Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Strategies for outcrossing and genetic manipulation of Drosophila compound autosome stocks.

Among all organisms, Drosophila melanogaster has the most extensive well-characterized collection of large-scale chromosome rearrangements. Compound chromosomes, rearrangements in which homologous chromosome arms share a centromere, have proven especially useful in genetic-based surveys of the entire genome. However, their potential has not been fully realized because compound autosome stocks are refractile to standard genetic manipulations: if outcrossed, they yield inviable aneuploid progeny. Here we describe two strategies, cold-shock and use of the bubR1 mutant alleles, to produce nullo gametes through nondisjunction. These gametes are complementary to the compound chromosome-bearing gametes and thus produce viable progeny. Using these techniques, we created a compound chromosome two C(2)EN stock bearing a red fluorescent protein-histone transgene, facilitating live analysis of these unusually long chromosomes.

Organized microtubule arrays in gamma-tubulin-depleted Drosophila spermatocytes.

To assess the role of gamma-tubulin in spindle assembly in vivo, we have followed meiosis progression by immunofluorescence and time-lapse video microscopy in gammaTub23C(PI) mutant spermatocytes. We have found that centrosomes associate with large numbers of astral microtubules even though gamma-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles. First, they are assembled midway through meiosis and elongate during anaphase. Second, they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone. Third, Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones. Finally, ectopic cytokinesis furrows are often formed at the distal end of the cone. Our results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a gamma-tubulin-independent manner in Drosophila spermatocytes. However, gamma-tubulin seems to be essential for spindle assembly in these cells. Finally, our results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles.

tef: a mutation that causes telomere fusion and severe genome rearrangements in Drosophila melanogaster.

Telomeres are the stable ends of linear chromosomes in eukaryotes. These complex protein-nucleic acid structures are essential to maintain genomic stability and the integrity of linear chromosomes. We identified a new mutation in Drosophila that causes a high frequency of end-to-end fusions of chromosomes during mitosis and meiosis. Linear chromosomal ends appear to be essential for fusions to take place. These fusions do not resolve, leading to cycles of chromosomal breakage and rejoining and severe genome rearrangements. The gene is essential for normal cell proliferation and mutant tissue shows significant apoptosis. Our analysis suggests that the function encoded by the mutant gene is required to protect the linear ends of chromosomes.

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis.

BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.

The Drosophila RAD21 cohesin persists at the centromere region in mitosis.

'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate (reviewed in [1,2]). Here we address this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins [3]. Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21-green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase-anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase-anaphase transition. We conclude that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains bound to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase-anaphase transition.

Microtubule binding of the drosophila DMAP-85 protein is regulated by phosphorylation in vitro.

The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule (MT) stability. Previously we isolated DMAP-85, a Drosophila MAP shown to be associated with stable MTs. In this work we show that DMAP-85 phosphorylated in cell-free early embryo extracts is released from MTs. MPM-2 antibodies recognize the phosphorylated protein. In vitro, DMAP-85 can be phosphorylated by the mitotic kinase Polo affecting its binding to MTs and creating MPM-2 epitopes on the protein. The results suggest that phosphorylation of DMAP-85 might affect its MT stabilizing activity during early mitotic cycles.

Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization.

Through mutational analysis in Drosopjila we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.

The human autoantigen MCP1 is required during early stages of DNA replication.

Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen that is associated with condensed chromosomes throughout mitosis. During interphase, this antigen shows a speckle distribution in the nucleus, excluding the nucleolus. Additionally, MCP1 binds tightly to the scaffold/matrix component of nuclei and isolated chromosomes. In order to determine the in-vivo localization of the antigen, we have expressed MCP1 fused to EGFP in tissue culture cells. The results demonstrate that MCP1 is located in the nucleus during interphase and during mitosis associates tightly to condensed chromosomes. Furthermore, microinjection of specific antibody confirms these results. We have used a specific monoclonal antibody (mAb 402) against MCP1 to assess the function of this antigen during cell cycle progression. HeLa and Ptk-2 cells that were microinjected into the nucleus and/or cytoplasm at G1/S and very early S phase were not able to progress and complete DNA replication. However, injection of mAb 402 at mid or late S phase does not prevent completion of DNA replication and subsequent progression into mitosis. Microinjection of mAb 402 in Ptk-2 cells synchronized in mitosis did not interfere with progression of mitosis and cells divided. Our results suggest that MCP1 is required at the G1/S transition and during early S phase.

Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila.

We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element-induced, near-null mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants. We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila.

