Elsa Logarinho

University of Porto | UP · Institute for Molecular and Cell Biology

Aneuploidy, an abnormal chromosome number, has been linked to aging and age-associated diseases, but the underlying molecular mechanisms remain unknown. Here we show, through direct live-cell imaging of young, middle-aged, and old-aged primary human dermal fibroblasts, that aneuploidy increases with aging due to general dysfunction of the mitotic machinery. Increased chromosome mis-segregation in elderly mitotic cells correlates with an early senescence-associated secretory phenotype (SASP) and repression of Forkhead box M1 (FoxM1), the transcription factor that drives G2/M gene expression. FoxM1 induction in elderly and Hutchison-Gilford progeria syndrome fibroblasts prevents aneuploidy and, importantly, ameliorates cellular aging phenotypes. Moreover, we show that senescent fibroblasts isolated from elderly donors' cultures are often aneuploid, and that aneuploidy is a key trigger into full senescence phenotypes. Based on this feedback loop between cellular aging and aneuploidy, we propose modulation of mitotic efficiency through FoxM1 as a potential strategy against aging and progeria syndromes.

Aging is a biological process characterized by the progressive deterioration of physiological functions known to be the main risk factor for chronic diseases and declining health. There has been an emerging connection between aging and aneuploidy, an aberrant number of chromosomes, even though the molecular mechanisms behind age-associated aneuploidy remain largely unknown. In recent years, several genetic pathways and biochemical processes controlling the rate of aging have been identified and proposed as aging hallmarks. Primary hallmarks that cause the accumulation of cellular damage include genomic instability, telomere attrition, epigenetic alterations and loss of proteostasis (López-Otín et al., Cell 153:1194–1217, 2013). Here we review the provocative link between these aging hallmarks and the loss of chromosome segregation fidelity during cell division, which could support the correlation between aging and aneuploidy seen over the past decades. Secondly, we review the systemic impacts of aneuploidy in cell physiology and emphasize how these include some of the primary hallmarks of aging. Based on the evidence, we propose a mutual causality between aging and aneuploidy, and suggest modulation of mitotic fidelity as a potential means to ameliorate healthy lifespan.

An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes.

The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado–Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.

Mitotic spindle bipolarity is essential for faithful segregation of chromosomes during cell division. Multipolar spindles are often seen in human cancers and are usually associated with supernumerary centrosomes that result from centrosome overduplication or cytokinesis failure. A less-understood path to multipolar spindle formation may arise due to loss of spindle pole integrity in response to spindle and/or chromosomal forces. Here we discuss the different routes leading to multipolar spindle formation, focusing on spindle multipolarity without centrosome amplification. We also present the distinct and common features between these pathways and discuss their therapeutic implications.

Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1-c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1-c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.

Loss of spindle-pole integrity during mitosis leads to multipolarity independent of centrosome amplification. Multipolar-spindle conformation favours incorrect kinetochore-microtubule attachments, compromising faithful chromosome segregation and daughter-cell viability. Spindle-pole organization influences and is influenced by kinetochore activity, but the molecular nature behind this critical force balance is unknown. CLASPs are microtubule-, kinetochore- and centrosome-associated proteins whose functional perturbation leads to three main spindle abnormalities: monopolarity, short spindles and multipolarity. The first two reflect a role at the kinetochore-microtubule interface through interaction with specific kinetochore partners, but how CLASPs prevent spindle multipolarity remains unclear. Here we found that human CLASPs ensure spindle-pole integrity after bipolarization in response to CENP-E- and Kid-mediated forces from misaligned chromosomes. This function is independent of end-on kinetochore-microtubule attachments and involves the recruitment of ninein to residual pericentriolar satellites. Distinctively, multipolarity arising through this mechanism often persists through anaphase. We propose that CLASPs and ninein confer spindle-pole resistance to traction forces exerted during chromosome congression, thereby preventing irreversible spindle multipolarity and aneuploidy.

