator is not localized to the chromosomes during interphase or on centrosomes during metaphase through telophase, the molecular interaction of the complex observed likely occurs on the spindle-like structure. Interestingly, the Megator deletion construct analysis in S2 cells indicate that the NH2-terminal coiled-coil containing domain has the ability to selfassemble into spherical structures in the cytoplasm. This is in contrast to the acidic COOH-terminal domain, which is targeted to the nucleus, implying the presence of a functional nuclear localization signal. Furthermore, the COOH-terminal domain is sufficient for localization to the nuclear rim as well as for spindle localization. Thus, an attractive hypothesis is that the COOH-terminal domain functions as a targeting and localization domain, whereas the NH2-terminal domain may be responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex. Supporting this notion is the finding that Megator spindles persist in the absence of microtubules depolymerized by cold or nocodazole treatment. The localization of Megator to at least three cellular compartments (nuclear rim, extrachromosomal nuclear space, spindle matrix complex) and reorganization during the cell cycle suggest that it is highly dynamic and that it may exist in several structural forms (Zimowska, 2002). This is underscored by the finding that 1 h after heat-shock treatment the amount of Megator protein in the extrachromosomal space diminishes, whereas accumulation occurs at a single chromosomal heat-shock puff, 93D; however, as this occurs Megator localization to the nuclear rim remains unchanged (Qi, 2004).

The colocalization of Megator with the Skeletor- and Chromator-defined spindle matrix during mitosis suggests that Megator may be involved in spindle matrix function. A spindle matrix has been hypothesized to provide a stationary substrate that anchors molecules during force production and microtubule sliding (Pickett-Heaps, 1997). Such a matrix could also be envisioned to have the added properties of helping to organize and stabilize the microtubule spindle. Previously, it has been demonstrated using RNAi assays in S2 cells that depletion of Chromator protein leads to abnormal spindle morphology and that chromosomes are scattered in the spindle, indicating defective spindle function in the absence of Chromator (Rath, 2004). However, it is not possible to infer a clear functional role for Megator based on the results obtained in the present study. When Megator levels are knocked down by RNAi in S2 cell cultures, the number of cells undergoing mitosis was greatly reduced. However, no cells were observed with obvious defects in tubulin spindle morphology or chromosome segregation defects, suggesting that depletion of Megator prevents cells from entering metaphase. This could be due to an essential function of Megator in maintaining nuclear structure and/or in maintaining the integrity of the nuclear rim and pore complexes during interphase or a necessary function for nuclear reorganization during prophase. Thus, if Megator plays multiple functional roles as its dynamic localization pattern suggests, it would prevent analysis of a mitotic function using RNAi approaches. That Megator is an essential protein necessary for viability is supported by the embryonic lethality observed as a consequence of P insertions in the Megator gene (Qi, 2004).

Studies using preparations spanning the evolutionary spectrum from lower eukaryotes to vertebrates have provided new and intriguing evidence that a spindle matrix may be a general feature of mitosis. This study shows that at least three proteins, Megator, Chromator, and Skeletor, from two different cellular compartments reorganize to form a putative spindle matrix during mitosis in Drosophila. Furthermore, the Megator and Skeletor defined fusiform spindle structure remains intact even in the absence of polymerized microtubules. The identification of several potential spindle matrix molecules in Drosophila together with P-element mutations in their genes should provide an avenue for further genetic and biochemical experiments. Especially, the future isolation and characterization of point mutations in Megator promises to provide the means to separate Megator's role in spindle matrix function from its role at other stages of the cell cycle (Qi, 2004).

EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila

Immunocytochemistry has been used to demonstrate that the EAST protein in Drosophila, which forms an expandable nuclear endoskeleton at interphase, redistributes during mitosis to colocalize with the spindle matrix proteins, Megator and Skeletor. EAST and Megator also colocalize to the intranuclear space surrounding the chromosomes at interphase. EAST is a novel protein that does not have any previously characterized motifs or functional domains. However, this study shows by immunoprecipitation experiments that EAST is likely to molecularly interact with Megator which has a large NH2-terminal coiled-coil domain with the capacity for self assembly. On the basis of these findings, it is proposed that Megator and EAST interact to form a nuclear endoskeleton and as well are important components of the putative spindle matrix complex during mitosis (Qi, 2005).

A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis

The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, it is demonstrated that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole. This 'internal' matrix is a distinct structure from the microtubule spindle and from a lamin B-containing spindle envelope. By injection of 2000-kDa dextran, it was shown that the disassembling nuclear envelope does not present a diffusion barrier. Furthermore, when microtubules are depolymerized with colchicine just before metaphase the spindle matrix contracts and coalesces around the chromosomes, suggesting that microtubules act as 'struts' stretching the spindle matrix. In addition, it was demonstrated that the spindle matrix protein Megator requires its coiled-coil amino-terminal domain for spindle matrix localization, suggesting that specific interactions between spindle matrix molecules are necessary for them to form a complex confined to the spindle region. The demonstration of an embedding spindle matrix lays the groundwork for a more complete understanding of microtubule dynamics and of the viscoelastic properties of the spindle during cell division (Yao, 2012).