The gene polo encodes a highly conserved serine/threonine protein kinase that has been implicated in several functions during cell division. Polo-like kinases are important positive regulators of cell cycle progression and have also been implicated in the exit from mitosis through the activation of the anaphase-promoting complex. Several data indicate that Plks are required for centrosome function, bipolar spindle organisation and cytokinesis. The intracellular localisation of Plks reflects their multiple roles in cell division, however, in vivo studies that describe the distribution of this protein during different stages of mitosis have never been performed. In the present work, we report the in vivo distribution of a GFP-POLO fusion protein expressed in stable transformants and analysed during the early embryonic development of Drosophila melanogaster. The GFP-POLO protein can be detected in unfertilised oocytes associated with the centromeric region of chromosomes of the polar body and followed until the formation of mitotic domains in later development. Detailed analysis of the dynamic localisation of GFP-POLO during syncytial mitotic cycles shows the timing of localisation to the centrosomes, centromeres and midbody. The results also indicate that GFP-POLO is present in astral microtubules early in mitosis, accumulates around the nuclear envelope until nuclear envelop breakdown and at metaphase associates to spindle microtubules. These in vivo studies show a highly dynamic association of POLO with multiple compartments of the mitotic apparatus. Furthermore, the wide distribution of the GFP-POLO protein to all compartments of the mitotic apparatus provides a valuable tool for future studies on cell cycle during development.

Molecular cloning, developmental expression, and cellular localization of the 70-kDa RPA-1 subunit of Drosophila melanogaster.

Replication protein A (RPA) is a highly conserved multifunctional heterotrimeric complex, involved in DNA replication, repair, recombination, and possibly transcription. Here, we report the cloning of the gene that codes for the largest subunit of the Drosophila melanogaster RPA homolog, dmRPA70. In situ hybridization showed that dmRPA70 RNA is present in developing embryos during the first 16 cycles. After this point, dm-RPA70 expression is downregulated in cells that enter a G1 phase and exit the mitotic cycle, becoming restricted to brief bursts of accumulation from late G1 to S phase. This pattern of regulated expression is also observed in the developing eye imaginal disc. In addition, we have shown that the presence of cyclin E is necessary and sufficient to drive the expression of dmRPA70 in embryonic cells arrested in G1 but is not required in tissues undergoing endoreduplication. Immunolocalization showed that in early developing embryos, the dmRPA70 protein associates with chromatin from the end of mitosis until the beginning of the next prophase in a dynamic speckled pattern that is strongly suggestive of its association with replication foci.

The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes.

The MPM2 antibody is a valuable tool for studying the regulation of mitotic events since it specifically recognises a subset of mitosis-specific phosphoproteins. Some MPM2 epitopes have been shown to be phosphorylated by p34(cdc2). However, recent results suggest that the newly emerging family of polo-like kinases (Plks) may also act as MPM2 kinases. In this study, we present evidence suggesting that the Drosophila POLO protein is required for the phosphorylation of MPM2 reactive epitopes. POLO displays a dynamic localisation pattern during mitosis, which parallels that of the MPM2 phosphoepitopes, since it is found in the centrosome and centromere from early prophase until late anaphase, the microtubule-overlapping region during anaphase, and the region on either side of the midbody during telophase. Centromere localisation is not dependent upon microtubules since it is retained in colchicine-arrested cells and is present in isolated chromosomes. Furthermore, the level of MPM2 immunoreactivity is directly correlated to the severity of the polo mutant alleles. In cells carrying a hypomorphic allele, the centrosomes of abnormal cells are small and fail to efficiently recruit MPM2 epitopes. In neuroblasts homozygous for a severe loss-of-function allele, the mitotic index is low and the MPM2 labelling is severely reduced or absent. Finally, rephosphorylation of MPM2 epitopes in detergent-extracted Schneider cells requires either POLO stably bound to the cytoskeletons or POLO present in soluble extracts. These results suggest that POLO is required for the phosphorylation of MPM2 epitopes in Drosophila, at the centrosomes, centromeres and the mitotic spindle, and thus might be involved in co-ordinating the mitotic changes of cellular architecture with the activity of the maturation promoting factor.

Molecular cloning of metaphase chromosome protein 1 (MCP1), a novel human autoantigen that associates with condensed chromosomes during mitosis.

Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human chromosome-associated proteins. Previous immunolocalization studies in human and murine tissue culture cells showed that some of these monoclonal antibodies recognize nuclear antigens that associate with condensed chromosomes during mitosis. One antibody was selected for screening a human HeLa S3 cDNA expression library, and cDNAs that code for an antigen of 31-33 kDa were isolated. Immunological, biochemical and cell fractionation data indicate that the 31- to 33-kDa antigen corresponds to the chromosome-associated protein recognized by the original monoclonal antibody. Sequence analysis shows that we isolated a novel human gene. Immunolocalization to human tissue culture cells shows that during interphase the antigen is dispersed in the nucleus and that during mitosis it associates exclusively with condensed chromosomes. A similar pattern of localization was also observed in mouse fibroblasts, suggesting that the antigen is conserved among different species. Finally, we show that part of the antigen remains bound to the scaffold/matrix component, even after high salt extraction.