Primers used in this study. (PDF)

DAF-16 targets sod-3 and mtl-1 are activated differentially in atx-3 mutants both at 20°C (A) and 25°C (B). At 20°C, sod-3 is up-regulated 90 minutes after heat shock while mtl-1 is up-regulated 15 minutes after the stress. At 25°C, sod-3 is up-regulated in all time points analyzed in atx-3 mutants while mtl-1 expression is increased starting at 15 min until 60 minutes after stimulus. *p<0.05. (PDF)

Lifespan results depicted in Figure 1 . (PDF)

Proteomic results from LC-ESI-MS/MS analysis of two ataxin-3 knockout strains in a basal condition and after heat shock. (XLS)

Lifespan results depicted in Figure 7 . (PDF)

Ataxin-3, the protein involved in Machado-Joseph disease, is able to bind ubiquitylated substrates and act as a deubiquitylating enzyme in vitro, and it has been involved in the modulation of protein degradation by the ubiquitin-proteasome pathway. C. elegans and mouse ataxin-3 knockout models are viable and without any obvious phenotype in a basal condition however their phenotype in stress situations has never been described. Considering the role of ataxin-3 in the protein degradation pathway, we analyzed the effects of heat shock, a known protein homeostasis stressor, in C. elegans ataxin-3 (ATX-3) knockout animals. We found that ATX-3 mutants have an exacerbated stress response and survive significantly better than wild type animals when subjected to a noxious heat shock stimulus. This increased thermotolerance of mutants was further enhanced by pre-exposure to a mild heat shock. At a molecular level, ATX-3 mutants have a distinct transcriptomic and proteomic profile with several molecular chaperones abnormally up-regulated during heat shock and recovery, consistent with the observed resistance phenotype. The improved thermotolerancein ATX-3 mutants is independent of heat shock factor 1, the maestro of the heat shock response, but fully dependent on DAF-16, a critical stress responsive transcription factor involved in longevity and stress resistance. We also show that the increased thermotolerance of ATX-3 mutants is mainly due to HSP-16.2, C12C8.1 and F44E5.5 given that the knockdown of these heat shock proteins using RNA interference causes the phenotype to revert. This report suggests that the absence of ATX-3 activates the DAF-16 pathway leading to an overexpression of molecular chaperones, which yields knockout animals with an improved capacity for dealing with deleterious stimuli.

Ataxin-3 (ATXN3) is a widely expressed protein that binds to ubiquitylated proteins, has deubiquitylating activity in vitro and is thought to modulate substrate degradation through the ubiquitin-proteasome pathway. Expansion of a polyglutamine tract in ATXN3 causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation and specific neuronal death. Although ATXN3 has been involved in transcriptional repression and in the ubiquitin-proteasome pathway, its biological function is still unknown. In this work, we show that depletion of ATXN3 using small-interference RNA (siRNA) causes a prominent phenotype in both human and mouse cell lines. A mild increase in ubiquitylation occurs and cells exhibit ubiquitin-positive foci, which is consistent with ATXN3 putative function as a deubiquitylating enzyme. In addition, siATXN3-silenced cells exhibit marked morphological changes such as rounder shape and loss of adhesion protrusions. At a structural level, the microtubule, microfilament and intermediate filament networks are severely compromised and disorganized. This cytoskeletal phenotype is reversible and dependent on ATXN3 levels. Cell-extracellular matrix connection is also affected in ATXN3-depleted cells as talin expression is reduced in the focal adhesions and lower levels of alpha-1 integrin subunit are expressed at their surface. Although the cytoskeletal and adhesion problems do not originate any major change in the cell cycle of siATXN3-depleted cells, cell death is increased in siATXN3 cultures compared to controls. In summary, in this work we show that the absence of ATXN3 leads to an overt cytoskeletal/adhesion defect raising the possibility that this protein may play a role in the cytoskeleton.

