In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.     

Spindle mechanics and dynamics during mitosis in Drosophila

Drosophila melanogaster is an excellent model for studying mitosis. Syncytial embryos are amenable to time-lapse imaging of hundreds of synchronously dividing spindles, allowing the quantitation of spindle and chromosome dynamics with unprecedented fidelity. Other Drosophila cell types, including neuroblasts, cultured cells, spermatocytes and oocytes, contain spindles that differ in their design, providing cells amenable to different types of experiments and allowing identification of common core mechanisms. The function of mitotic proteins can be studied using mutants, inhibitor microinjection and RNA interference (RNAi) to identify the full inventory of mitotic proteins encoded by the genome. Here, we review recent advances in understanding how ensembles of mitotic proteins coordinate spindle assembly and chromosome motion in this system.     

In vivo dynamics of Drosophila nuclear envelope components

Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells.     

In vivo protein trapping in Drosophila.

The deciphering of complete genome sequences has opened a post-genomic proteomics era. While the sequence of many proteins is now known, attention will increasingly focus on the complex interaction networks that regulate their activity, and the analysis of protein distribution in the cell will be crucial to elucidating their function. A new generation of gene trapping devices, protein trap transposons, offers a way of analysing In vivo the dynamics of sub-cellular distribution of a large number of proteins. Many transgenic lines have already been established and are available. Screens focusing on particular cell compartments can be devised.     

Ran localizes around the microtubule spindle in vivo during mitosis in Drosophila embryos

The GTPase Ran regulates multiple cellular functions throughout the cell cycle, including nucleocytoplasmic transport, nuclear membrane assembly, and spindle assembly. Ran mediates spindle assembly by affecting multiple spindle assembly pathways: microtubule dynamics, microtubule motor activity, and spindle pole assembly. Ran is predicted to facilitate spindle assembly by remaining in the GTP-bound state around the chromatin in mitosis. Here, we directly test the central tenet of this hypothesis in vivo by determining the cellular localization of Ran pathway components in Drosophila embryos. We find that, during mitosis, RCC1, the nucleotide exchange factor for Ran, is associated with chromatin, while Ran and RanL43E, an allele locked in the GTP-bound state, localize around the spindle. In contrast, nuclear proteins redistribute throughout the embryo upon nuclear envelope breakdown (NEB). Thus, in vivo RanGTP has the correct spatial localization within the cell to modulate spindle assembly.     

Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila

The metaphase-anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells. A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle. Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy. Here we show that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies. Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants - they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis. These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast.     

In vivo function of a novel Siah protein in Drosophila

The Siah proteins, mammalian homologues of the Drosophila Sina protein, function as E3 ubiquitin ligase enzymes and target a wide range of cellular proteins for degradation. Here, I investigate the in vivo function of the fly protein, Sina-Homologue (SinaH), which is highly similar to Sina. Flies that completely lack SinaH are viable and in combination with a mutation in the gene, Ebi, show an extra dorsal central bristle phenotype. I also show that SinaH and Ebi can interact with each other both in vivo and in vitro suggesting that they act in the same physical complex. Flies that lack both Sina and Sina-Homologue were also created and show visible eye and bristle phenotypes, which can be explained by an inability to degrade the neuronal repressor, Tramtrack. I find no evidence for redundancy in the function of Sina and SinaH.     

rough deal: a gene required for proper mitotic segregation in Drosophila

We describe a genetic locus rough deal (rod) in Drosophila melanogaster, identified by mutations that interfere with the faithful transmission of chromosomes to daughter cells during mitosis. Five mutant alleles were isolated, each associated with a similar set of mitotic abnormalities in the dividing neuroblasts of homozygous mutant larvae: high frequencies of aneuploid cells and abnormal anaphase figures, in which chromatids may lag, form bridges, or completely fail to separate. Surviving homozygous adults are sterile, and show cuticular defects associated with cell death, i.e., roughened eyes, sparse abdominal bristles, and notched wing margins. The morphological process of spermatogenesis is largely unaffected and motile sperm are produced, but meiocyte aneuploidy is common. The nature of the observed abnormalities in mitotic cells suggests that the reduced fidelity of chromosome transmission to the daughter cells is due to a failure in a mechanism involved in assuring the proper release of sister chromatids.     

