RESUMO
The number of chromosomes varies tremendously across species. It is not clear whether having more or fewer chromosomes could be advantageous. The probability of non-disjunction should theoretically decrease with smaller karyotypes, but too long chromosomes should enforce spatial constraint for their segregation during the mitotic anaphase. Here, we propose a new experimental cell system to acquire novel insights into the mechanisms underlying chromosome segregation. We collected the endemic Australian ant Myrmecia croslandi, the only known species with the simplest possible karyotype of a single chromosome in the haploid males (and one pair of chromosomes in the diploid females), since males are typically haploid in hymenopteran insects. Five colonies, each with a queen and a few hundreds of workers, were collected in the Canberra district (Australia), underwent karyotype analysis to confirm the presence of a single pair of chromosomes in worker pupae, and were subsequently maintained in the laboratory in Paris (France). Starting from dissociated male embryos, we successfully conducted primary cell cultures comprised of single-chromosome cells. This could be developed into a unique model that will be of great interest for future genomic and cell biology studies related to mitosis.
Assuntos
Formigas , Cromossomos de Insetos , Animais , Formigas/genética , Masculino , Feminino , Cultura Primária de Células , Cariotipagem , Cariótipo , Haploidia , Segregação de CromossomosRESUMO
Wolbachia is an intracellular endosymbiont present in most arthropod and filarial nematode species. Transmission between hosts is primarily vertical, taking place exclusively through the female germ line, although horizontal transmission has also been documented. The results of several studies indicate that Wolbachia spp. can undergo transfer between somatic and germ line cells during nematode development and in adult flies. However, the mechanisms underlying horizontal cell-to-cell transfer remain largely unexplored. Here, we establish a tractable system for probing horizontal transfer of Wolbachia cells between Drosophila melanogaster cells in culture using fluorescence in situ hybridization (FISH). First, we show that horizontal transfer is independent of cell-to-cell contact and can efficiently take place through the culture medium within hours. Further, we demonstrate that efficient transfer utilizes host cell phagocytic and clathrin/dynamin-dependent endocytic machinery. Lastly, we provide evidence that this process is conserved between species, showing that horizontal transfer from mosquito to Drosophila cells takes place in a similar fashion. Altogether, our results indicate that Wolbachia utilizes host internalization machinery during infection, and this mechanism is conserved across insect species.IMPORTANCE Our work has broad implications for the control and treatment of tropical diseases. Wolbachia can confer resistance against a variety of human pathogens in mosquito vectors. Elucidating the mechanisms of horizontal transfer will be useful for efforts to more efficiently infect nonnatural insect hosts with Wolbachia as a biological control agent. Further, as Wolbachia is essential for the survival of filarial nematodes, understanding horizontal transfer might provide new approaches to treating human infections by targeting Wolbachia Finally, this work provides a key first step toward the genetic manipulation of Wolbachia.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/microbiologia , Wolbachia/fisiologia , Animais , Clatrina/metabolismo , Drosophila melanogaster/fisiologia , Dinaminas/metabolismo , Células Germinativas/microbiologia , Hibridização in Situ Fluorescente , Wolbachia/citologiaRESUMO
Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4's tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of ß-sheets fold. This unique feature of the TCP domain suggests that it could provide an "extended surface-like" platform to tether the Sas-4-PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9-10th ß-strands (ß9-10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 ß9-10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the ß9-10 surface in mediating protein-protein interactions for efficient Sas-4-PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.
Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/patologia , Citoplasma/metabolismo , Drosophila melanogaster/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Microcefalia/genética , Proteínas Associadas aos Microtúbulos , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Testículo/metabolismoRESUMO
Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts.
Assuntos
Albendazol/análogos & derivados , Brugia Malayi/microbiologia , Drosophila melanogaster/microbiologia , Filariose/tratamento farmacológico , Wolbachia/efeitos dos fármacos , Albendazol/farmacologia , Animais , Brugia Malayi/efeitos dos fármacos , Linhagem Celular , Drosophila melanogaster/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microtúbulos/efeitos dos fármacos , SimbioseRESUMO
Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. New replacement cells-the progeny of basal stem cells-are born without barrier-forming structures such as a specialized apical membrane and occluding junctions. Here, we investigate how new progeny acquire barrier structures as they integrate into the intestinal epithelium of adult Drosophila. We find they gestate their future apical membrane in a sublumenal niche created by a transitional occluding junction that envelops the differentiating cell and enables it to form a deep, microvilli-lined apical pit. The transitional junction seals the pit from the intestinal lumen until differentiation-driven, basal-to-apical remodelling of the niche opens the pit and integrates the now-mature cell into the barrier. By coordinating junctional remodelling with terminal differentiation, stem cell progeny integrate into a functional, adult epithelium without jeopardizing barrier integrity.
