RESUMO
Expression of the Drosophila cancer-germline (CG), X-linked, head-to-head gene pair TrxT and dhd is normally germline-specific but becomes upregulated in brain tumours caused by mutation in l(3)mbt. Here, we show that TrxT and dhd play a major synergistic role in the emergence of l(3)mbt tumour-linked transcriptomic signatures and tumour development, which is remarkable, taking into account that these two genes are never expressed together under normal conditions. We also show that TrxT, but not dhd, is crucial for the growth of l(3)mbt allografts, hence suggesting that the initial stages of tumour development and long-term tumour growth may depend on different molecular pathways. In humans, head-to-head inverted gene pairs are abundant among CG genes that map to the X chromosome. Our results identify a first example of an X-linked, head-to-head CG gene pair in Drosophila, underpinning the potential of such CG genes, dispensable for normal development and homoeostasis of somatic tissue, as targets to curtail malignant growth with minimal impact on overall health.
Assuntos
Neoplasias Encefálicas , Encéfalo , Proteínas de Drosophila , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/crescimento & desenvolvimento , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Humanos , Regulação Neoplásica da Expressão Gênica , TranscriptomaRESUMO
Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting ß-tubulin, suggesting Mgr function is required for tubulin stability. Instability of ß-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic ß-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Chaperonas Moleculares/metabolismo , Subunidades Proteicas/metabolismo , Tubulina (Proteína)/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Sequência Conservada , Drosophila melanogaster/citologia , Humanos , Microtúbulos/metabolismo , Mutação/genética , Ligação Proteica , Estabilidade Proteica , Proteólise , Fuso Acromático/metabolismoRESUMO
Ewing sarcoma (EwS) is a cancer that arises in the bones and soft tissues, typically driven by the Ewing's sarcoma breakpoint region 1-Friend leukemia virus integration 1 (EWS-FLI) oncogene. Implementation of genetically modified animal models of EwS has proved difficult largely owing to EWS-FLI's high toxicity. The EWS-FLI1FS frameshift variant that circumvents toxicity but is still able to perform key oncogenic functions provided the first study model in Drosophila. However, the quest for Drosophila lines expressing full-length, unmodified EWS-FLI remained open. Here, we show that EWS-FLI1FS's lower toxicity is owed to reduced protein levels caused by its frameshifted C-terminal peptide, and report new strategies through which we have generated Drosophila lines that express full-length, unmodified EWS-FLI. Using these lines, we have found that the upregulation of transcription from GGAA-microsatellites (GGAAµSats) presents a positive linear correlation within a wide range of EWS-FLI protein concentrations. In contrast, rather counterintuitively, GGAAµSats-independent transcriptomic dysregulation presents relatively minor differences across the same range, suggesting that GGAAµSat-dependent and -independent transcriptional upregulation present different kinetics of response with regards to changing EWS-FLI protein concentration. Our results underpin the functional relevance of varying EWS-FLI expression levels and provide experimental tools to investigate, in Drosophila, the effect of the EWS-FLI 'high' and 'low' states that have been reported and are suspected to be important for EwS in humans.
Assuntos
Proteínas de Fusão Oncogênica , Proteína Proto-Oncogênica c-fli-1 , Proteína EWS de Ligação a RNA , Animais , Proteína EWS de Ligação a RNA/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Animais Geneticamente Modificados , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMO
Stem cell asymmetric division requires tight control of spindle orientation. To study this key process, we have recorded Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles. We have found that early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function.
Assuntos
Divisão Celular/fisiologia , Centrossomo/metabolismo , Drosophila/embriologia , Sistema Nervoso/embriologia , Fuso Acromático/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Polaridade Celular/fisiologia , Células Cultivadas , Centríolos/genética , Centríolos/metabolismo , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Regulação para Baixo/fisiologia , Drosophila/citologia , Drosophila/metabolismo , Larva/citologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Sistema Nervoso/metabolismo , Sistema Nervoso/ultraestrutura , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/metabolismo , Região Organizadora do Nucléolo/ultraestrutura , Fuso Acromático/ultraestrutura , Células-Tronco/ultraestruturaRESUMO
Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. Here, we show that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients.