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The transcript levels of the genes encoding other josephin-domain containing proteins Josd1, and Josd2 for mock and Mjd siRNA transfected C2C12 cells at Day 0 and Day 1 of differentiation, were measured by quantitative real-time RT-PCR, and showed to be similar. The results were normalised for the Hprt1 gene and correspond to the mean of three independent transfections +/− SEM (error bars). (0.09 MB TIF)

Mouse ataxin-3 conserves the deubiquitinating activity observed for the human ataxin-3. A) Scheme of the recombinant His-tagged proteins used in this study: mATX3, the full-length protein; mATX3:C14A, the full-length protein carrying the C14A point mutation; mATX3:jos, the Josephin domain of mATX3; mATX3:jos:C14A, the Josephin domain with the C14A mutation; and mATX3:UIMs, the C-terminal of mATX3 containing the three UIMs. B) Polyubiquitin immunoblotting showing the deubiquitinating activity of each used protein. mATX3, as well as its Josephin domain for itself (mATX3:jos) were able to cleave both K48 and K63-linked polyubiquitin chains, preferentially with two or more ubiquitins. The mutation of the catalytic cysteine in these two proteins (mATX3:C14A and mATX3:jos:C14A) abolished their DUB activity. The C-terminal of mATX3 containing the UIMs (mATX3:UIMs) is not capable to hydrolyse polyubiquitin chains. (0.60 MB TIF)

Proteins showing altered levels upon Mjd siRNA in undifferentiated C2C12 cells (Day 0). Cut-off (sketched line): proteins with fold changes ≥20% (1.2< ratio <0.8) considering a statistical significant p-value <0.05. Functional networks: (A) cellular movement; (B) drug metabolism; (C) protein synthesis; (D) skeletal and muscular system development and function, tissue morphology; (E) cellular development, embryonic development, cellular assembly and organization; (F) small molecule biochemistry, gene expression. (0.06 MB PDF)

Primers used in this study. (0.02 MB PDF)

During myogenesis several transcription factors and regulators of protein synthesis and assembly are rapidly degraded by the ubiquitin-proteasome system (UPS). Given the potential role of the deubiquitinating enzyme (DUB) ataxin-3 in the UPS, and the high expression of the murine ataxin-3 homolog in muscle during embryogenesis, we sought to define its role in muscle differentiation. Using immunofluorescence analysis, we found murine ataxin-3 (mATX3) to be highly expressed in the differentiated myotome of E9.5 mouse embryos. C2C12 myoblasts depleted of mATX3 by RNA interference exhibited a round morphology, cell misalignment, and a delay in differentiation following myogenesis induction. Interestingly, these cells showed a down-regulation of alpha5 and alpha7 integrin subunit levels both by immunoblotting and immunofluorescence. Mouse ATX3 was found to interact with alpha5 integrin subunit and to stabilize this protein by repressing its degradation through the UPS. Proteomic analysis of mATX3-depleted C2C12 cells revealed alteration of the levels of several proteins related to integrin signaling. Ataxin-3 is important for myogenesis through regulation of integrin subunit levels.

Buruli ulcer, caused by Mycobacterium ulcerans infections, is a necrotizing skin disease whose pathogenesis is associated with the exotoxin mycolactone. Despite the relevance of this emergent disease, little is known on the immune response against the pathogen. Following the recent demonstration of an intramacrophage growth phase for M. ulcerans, we investigated the biological relevance of IFN-gamma and the antimycobacterial mechanisms activated by this cytokine in M. ulcerans-infected macrophages. Three M. ulcerans strains were tested: 5114 (mutant mycolactone-negative, avirulent strain); 94-1327 (intermediate virulence); and 98-912 (high virulence). We show in this study that IFN-gamma is expressed in mouse-infected tissues and that IFN-gamma-deficient mice display increased susceptibility to infection with strains 5114 and, to a lesser extent, 94-1327, but not with the highly virulent strain. Accordingly, IFN-gamma-activated cultured macrophages controlled the proliferation of the avirulent and the intermediate virulent strains. Addition of mycolactone purified from strain 98-912 to cultures of IFN-gamma-activated macrophages infected with the mycolactone-negative strain led to a dose-dependent inhibition of the IFN-gamma-induced protective mechanisms, involving phagosome maturation/acidification and increased NO production, therefore resulting in increased bacterial burdens. Our findings suggest that the protection mediated by IFN-gamma in M. ulcerans-infected macrophages is impaired by the local buildup of mycolactone.

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42's expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.