Assuntos
Mucosa Intestinal , Intestinos , Epitélio , Membrana Celular , Mucosa Intestinal/metabolismo , Células-Tronco/metabolismoRESUMO
Centrioles are cylinders made of nine microtubule (MT) triplets present in many eukaryotes. Early studies, where centrosomes were seen at the poles of the mitotic spindle led to their coining as "the organ for cell division". However, a variety of subsequent observational and functional studies showed that centrosomes might not always be essential for mitosis. Here we review the arguments in this debate. We describe the centriole structure and its distribution in the eukaryotic tree of life and clarify its role in the organization of the centrosome and cilia, with an historical perspective. An important aspect of the debate addressed in this review is how centrioles are inherited and the role of the spindle in this process. In particular, germline inheritance of centrosomes, such as their de novo formation in parthenogenetic species, poses many interesting questions. We finish by discussing the most likely functions of centrioles and laying out new research avenues.
Assuntos
Centríolos/fisiologia , Mitose , Animais , Divisão Celular , Linhagem Celular , Centrossomo/fisiologia , Cílios/classificação , Cílios/metabolismo , Drosophila , Microtúbulos/metabolismo , Partenogênese , Fuso Acromático/fisiologiaRESUMO
The Drosophilamelanogaster cell line 1182-4, which constitutively lacks centrioles, was established many years ago from haploid embryos laid by females homozygous for the maternal haploid (mh) mutation. This was the first clear example of animal cells regularly dividing in the absence of this organelle. However, the cause of the acentriolar nature of the 1182-4 cell line remained unclear and could not be clearly assigned to a particular genetic event. Here, we detail historically the longstanding mystery of the lack of centrioles in this Drosophila cell line. Recent advances, such as the characterization of the mh gene and the genomic analysis of 1182-4 cells, allow now a better understanding of the physiology of these cells. By combining these new data, we propose three reasonable hypotheses of the genesis of this remarkable phenotype.
Assuntos
Centríolos/metabolismo , Drosophila melanogaster/citologia , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Genoma de Inseto , Modelos BiológicosRESUMO
Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets Wolbachia, a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in L. sigmodontis, Brugia malayi, and Onchocerca ochengi in vivo preclinical models of filarial disease and in vitro selectivity against Loa loa (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.
Assuntos
Antibacterianos/uso terapêutico , Descoberta de Drogas , Filariose/tratamento farmacológico , Filariose/parasitologia , Filarioidea/fisiologia , Quinazolinas/uso terapêutico , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Modelos Animais de Doenças , Feminino , Filarioidea/efeitos dos fármacos , Filarioidea/microbiologia , Ensaios de Triagem em Larga Escala , Camundongos , Fenótipo , Quinazolinas/química , Quinazolinas/farmacologia , Bibliotecas de Moléculas Pequenas , Wolbachia/efeitos dos fármacosRESUMO
Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4's localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.
Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Proteínas de Drosophila/química , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Larva/citologia , Masculino , Meiose , Proteínas Associadas aos Microtúbulos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Espermatócitos/citologia , Espermatócitos/metabolismoRESUMO
Wolbachia are gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use a Wolbachia-infected Drosophila cell line and genome-wide RNA interference (RNAi) screening to identify host factors that influence Wolbachia titer. By screening an RNAi library targeting 15,699 transcribed host genes, we identified 36 candidate genes that dramatically reduced Wolbachia titer and 41 that increased Wolbachia titer. Host gene knockdowns that reduced Wolbachia titer spanned a broad array of biological pathways including genes that influenced mitochondrial function and lipid metabolism. In addition, knockdown of seven genes in the host ubiquitin and proteolysis pathways significantly reduced Wolbachia titer. To test the in vivo relevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels of Wolbachia in the Drosophila oocyte. The presence of Wolbachia in either cell lines or oocytes dramatically alters the distribution and abundance of ubiquitinated proteins. Functional studies revealed that maintenance of Wolbachia titer relies on an intact host Endoplasmic Reticulum (ER)-associated protein degradation pathway (ERAD). Accordingly, electron microscopy studies demonstrated that Wolbachia is intimately associated with the host ER and dramatically alters the morphology of this organelle. Given Wolbachia lack essential amino acid biosynthetic pathways, the reliance of Wolbachia on high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioning Wolbachia with amino acids. In addition, the reliance of Wolbachia on the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism for Wolbachia's potent ability to prevent RNA virus replication.