In vivo proteome dynamics during early bovine myogenesis

Myogenesis is a complex process of which the underlying mechanisms are conserved between species, including birds and mammals. Despite a good understanding of the stages of myogenesis, many of the mechanisms involved in the regulation of proliferation of the successive myoblast generations, the cellular transitions cell proliferation/alignment of myoblasts/fusion of myoblasts into myotubes/differentiation of myofibres and the control of total myofibre number still remain unknown. An in vivo proteomic analysis of the semitendinosus muscle from Charolais foetuses, at three specific stages of myogenesis (60, 110 and 180 days postconception), was conducted using 2-DE and MS. Expression profiles of more than 170 proteins were revealed and analysed using two way hierarchical clustering and statistical analysis. Our studies identify, for the first time, distinct proteins of varied biological functions and protein clusters with myogenic processes, such as the control of cell cycle activity and apoptosis, the establishment of cellular metabolism and muscle contractile properties and muscle cell reorganisation. These results are of fundamental interest to the field of myogenesis in general, and more specifically to the control of muscle development in meat producing animals.     

A role for Drosophila Polo protein in chromosome resolution and segregation during mitosis

Correct chromosome structure is essential to ensure faithful segregation during mitosis. Chromosome condensation occurs at the same time as cohesion is released from the arms of the sister chromatids. It is not until metaphase-anaphase transition that chromosomes lose cohesion completely, by proteolysis of the component of the cohesin complex Scc1 (Sister chromatid cohesion 1). It has been shown in vertebrates that the Polo-like kinase, Plk1, is important for this process by inducing the destabilization of Scc1 from the chromosome arms. It is still unclear if this process is conserved in other high eukaryotes, namely in Drosophila. Here we analysed the consequences over chromosome resolution of the downregulation of Drosophila Polo, both by mutant analysis and by RNAi-depletion in S2 cells. We show that the depletion of Polo results in a strong a prometa/metaphase arrest with the spindle checkpoint activated in response to lack of tension. In addition, the checkpoint protein ROD fails to stream over the kinetochore microtubules in the lack of Polo activity. We also show that loss of Polo causes strong defects in chromosome resolution, a phenotype we partially rescued by depleting Scc1. Importantly, we show Scc1 fails to accumulate on the kinetochores during mitosis and remains on the chromosome arms in the absence of Polo. We therefore propose an alternative role for Drosophila Polo in Scc1 redistribution during mitosis.     

Assembly of Drosophila centromeric chromatin proteins during mitosis

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.     

A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.     

Interaction of checkpoint kinase 1 and the X-linked inhibitor of apoptosis during mitosis

Senescence marker protein-30 (SMP30) was originally identified as a novel protein in the rat liver, the expression of which decreases androgen-independently with aging. We have now characterized a unique property of SMP30, the hydrolysis of diisopropyl phosphorofluoridate (DFP), which is similar to the chemical warfare nerve agents sarine, soman and tabun. Hydrolysis of DFP was stimulated equally well by 1 mM MgCl₂, MnCl₂ or CoCl₂, to a lesser extent by 1 mM CdCl₂ but not at all by 1 mM CaCl₂. No ⁴⁵Ca²⁺-binding activity was detected for purified SMP30, suggesting that SMP30 is not a calcium-binding protein, as others previously stated. Despite the sequence similarity between SMP30 and a serum paraoxonase (PON), the inability of SMP30 to hydrolyze PON-specific substrates such as paraoxon, dihydrocoumarin, γ-nonalactone, and δ-dodecanolactone indicate that SMP30 is distinct from the PON family. We previously established SMP30 knockout mice and have now tested DFPase activity in their livers. The livers from wild-type mice contained readily detectable DFPase activity, whereas no such enzyme activity was found in livers from SMP30 knockout mice. Moreover, the hepatocytes of SMP30 knockout mice were far more susceptible to DFP-induced cytotoxicity than those from the wild-type. These results indicate that SMP30 is a unique DFP hydrolyzing enzyme in the liver and has an important detoxification effect on DFP. Consequently, a reduction of SMP30 expression might account for the age-associated deterioration of cellular functions and enhanced susceptibility to harmful stimuli in aged tissue.     

Armadillo levels are reduced during mitosis in Drosophila

The Armadillo protein of Drosophila melanogaster is both a structural component of adherens junctions at apical cell membranes and also a key cytoplasmic transducer of the Wingless signalling pathway. We have used the Gal4-UAS system to over-express Armadillo in the Drosophila wing: this hyperactivates the Wingless pathway and leads to the formation of ectopic, supernumerary wing bristles. Here, we report that this adult phenotype is dominantly enhanced by mutations in cdc25(string) and, conversely, is suppressed by co-expression of Cdc25(String). Furthermore, we show that the steady state levels of Armadillo protein produced from the UAS transgene are also sensitive to cdc25(string) dosage in the cells of the larval imaginal wing disc. Consistent with the role of Cdc25(String) in promoting mitosis and with our genetic interaction data, we find a strong correlation between progression through mitosis and a reduction in Armadillo levels. Significantly, this is true whether Armadillo is over-expressed or not, and both cytoplasmic (signalling) and membrane-associated (junctional) Armadillo appears to be affected. We conclude that this phenomenon may reduce the efficacy of Wingless signalling and/or intercellular adhesion during cell division.