Ataxin-3 is the protein involved in Machado-Joseph disease, a neurodegenerative disorder caused by a polyglutamine expansion. Ataxin-3 binds ubiquitylated proteins and acts as a deubiquitylating enzyme in vitro. It was previously proposed that ataxin-3, along with the VCP/p97 protein, escorts ubiquitylated substrates for proteasomal degradation, although other players of this escort complex were not identified yet. In this work, we show that the Caenorhabditis elegans ataxin-3 protein (ATX-3) interacts with both VCP/p97 worm homologs, CDC-48.1 and CDC-48.2 and we map the interaction domains. We describe a motility defect in both ATX-3 and CDC-48.1 mutants and, in addition, we identify a new protein interactor, UBXN-5, potentially an adaptor of the CDC-48-ATX-3 escort complex. CDC-48 binds to both ATX-3 and UBXN-5 in a non-competitive manner, suggesting the formation of a trimolecular complex. Both CDC-48 and ATX-3, but not UBXN-5, were able to bind K-48 polyubiquitin chains, the standard signal for proteasomal degradation. Additionally, we describe several common interactors of ATX-3 and UBXN-5, some of which can be in vivo targets of this complex.

The spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes accomplish proper bipolar attachments to the mitotic spindle and come under tension, thereby ensuring the fidelity of chromosome segregation. Despite significant advances in our understanding of SAC signalling, a clear link between checkpoint signalling and the molecular mechanisms underlying chromosome attachment to microtubules has not been established so far. However, independent studies from many groups have interestingly found that the bone-a-fide Bub1, BubR1 and Bub3 SAC proteins are themselves required for proper kinetochoremicrotubule (K-MT) interactions. Here, we review these findings and discuss the specific contribution of each of these proteins in the regulation of K-MT attachment, taking into consideration their interdependencies for kinetochore localization as well as their relationship with other proteins with a known role in chromosome attachment and congression.

The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.

Machado-Joseph disease (MJD/SCA3) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG tract in the coding portion of the ATXN3 gene. The presence of ubiquitin-positive aggregates of the defective protein in affected neurons is characteristic of this and most of the polyglutamine disorders. Recently, the accumulation of the neural precursor cell expressed developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, in the inclusions of MJD brains was reported. Here, we report a new molecular interaction between wild-type ataxin-3 and NEDD8, using in vitro and in situ approaches. Furthermore, we show that this interaction is not dependent on the ubiquitin-interacting motifs in ataxin-3, since the presence of the Josephin domain is sufficient for the interaction to occur. The conservation of the interaction between the Caenorhabditis elegans ataxin-3 homologue (atx-3) and NEDD8 suggests its biological and functional relevance. Molecular docking studies of the NEDD8 molecule to the Josephin domain of ataxin-3 suggest that NEDD8 interacts with ataxin-3 in a substrate-like mode. In agreement, ataxin-3 displays deneddylase activity against a fluorogenic NEDD8 substrate.

The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.

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.

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.

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.

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.

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.

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.

We report the molecular cloning and characterisation of the first CK1(casein kinase) gene of Drosophila melanogaster (dmCK1). The protein sequence (DMCK1) shares significant homology with other mammalian CK1 protein kinases of the alpha sub-class. The dmCK1 gene is expressed only in adult females and during early embryonic development as a single transcript. Western blot analysis of total protein extracts of different stages of development show that the gene product is likewise present during early embryogenesis and in adult females. Kinase activity studies show that DMCK1 is active when in vitro translated but inactive when immunoprecipitated from total early embryo extracts. However, after dephosphorylation treatment the immunoprecipitates show high kinase activity. More significantly, DMCK1 kinase activity present in the immunoprecipitates can be specifically activated by gamma-irradiation of early embryos. Also, when DMCK1 is immunoprecipitated after irradiation it appears to undergo phosphorylation. Immunolocalization of DMCK1 in early embryos shows that the protein is predominantly cytoplasmic but after irradiation there is a significant relocalization to the interphase nucleus. The results suggest a possible requirement of the Drosophila CK1 alpha for mechanisms associated with DNA repair during early embryogenesis.