Search PubMed for articles about Drosophila Megator

Cordes, V. C., et al. (1997). Identification of protein p270/Tpr as a constitutive component of the nuclear pore complex-attached intranuclear filaments. J. Cell Biol. 136: 515-529. PubMed ID: 9024684

Fabian, L., Xia, X., Venkitaramani, D. V., Johansen, K. M., Johansen, J., Andrew, D. J. and Forer, A. (2007). Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator. J. Cell Sci. 120(Pt 13): 2190-204. PubMed ID: 17591688

Frosst, P., Guan, T., Subauste, C., Hahn, K. and Gerace, L. (2002). Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export. J. Cell Biol. 156: 617-630. PubMed ID: 11839768

Iouk, T., et al. (2002). The yeast nuclear pore complex functionally interacts with components of the spindle assembly checkpoint. J. Cell Biol. 159: 807-819. PubMed ID: 12473689

Johansen, K. M., and Johansen, J. (2007). Cell and molecular biology of the spindle matrix. Int. Rev. Cytol. 263: 155-206. PubMed ID: 17725967

Katsani, K. R., Karess, R. E., Dostatni, N. and Doye, V. (2008). In vivo dynamics of Drosophila nuclear envelope components. Mol. Biol. Cell. 19: 3652-3666. PubMed ID: 18562695

Lee, S. H., et al. (2008). Tpr directly binds to Mad1 and Mad2 and is important for the Mad1-Mad2-mediated mitotic spindle checkpoint. Genes Dev. 22: 2926-2931. PubMed ID: 18981471

Lince-Faria, M., (2009). Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator. J. Cell Biol. 184(5): 647-57. PubMed ID: 19273613

Loiodice, I., et al. (2004). The entire Nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis. Mol. Biol. Cell. 15: 3333-3344. PubMed ID: 15146057

Maiato, H., et al. (2006). The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells. Chromosoma 115: 469-480. PubMed ID: 16909258

Mitchell, P. J. and Cooper, C. S. (1992). The human tpr gene encodes a protein of 2094 amino acids that has extensive coiled-coil regions and an acidic C-terminal domain. Oncogene 7: 2329-2333. PubMed ID: 1437155

Musacchio, A. and Salmon, E.D. (2007). The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8: 379-393. PubMed ID: 17426725

Pickett-Heaps, J. D., Forer, A. and Spurck, T. (1997). Traction fiber: toward a "tensegral" model of the spindle. Cell Motil. Cytoskeleton 37: 1-6. PubMed ID: 9142434

Qi, H., et al. (2004). Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila. Mol. Biol. Cell 15(11): 4854-65. PubMed ID: 15356261

Qi, H., et al. (2005). EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila. J. Cell Biochem. 95(6): 1284-91. PubMed ID: 15962301

Rath, U., Wang, D., Ding, Y., Xu, Y.-Z., Qi, H., Blacketer, M. J., Girton, J., Johansen, J. and Johansen, K. M. (2004). Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein Skeletor. J. Cell. Biochem. 93(5): 1033-47. PubMed ID: 15389869

Sauer, G., et al. (2005). Proteome analysis of the human mitotic spindle. Mol. Cell. Proteomics. 4: 35-43. PubMed ID: 15561729

Schweizer, N., Pawar, N., Weiss, M. and Maiato, H. (2015). An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region. J Cell Biol 210: 695-704. PubMed ID: 26304726

Scott, R. J., et al. (2005). Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell. 16: 4362-4374. PubMed ID: 16000377

Strambio-de-Castillia, C., Blobel, G. and Rout, M. P. (1999). Proteins connecting the nuclear pore complex with the nuclear interior. J. Cell Biol. 144: 839-855. PubMed ID: 10085285

Tighe, A., Staples, O. and Taylor, S. (2008). Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores. J. Cell Biol. 181: 893-901. PubMed ID: 18541701

Xu, X. M., et al. (2007). NUCLEAR PORE ANCHOR, the Arabidopsis homolog of Tpr/Mlp1/Mlp2/megator, is involved in mRNA export and SUMO homeostasis and affects diverse aspects of plant development. Plant Cell. 19: 1537-1548. PubMed ID: 17513499

Yao, C., Rath, U., Maiato, H., Sharp, D., Girton, J., Johansen, K. M. and Johansen, J. (2012). A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis. Mol Biol Cell 23: 3532-3541. PubMed ID:22855526

Zimowska, G., et al. (1997). A Drosophila Tpr protein homolog is localized both in the extrachromosomal channel network and to nuclear pore complexes. J. Cell Sci. 110: 927-944. PubMed ID: 9152019

Zimowska, G. and Paddy, M. R. (2002). Structures and dynamics of Drosophila Tpr inconsistent with a static, filamentous structure. Exp. Cell Res. 276: 223-232. PubMed ID: 12027452

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date revised: 30 October 2015

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