Assuntos
Drosophila/genética , Genoma de Inseto , Interações Hospedeiro-Patógeno/genética , Proteólise , Wolbachia/patogenicidade , Animais , Linhagem Celular , Drosophila/metabolismo , Drosophila/microbiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Degradação Associada com o Retículo Endoplasmático , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Interferência de RNARESUMO
Hundreds of Drosophila cell lines have been established in the labs of many researchers over the last decades and have been important tools for research. Although these cells often deviate from normal cell physiology and genetic composition, such systems nonetheless are powerful models for biochemical, cell biological, and genetics studies that are experimentally difficult in vivo. While published descriptions of cell line generation are available in the literature, how to generate new Drosophila cell lines can be challenging for beginners. Here, we describe a detailed, simple protocol to establish new Drosophila cell lines.
Assuntos
Técnicas de Cultura de Células , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Efeito Fundador , Animais , Linhagem Celular , Drosophila melanogaster/fisiologia , Embrião não Mamífero/fisiologia , Cultura Primária de CélulasRESUMO
A protein-trap screen using the Drosophila neuromuscular junction (NMJ) as a model synapse was performed to identify genes that control synaptic structure or plasticity. We found that Shaggy (Sgg), the Drosophila homolog of the mammalian glycogen synthase kinases 3 alpha and beta, two serine-threonine kinases, was concentrated at this synapse. Using various combinations of mutant alleles of shaggy, we found that Shaggy negatively controlled the NMJ growth. Moreover, tissue-specific expression of a dominant-negative Sgg indicated that this kinase is required in the motoneuron, but not in the muscle, to control NMJ growth. Finally, we show that Sgg controlled the microtubule cytoskeleton dynamics in the motoneuron and that Futsch, a microtubule-associated protein, was required for Shaggy function on synaptic growth.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Quinase 3 da Glicogênio Sintase/fisiologia , Junção Neuromuscular/enzimologia , Junção Neuromuscular/crescimento & desenvolvimento , Animais , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Drosophila melanogaster/ultraestrutura , Quinase 3 da Glicogênio Sintase/análise , Quinase 3 da Glicogênio Sintase/genética , Larva/enzimologia , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Neurônios Motores/enzimologia , Mutação , Fatores de Crescimento Neural/genética , Plasticidade Neuronal , Terminações Pré-Sinápticas/enzimologia , Terminações Pré-Sinápticas/ultraestruturaRESUMO
In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT) assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs "inside-out" from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC) in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.
RESUMO
Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain gamma-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle-regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles.
Assuntos
Citoesqueleto/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Anáfase/fisiologia , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Centríolos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Dineínas/genética , Dineínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Modelos Biológicos , Interferência de RNA , Fuso Acromático/fisiologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Importin-beta is an essential component of nuclear protein import, spindle formation and nuclear envelope assembly. Formerly, the function of the Drosophila Ketel gene, which encodes importin-beta and is essential for the survival to adulthood, seemed to be required only in the mitotically active cells. We report here that importin-beta function is required in every cell and that this protein possesses an exceptionally long life span. Mosaic analysis, using gynanders, indicated that zygotic function of the Ketel gene is essential in a large group of cells in the embryos. Expression of a UAS-Ketel transgene by different tissue specific Gal4 drivers on ketel(null)/- hemizygous background revealed the requirement of Ketel gene function in the ectoderm. Elimination of the Ketel gene function using a UAS-Ketel-RNAi transgene driven by different Gal4 drivers confirmed the indispensability of the Ketel gene in the ectoderm. Using GFP-tagged importin-beta (encoded by a ketel(GFP) allele) we revealed that the maternally provided GFP-importin-beta molecules persist up to larval life. The zygotic Ketel gene is expressed in every cell during early gastrulation. Although the gene is then turned off in the non-dividing cells, the produced importin-beta molecules persist long and carry out nuclear protein import throughout the subsequent stages of development. In the continuously dividing diploid cells, the Ketel gene is constitutively expressed to fulfill all three functions of importin-beta.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Gástrula/crescimento & desenvolvimento , Zigoto/metabolismo , beta Carioferinas/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Sobrevivência Celular , Drosophila/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , beta Carioferinas/genéticaRESUMO
The assembly of a functional bipolar mitotic spindle requires an exquisite regulation of microtubule behavior in time and space. To characterize new elements of this machinery we carried out a GFP based "protein trap" screen and selected fusion proteins which localized to the spindle apparatus. By this method we identified Shaggy, the Drosophila homologue of glycogen synthase kinase-3beta (GSK-3beta), as a component of centrosomes. GSK-3beta acting in the Wingless signaling pathway is involved in a vast range of developmental processes, from pattern formation to cell-fate specification, and is a key factor for cell proliferation in most animals. We exploited our Shaggy::GFP Drosophila line to analyze the subcellular localizations of GSK-3beta/Shaggy and shed light on its multiple roles during embryogenesis. We found that Shaggy becomes enriched transiently in a variety of specialized cytoskeletal structures of the embryo, including centrosomes throughout mitosis, suggesting that this kinase is involved in the regulation of many aspects of the cytoskeleton function.