Assuntos
Centrossomo/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Proteínas de Drosophila/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Animais , Linhagem Celular , Centrossomo/química , Síndrome de Chediak-Higashi , Grânulos Citoplasmáticos/química , Drosophila/química , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/análise , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Humanos , Lisossomos , Proteínas Associadas aos Microtúbulos/genética , Mutação , Oócitos/química , Fuso Acromático/química , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: Centrosomes, the major organizers of the microtubule network in most animal cells, are composed of centrioles embedded in a web of pericentriolar material (PCM). Recruitment and stabilization of PCM on the centrosome is a centriole-dependent function. Compared to the considerable number of PCM proteins known, the molecular characterization of centrioles is still very limited. Only a few centriolar proteins have been identified so far in Drosophila, most related to centriole duplication. RESULTS: We have cloned asterless (asl) and found that it encodes a 120 kD highly coiled-coil protein that is a constitutive pancentriolar and basal body component. Loss of asl function impedes the stabilization/maintenance of PCM at the centrosome. In embryos deficient for Asl, development is arrested right after fertilization. Asl shares significant homology with Cep 152, a protein described as a component of the human centrosome for which no functional data is yet available. CONCLUSIONS: The cloning of asl offers new insight into the molecular composition of Drosophila centrioles and a possible model for the role of its human homolog. In addition, the phenotype of asl-deficient flies reveals that a functional centrosome is required for Drosophila embryo development.
Assuntos
Centríolos/fisiologia , Centrossomo/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Genes de Insetos , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The notable male predominance across many human cancer types remains unexplained. Here, we show that Drosophila l(3)mbt brain tumors are more invasive and develop as malignant neoplasms more often in males than in females. By quantitative proteomics, we have identified a signature of proteins that are differentially expressed between male and female tumor samples. Prominent among them is the conserved chromatin reader PHD finger protein 7 (Phf7). We show that Phf7 depletion reduces sex-dependent differences in gene expression and suppresses the enhanced malignant traits of male tumors. Our results identify potential regulators of sex-linked tumor dimorphism and show that these genes may serve as targets to suppress sex-linked malignant traits.
Assuntos
Neoplasias Encefálicas/patologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Código das Histonas/genética , Proteínas de Homeodomínio/genética , Animais , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica , Masculino , Proteínas Quinases/genética , Fatores SexuaisRESUMO
Centrobin homologues identified in different species localize on daughter centrioles. In Drosophila melanogaster sensory neurons, Centrobin (referred to as CNB in Drosophila) inhibits basal body function. These data open the question of CNB's role in spermatocytes, where daughter and mother centrioles become basal bodies. In this study, we report that in these cells, CNB localizes equally to mother and daughter centrioles and is essential for C-tubules to attain the right position and remain attached to B-tubules as well as for centrioles to grow in length. CNB appears to be dispensable for meiosis, but flagellum development is severely compromised in Cnb mutant males. Remarkably, three N-terminal POLO phosphorylation sites that are critical for CNB function in neuroblasts are dispensable for spermatogenesis. Our results underpin the multifunctional nature of CNB that plays different roles in different cell types in Drosophila, and they identify CNB as an essential component for C-tubule assembly and flagellum development in Drosophila spermatogenesis.
Assuntos
Proteínas de Ciclo Celular/genética , Centríolos/genética , Proteínas de Drosophila/genética , Proteínas Serina-Treonina Quinases/genética , Espermatogênese/genética , Animais , Axonema/genética , Corpos Basais/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Flagelos/genética , Masculino , Mitose/genética , Células Receptoras Sensoriais/metabolismo , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismoRESUMO
Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.
Assuntos
Drosophila/genética , Microtúbulos/ultraestrutura , Espermatócitos/ultraestrutura , Fuso Acromático , Animais , Animais Geneticamente Modificados , Divisão Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Centrossomo/ultraestrutura , Cromatina/química , Citocinese , Demecolcina/farmacologia , Feminino , Corantes Fluorescentes/farmacologia , Masculino , Metáfase , Microscopia Confocal , Dados de Sequência Molecular , Espermatócitos/metabolismo , Fatores de Tempo , TransgenesRESUMO
Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is meiotic W68 (mei-W68), the Drosophila orthologue of the human cancer/testis gene Sporulation-specific protein 11 (SPO11), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that Drosophila mei-W68/SPO11 drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a SPO11 orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like Drosophila.