Human autoantibodies reveal titin as a chromosomal protein.

Assembly of the higher-order structure of mitotic chromosomes is a prerequisite for proper chromosome condensation, segregation and integrity. Understanding the details of this process has been limited because very few proteins involved in the assembly of chromosome structure have been discovered. Using a human autoimmune scleroderma serum that identifies a chromosomal protein in human cells and Drosophila embryos, we cloned the corresponding Drosophila gene that encodes the homologue of vertebrate titin based on protein size, sequence similarity, developmental expression and subcellular localization. Titin is a giant sarcomeric protein responsible for the elasticity of striated muscle that may also function as a molecular scaffold for myofibrillar assembly. Molecular analysis and immunostaining with antibodies to multiple titin epitopes indicates that the chromosomal and muscle forms of titin may vary in their NH2 termini. The identification of titin as a chromosomal component provides a molecular basis for chromosome structure and elasticity.

Pattern of chromosomal localization of the Hoppel transposable element family in the Drosophila melanogaster subgroup.

We have isolated a Hoppel-like transposon from heterochromatin of the second chromosome of Drosophila melanogaster and used a conserved DNA sequence between the different elements of this family to determine their distribution in both mitotic and polytene chromosomes. The hybridization pattern of polytene chromosomes extends throughout the entire chromocentre, as well as a substantial portion of the fourth chromosome. Analysis of different wild-type strains of D. melanogaster shows variation in euchromatic insertion sites, although most insertions are found near the chromocentre. The positions and the number of heterochromatic clusters of Hoppel on mitotic chromosomes are conserved among the several strains analysed. Accurate mapping of this element was achieved by in situ hybridization on D. melanogaster mitotic chromosomes that had previously been banded with Hoechst 33258. To evaluate the evolutionary stability of this pattern, different species were analysed by in situ hybridization and Southern blotting. We conclude that Hoppel has a conserved distribution in mitotic heterochromatin within the D. melanogaster subgroup, established around 5 million years ago. The overall conservation of heterochormatic organization supports the notion that heterochormatin does perform important structural and functional roles.

Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod.

We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways.

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 alpha-tubulin shows several abnormalities of the meiotic spindle, including a significantly reduced central spindle. Our results suggest that polo has multiple functions during spermatogenesis.

Interactions between mgr, asp, and polo: asp function modulated by polo and needed to maintain the poles of monopolar and bipolar spindles.

We describe genetic interactions between mutations in mgr, asp, and polo, genes required for the correct behaviour of the spindle poles in Drosophila. The phenotype of a polo1 mgr double mutant is more similar to mgr than polo1, but the frequency of circular monopolar figures (CMFs) seen with either mutant alone is additive, suggesting that the two gene products are required for independent functions in the formation of bipolar spindles. The aspE3 mgr double mutant arrests much earlier in development than either mutant alone, indicative of a strong block to cell proliferation. We discuss whether the lack of microtubular structures in these cells reflects an extended mitotic arrest, or if it is a more direct consequence of the double mutant combination. A polo1 aspE3 double mutant shows a dramatic synergistic increase in mitotic frequency. The loss of CMFs normally associated with the polo1phenotype suggests that the Asp microtubule-associated protein is required to maintain the structure of spindle poles. We speculate that Asp protein might be a substrate for the serine-threonine protein kinase encoded by polo.

Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster.

The progression of cells from metaphase to anaphase is thought to be regulated by a checkpoint that delays entry into anaphase until all chromosomes reach a stable bi-polar attachment at the metaphase plate. Previous work has suggested that the 3F3/2 kinetochore phosphoepitopes are involved in this checkpoint system. We show that the 3F3/2 centromere phosphoepitopes are present in Kc cells, third instar larval neuroblasts and isolated chromosomes of Drosophila melanogaster. In tissue culture cells and neuroblasts isolated from third instar larvae, centromere labelling is detected from early prophase to the metaphase-anaphase transition but absent once cells center anaphase. During anaphase, the antibody stains the spindle mid zone and during telophase the midbody is labelled until cells separate. In both cell types, the 3F3/2 antibody stains the centrosome from prophase to late telophase. The 3F3/2 staining is retained in Kc cells and third instar larval neuroblasts arrested at the prometaphase state with microtubule inhibitors. Also, two mitotic mutants that show abnormal spindle morphology retain the centromere labelling in a metaphase-like configuration, suggesting that they activate the metaphase-anaphase checkpoint. Finally, mitotic chromosomes isolated in the presence of a phosphatase inhibitor show phosphoepitopes at the primary constriction on the surface of each chromatid, however, chromosomes isolated in the absence of a phosphatase inhibitor do not. Incubation of these chromosomes with ATP causes the rephosphorylation of the phosphoepitopes at the centromere.