Assuntos
Centrossomo/enzimologia , Citoesqueleto/enzimologia , Proteínas de Drosophila/metabolismo , Drosophila/enzimologia , Quinase 3 da Glicogênio Sintase/metabolismo , Fuso Acromático/enzimologia , Animais , Proliferação de Células , Centrossomo/fisiologia , Centrossomo/ultraestrutura , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Drosophila/embriologia , Drosophila/genética , Drosophila/ultraestrutura , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Proteína Wnt1RESUMO
In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.
Assuntos
Proteínas de Drosophila/análise , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microtúbulos/análise , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/química , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Embrião não Mamífero/química , Embrião não Mamífero/citologia , Genes de Insetos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Proteínas dos Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The nuclear-cytoplasmic shuttling heterogeneous nuclear RNA-binding protein (hnRNP) Squid (Sqd) is required during Drosophila melanogaster oogenesis, where it plays a critical role in the regulation of the TGFalpha-like molecule Gurken (Grk). Three Sqd isoforms have been described, SqdA, S and B, and two of these, SqdA and SqdS, differentially function in grk mRNA nuclear export, cytoplasmic transport and translational control during oogenesis. Here, we report that Sqd is also required for the regulation of oskar (osk) mRNA, functioning in the cytoplasmic localization of the osk transcript. In oocytes from sqd females, osk mRNA is not efficiently localized to the posterior pole, but rather accumulates at the anterior cortex. Furthermore, anterior patterning defects observed in embryos from sqd females expressing only the SqdS protein isoform suggest that Sqd may also play a role in the translational regulation of the mislocalized osk mRNA. These findings provide additional support for models of mRNA regulation in which cytoplasmic events, such as localization and translational regulation, are coupled. These results also place Sqd among an emerging class of proteins, including such other members as Bruno (Bru) and Hrb27C/Hrp48, which function in multiple aspects of both grk and osk mRNA regulation during Drosophila oogenesis.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Oogênese/fisiologia , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Padronização Corporal , Reagentes de Ligações Cruzadas/farmacologia , Drosophila/anatomia & histologia , Proteínas de Drosophila/genética , Feminino , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Mutação , Oócitos/fisiologia , Ovário/citologia , Ovário/fisiologia , Ovário/efeitos da radiação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismo , Raios UltravioletaRESUMO
In this study, we analyze for the first time endoplasmic reticulum (ER) dynamics and organization during oogenesis and embryonic divisions of Drosophila melanogaster using a Protein Disulfide Isomerase (PDI) GFP chimera protein. An accumulation of ER material into the oocyte takes place during the early steps of oogenesis. The compact organization of ER structures undergoes a transition to an expanded reticular network at fertilization. At the syncytial stage, this network connects to the nuclear envelope as each nucleus divides. Time-lapse confocal microscopy on PDI transgenic embryos allowed us to characterize a rapid redistribution of the ER during the mitotic phases. The ER network is massively recruited to the spindle poles in prophase. During metaphase most of the ER remains concentrated at the spindle poles and shortly thereafter forms several layers of membranes along the ruptured nuclear envelope. Later, during telophase an accumulation of ER material occurs at the spindle equator. We also analyzed the subcellular organization of the ER network at the ultrastructural level, allowing us to corroborate the results from confocal microscopy studies. This dynamic redistribution of ER suggests an unexpected regulatory function for this organelle during mitosis.