Salud Llamazares

Ectopic Expression of Germline Genes Drives Malignant Brain Tumor Growth in Drosophila

Curing Insect Brain Tumors Mammalian tumors often show ectopic expression of genes that normally function only in the germ line. The possible contribution of cancer germline (CG) genes to malignancy is unknown. Janic et al. (p. 1824; see the Perspective by Wu and Ruvkun) found that CG genes are also expressed in Drosophila brain tumors caused by mutants in the gene lethal (3) malignant brain tumor. Moreover, inactivation of some of these genes suppressed fly tumor growth. Because some Drosophila CG genes are orthologs of human CG genes, it is possible that inactivation of germline genes may show human tumor suppressor activity. Inactivation of germline genes suppresses brain tumor growth in Drosophila. 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.

Drosophila neuroblasts retain the daughter centrosome

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.

Asterless is a centriolar protein required for centrosome function and embryo development in Drosophila

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.

ESSENTIAL ROLE FOR GAMMA -TUBULIN IN THE ACENTRIOLAR FEMALE MEIOTIC SPINDLE OF DROSOPHILA

Microtubule nucleation in vivo requires γ‐tubulin, a highly conserved component of microtubule‐organizing centers. In Drosophila melanogaster there are two γ‐tubulin genes, γTUB23C and γTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of γ‐tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C γ‐tubulin isoform of D.melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires γ‐tubulin.

Centrobin controls mother–daughter centriole asymmetry in Drosophila neuroblasts

During interphase in Drosophila neuroblasts, the Centrobin (CNB)-positive daughter centriole retains pericentriolar material (PCM) and organizes an aster that is a key determinant of the orientation of cell division. Here we show that daughter centrioles depleted of CNB cannot fulfil this function whereas mother centrioles that carry ectopic CNB can. CNB co-precipitates with a set of centrosomal proteins that include γ-TUB, ANA2, CNN, SAS-4, ASL, DGRIP71, POLO and SAS-6. Following chemical inhibition of POLO or removal of three POLO phosphorylation sites present in CNB, the interphase microtubule aster is lost. These results demonstrate that centriolar CNB localization is both necessary and sufficient to enable centrioles to retain PCM and organize the interphase aster in Drosophila neuroblasts. They also reveal an interphase function for POLO in this process that seems to have co-opted part of the protein network involved in mitotic centrosome maturation.

Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes.

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.

Loss of Centrobin Enables Daughter Centrioles to Form Sensory Cilia in Drosophila

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.

Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability

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 β-tubulin, suggesting Mgr function is required for tubulin stability. Instability of β-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic β-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.

16 Methods in Drosophila Cell Cycle Biology

Publisher Summary The chapter focuses on fluorescent in situ hybridization, immunofluorescence, and primary cultures. As far as fluorescent in situ hybridization is concerned, it includes protocols for the application of this technique to polytene and diploid cells both in squashed and whole-mounted tissues. It deals with immunofluorescence techniques includes protocols for the staining of embryos, third-instar larval brains, and mature oocytes. Finally, a newly developed version of a protocol for primary culture of cells from third-instar larval brains is also included. In situ hybridization to diploid cells provides the only route toward mapping and characterizing the behavior of heterochromatic sequences of Drosophila, which are both underrepresented and aggregated in polytene cells. It includes two protocols for in situ hybridization to diploid cells from squashed and whole-mounted third-instar larval brains. For genome mapping, squashed preparations are by far the best choice because they are easy to make and provide the highest possible resolution.

An in vivo genetic screen in Drosophila identifies the orthologue of human cancer/testis gene SPO11 among a network of targets to inhibit lethal(3)malignant brain tumour growth

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.