Assuntos
Neoplasias Encefálicas/genética , Proteínas de Drosophila/genética , Endodesoxirribonucleases/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Dano ao DNA , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Endodesoxirribonucleases/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MasculinoRESUMO
Sensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs, but ciliary dysfunction also has dramatic consequences in this organism because it impairs the mechanosensory properties of bristles and chaetae and leads to uncoordination, a crippling condition that causes lethality shortly after eclosion. The cilium is defined by the ciliary membrane, a protrusion of the cell membrane that envelops the core structure known as the axoneme, a microtubule array that extends along the cilium from the basal body. In vertebrates, basal body function requires centriolar distal and subdistal appendages and satellites. Because these structures are acquired through centriole maturation, only mother centrioles can serve as basal bodies. Here, we show that although centriole maturity traits are lacking in Drosophila, basal body fate is reserved to mother centrioles in Drosophila type I neurons. Moreover, we show that depletion of the daughter-centriole-specific protein Centrobin (CNB) enables daughter centrioles to dock on the cell membrane and to template an ectopic axoneme that, although structurally defective, protrudes out of the cell and is enveloped by a ciliary membrane. Conversely, basal body capability is inhibited in mother centrioles modified to carry CNB. These results reveal the crucial role of CNB in regulating basal body function in Drosophila ciliated sensory organs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Cílios/metabolismo , Drosophila/metabolismo , Animais , Axonema/metabolismo , Corpos Basais/metabolismo , Drosophila/citologia , Neurônios/citologia , Organelas/metabolismoRESUMO
During asymmetric mitosis, both in male Drosophila germline stem cells and in mouse embryo neural progenitors, the mother centrosome is retained by the self-renewed cell; hence suggesting that mother centrosome inheritance might contribute to stemness. We test this hypothesis in Drosophila neuroblasts (NBs) tracing photo converted centrioles and a daughter-centriole-specific marker generated by cloning the Drosophila homologue of human Centrobin. Here we show that upon asymmetric mitosis, the mother centrosome is inherited by the differentiating daughter cell. Our results demonstrate maturation-dependent centrosome fate in Drosophila NBs and that the stemness properties of these cells are not linked to mother centrosome inheritance.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/genética , Proteínas de Drosophila/metabolismo , Células-Tronco Neurais/citologia , Proteínas Recombinantes/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Centríolos/metabolismo , Centríolos/ultraestrutura , Clonagem Molecular , Drosophila/citologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Feminino , Expressão Gênica , Humanos , Padrões de Herança/genética , Larva/citologia , Larva/genética , Larva/metabolismo , Masculino , Microscopia Confocal , Mitose , Células-Tronco Neurais/metabolismo , Marcadores de Fotoafinidade/análise , Proteínas Recombinantes/genéticaRESUMO
Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. Here we show that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. Our results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species.
Assuntos
Neoplasias Encefálicas/genética , Transformação Celular Neoplásica , Drosophila melanogaster , Regulação Neoplásica da Expressão Gênica , Genes de Insetos , Células Germinativas/fisiologia , Animais , Proteínas Argonautas , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Neoplasias Encefálicas/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Animais , Transplante de Neoplasias , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Transplante Homólogo , Regulação para CimaRESUMO
The Drosophila protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. An unusual sequence arrangement of two AG dinucleotides and an intervening polypyrimidine (Py)-tract at the 3' end of intron 2 is important for Sxl autoregulation. Here we show that U2AF interacts with the Py-tract and downstream AG, whereas the spliceosomal protein SPF45 interacts with the upstream AG and activates it for the second catalytic step of the splicing reaction. SPF45 represents a new class of second step factors, and its interaction with SXL blocks splicing at the second step. These results are in contrast with other known mechanisms of splicing regulation, which target early events of spliceosome assembly. A similar role for SPF45 is demonstrated in the activation of a cryptic 3' ss generated by a mutation that causes human beta-thalassemia.