Involvement of the rolled/MAP kinase gene in Drosophila mitosis: interaction between genes for the MAP kinase cascade and abnormal spindle

We have found that mutations that lead to loss of rolled/MAP kinase function result in a reduced mitotic index in the larval central nervous system, consistent with an interphase block to cell cycle progression, associated with a low frequency of cells showing chromosome over-condensation in mitosis and abnormal anaphase figures. In contrast to wild-type tissue, such rolled mutants do not show a significant increase in accumulation of mitotic cells when treated with colchicine. We have studied double mutant combinations between mutations affecting the activity of rolled/MAP kinase and several genes that are essential to the establishment of a bipolar spindle during progression through mitosis, and find no interactions with mutations in polo, mgr,or aurora. However, partial loss-of-function mutations in rolled enhance the abnormal spindle (asp) phenotype, whereas gain-of function mutations in rolled or in the gene encoding its activating kinase Dsor1, act as suppressors. We discuss these findings in relation to the proposed role of MAP kinase in mediating the spindle integrity checkpoint. Received: 27 October 1997 / Accepted: 18 December 1997     

Involvement of the rolled/MAP kinase gene in Drosophila mitosis: interaction between genes for the MAP kinase cascade and abnormal spindle

We have found that mutations that lead to loss of rolled/MAP kinase function result in a reduced mitotic index in the larval central nervous system, consistent with an interphase block to cell cycle progression, associated with a low frequency of cells showing chromosome over-condensation in mitosis and abnormal anaphase figures. In contrast to wild-type tissue, such rolled mutants do not show a significant increase in accumulation of mitotic cells when treated with colchicine. We have studied double mutant combinations between mutations affecting the activity of rolled/MAP kinase and several genes that are essential to the establishment of a bipolar spindle during progression through mitosis, and find no interactions with mutations in polo, mgr,or aurora. However, partial loss-of-function mutations in rolled enhance the abnormal spindle (asp) phenotype, whereas gain-of function mutations in rolled or in the gene encoding its activating kinase Dsor1, act as suppressors. We discuss these findings in relation to the proposed role of MAP kinase in mediating the spindle integrity checkpoint.     

The POLO kinase is required at multiple stages during spermatogenesis in Drosophila melanogaster

The polo gene of Drosophila melanogaster is the founding member of the polo-like kinase family which is conserved among eukaryotes. POLO has been implicated in the organisation and function of the mitotic apparatus. Furthermore, POLO has been shown to be required for normal spermatogenesis. To characterize further the role of POLO in spermatogenesis, polo mutants were analysed by immunostaining with specific antibodies and phase contrast microscopy. Immunofluorescence shows that POLO localises to the centrosomes, the centromere/kinetochore and the spindle midzone. The meiotic phenotype of various mutant allelic combinations was also studied in detail. Observation of mutant live testes indicates cytological abnormalities in all meiotic cell types, including variable DNA content and multipolar spindles. Primary spermatocytes in polo mutant testes contain an abnormal DNA content, suggesting failure of chromosome segregation during gonial division. Immunostaining of polo mutant cells with α-tubulin shows several abnormalities of the meiotic spindle, including a significantly reduced central spindle. Our results suggest that polo has multiple functions during spermatogenesis. Received: 5 August 1998; in revised form: 3 September 1998 / Accepted: 3 September, 1998     

The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.     

The Drosophila Gene abnormal spindle Encodes a Novel Microtubule-associated Protein That Associates with the Polar Regions of the Mitotic Spindle

abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp¹ directs the synthesis of a COOH terminally truncated or internally deleted peptide of ~124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH₂-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34^cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.     

Metaphase arrest with centromere separation in polo mutants of Drosophila

The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Here we report two strongly hypomorphic mutations of polo that arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20-50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the abnormal spindle protein (Asp), but neither with gamma-tubulin nor CP190. We discuss roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase-anaphase checkpoint.     

An Asp–CaM complex is required for centrosome–pole cohesion and centrosome inheritance in neural stem cells

The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, our understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. Here we report that Drosophila melanogaster calmodulin (CaM) regulates the ability of the microcephaly-associated protein, abnormal spindle (Asp), to cross-link spindle microtubules. Both proteins colocalize on spindles and move toward spindle poles, suggesting that they form a complex. Our binding and structure–function analysis support this hypothesis. Disruption of the Asp–CaM interaction alone leads to unfocused spindle poles and centrosome detachment. This behavior leads to randomly inherited centrosomes after neuroblast division. We further show that spindle polarity is maintained in neuroblasts despite centrosome detachment, with the poles remaining stably associated with the cell cortex. Finally, we provide evidence that CaM is required for Asp’s spindle function; however, it is completely dispensable for Asp’s role in microcephaly suppression.     

Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.     

The role of Patronin in Drosophila mitosis

Background

The calmodulin-regulated spectrin-associated proteins (CAMSAPs) belong to a conserved protein family, which includes members that bind the polymerizing mcrotubule (MT) minus ends and remain associated with the MT lattice formed by minus end polymerization. Only one of the three mammalian CAMSAPs, CAMSAP1, localizes to the mitotic spindle but its function is unclear. In Drosophila, there is only one CAMSAP, named Patronin. Previous work has shown that Patronin stabilizes the minus ends of non-mitotic MTs and is required for proper spindle elongation. However, the precise role of Patronin in mitotic spindle assembly is poorly understood.

Results

Here we have explored the role of Patronin in Drosophila mitosis using S2 tissue culture cells as a model system. We show that Patronin associates with different types of MT bundles within the Drosophila mitotic spindle, and that it is required for their stability. Imaging of living cells expressing Patronin-GFP showed that Patronin displays a dynamic behavior. In prometaphase cells, Patronin accumulates on short segments of MT bundles located near the chromosomes. These Patronin “seeds” extend towards the cell poles and stop growing just before reaching the poles. Our data also suggest that Patronin localization is largely independent of proteins acting at the MT minus ends such as Asp and Klp10A.

Conclusion

Our results suggest a working hypothesis about the mitotic role of Patronin. We propose that Patronin binds the minus ends within MT bundles, including those generated from the walls of preexisting MTs via the augmin-mediated pathway. This would help maintaining MT association within the mitotic bundles, thereby stabilizing the spindle structure. Our data also raise the intriguing possibility that the minus ends of bundled MTs can undergo a limited polymerization.

     

Sinup is essential for the integrity of centrosomes and mitotic spindles in zebrafish embryos

Fish lineage-specific gene, sinup [Siaz-interacting nuclear protein], modulates neural plate formation in embryogenesis and shares homology with human TPX2 protein, a member of the vertebrate mitogen-activating protein family. In spite of the presence of the TPX2 domain in Sinup, its cellular function has been unknown. As an initial approach to this question, we expressed Sinup by injecting sinup-EGFP mRNAs into zebrafish embryos at the one- to two-cell stage. First of all, Sinup-EGFP was associated with centrosomes and mitotic spindles. In particular, Sinup was localized to the spindle poles and midbody microtubules during the period between anaphase and cytokinesis. Second, various deleted mutants of Sinup-EGFP failed to be associated with the centrosomes and mitotic spindles. Third, a Sinup mutant, where the 144th Serine residue was converted to alanine, not only disturbed the mitotic spindle organization, such as multipolar spindles, fragmented spindle poles, and flattened spindles, but also arrested the cell cycle at metaphase and cell movement. Finally, Sinup is phosphorylated by Aurora A and the 144th Serine mutant of Sinup is partially phosphorylated by Aurora A kinase. We thus propose that Sinup is an essential element for the integrity of centrosomes and mitotic spindle fibers as well as for the normal process of cell cycle and cellular movement in vertebrate embryos.     

ZYG-9, A Caenorhabditis elegans Protein Required for Microtubule Organization and Function, Is a Component of Meiotic and Mitotic Spindle Poles

We describe the molecular characterization of zyg-9, a maternally acting gene essential for microtubule organization and function in early Caenorhabditis elegans embryos. Defects in zyg-9 mutants suggest that the zyg-9 product functions in the organization of the meiotic spindle and the formation of long microtubules. One-cell zyg-9 embryos exhibit both meiotic and mitotic spindle defects. Meiotic spindles are disorganized, pronuclear migration fails, and the mitotic apparatus forms at the posterior, orients incorrectly, and contains unusually short microtubules. We find that zyg-9 encodes a component of the meiotic and mitotic spindle poles. In addition to the strong staining of spindle poles, we consistently detect staining in the region of the kinetochore microtubules at metaphase and early anaphase in mitotic spindles. The ZYG-9 signal at the mitotic centrosomes is not reduced by nocodazole treatment, indicating that ZYG-9 localization to the mitotic centrosomes is not dependent upon long astral microtubules. Interestingly, in embryos lacking an organized meiotic spindle, produced either by nocodazole treatment or mutations in the mei-1 gene, ZYG-9 forms a halo around the meiotic chromosomes. The protein sequence shows partial similarity to a small set of proteins that also localize to spindle poles, suggesting a common activity of the proteins.     

Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.     

A novel fission yeast gene, kms1 +, is required for the formation of meiotic prophase-specific nuclear architecture

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1 ⁺ gene is involved in SPB function. However, the kms1 ⁺ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins. Received: 5 September 1996 / Accepted: 21 November 1996     

Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein

We previously described a dominant negative secY ^-d 1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the product of a multicopy suppressor of the secY ^-d 1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240→Asp). The secY249 mutation (Ala249→Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity␣to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a “hyper reversion” with respect to the SecY-SecE interaction. Protein export in this mutant was no longer sensitive to SecY^-d1. When the secY ^-d 1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY ^-d 1–241) showed an increased ability to interfere with protein export. On the basis of our model for SecY^-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex. Received: 20 October 1997 / Accepted: 1 December 1997     

Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation

Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization.     

Novel mad2 alleles isolated in a Schizosaccharomyces pombe gamma-tubulin mutant are defective in metaphase arrest activity, but remain functional for chromosome stability in unperturbed mitosis

A previously isolated fission yeast gamma-tubulin mutant containing apparently stabilized microtubules proliferated at an approximately identical rate as wild type, yet the mutant mitosis spindle dynamics were aberrant, particularly the kinetochore microtubule dynamics. Progression through mitosis in the mutant, however, resulted in mostly accurate chromosome segregation. In the absence of the spindle assembly checkpoint gene, mad2+, the spindle dynamics in the gamma-tubulin mutant were greatly compromised, leading to a high incidence of chromosome missegregation. Unlike in wild-type cells, green fluorescent protein (GFP)-tagged Mad2 protein often accumulated near one of the poles of an elongating spindle in the gamma-tubulin mutant. We isolated novel mad2 mutants that were defective in arresting mitotic progression upon gross perturbation of the spindle formation but remained functional for the viability of the gamma-tubulin mutant. Further, the mad2 mutations did not appreciably destabilize minichromosomes in unperturbed mitoses. When overexpressed ectopically, these mutant Mad2 proteins sequestered wild-type Mad2, preventing its function in mitotic checkpoint arrest, but not in minichromosome stability. These results indicated that the Mad2 functions required for checkpoint arrest and chromosome stability in unperturbed mitosis are genetically discernible. Immunoprecipitation studies demonstrated that GFP-fused mutant Mad2 proteins formed a Mad1-containing complex with altered stability compared to that formed with wild-type Mad2, providing clues to the novel mad2 mutant phenotype.     

Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr⁷²) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr⁷², we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr⁷² does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr⁷² regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.     

Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.     

Apoptotic cleavage of NuMA at the C-terminal end is related to nuclear disruption and death amplification

NuMA is a nuclear matrix protein in interphase and distributes to the spindle poles during mitosis. While the essential function of NuMA for mitotic spindle assembly is well established, a structural role of NuMA in interphase nucleus has also been proposed. Several observations suggest that the apoptotic degradation of NuMA may relate to chromatin condensation and micronucleation. Here we demonstrate that four apoptotic cleavage sites are clustered at a junction between the globular tail and the central coiled-coil domains of NuMA. Cleavage of a caspase-6-sensitive site at D¹⁷⁰⁵ produced the R-form, a major tail-less product of NuMA during apoptosis. The other two cleavage sites were defined at D¹⁷²⁶ and D¹⁷⁴⁷ that were catalyzed, respectively, by caspase-3 and an unknown aspartase. A NuMA deletion mutant missing the entire cleavage region of residues 1701–1828 resisted degradation and protected cells from nuclear disruption upon apoptotic attack. Under such conditions, cytochrome c was released from mitochondria, but the subsequent apoptotic events such as caspase-3 activation, poly(ADP-ribose) polymerase degradation, and DNA fragmentation were attenuated. Conversely, the tail-less NuMA alone, a mutant mimicking the R-form, induced chromatin condensation and activated the death machinery. It supports that intact NuMA is a structural element in maintaining nuclear integrity.