RESUMEN
The conserved Ser/Thr protein phosphatase 5 (PP5) is involved in the regulation of key cellular processes, including DNA damage repair and cell division in eukaryotes. As a co-chaperone of Hsp90, PP5 has been shown to modulate the maturation and activity of numerous oncogenic kinases. Here, we identify a novel substrate of PP5, the Polo-like kinase 4 (Plk4), which is the master regulator of centriole duplication in animal cells. We show that PP5 specifically interacts with Plk4, and is able to dephosphorylate the kinase in vitro and in vivo, which affects the interaction of Plk4 with its partner proteins. In addition, we provide evidence that PP5 and Plk4 co-localize to the centrosomes in Drosophila embryos and cultured cells. We demonstrate that PP5 is not essential; the null mutant flies are viable without a severe mitotic phenotype; however, its loss significantly reduces the fertility of the animals. Our results suggest that PP5 is a novel regulator of the Plk4 kinase in Drosophila.
Asunto(s)
Centriolos , Centrosoma , Animales , Centriolos/metabolismo , Centrosoma/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Drosophila/genética , Drosophila/metabolismoRESUMEN
Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecular mechanisms regulating cytoskeletal rearrangements. In this study, we investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium. We show that Shot is essential for the efficient final zippering of the opposing epithelial margins. By using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin-microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. Fluorescence recovery after photobleaching (FRAP) analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule-binding activities of Shot are simultaneously required in the same molecule, indicating that Shot is engaged as a physical crosslinker in this process. We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation, which promotes zippering by initiating contact between opposing epithelial cells.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliales/metabolismo , Proteínas de Microfilamentos/metabolismo , Microtúbulos/metabolismo , Morfogénesis , Actinas/metabolismo , Animales , Proteínas de Drosophila/química , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Células Epiteliales/citología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Microfilamentos/química , Mutación/genética , Dominios Proteicos , Seudópodos/metabolismoRESUMEN
Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , ARN Mensajero/genética , Animales , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosforilación , Unión Proteica , Transporte de ARN/genética , ARN Mensajero/metabolismo , Ribonucleoproteínas/genéticaRESUMEN
Until recently it was widely accepted that the dynamic cytoskeletal matrix is exclusive to the cytoplasm of eukaryotes, evolving before the emergence of the cell nucleus to enable phagocytosis, cell motility and the sophisticated functioning of the endomembrane system within the cytosol. The discovery of the existence of a prokaryotic cytoskeleton has changed this picture significantly. As a result, the idea has taken shape that the appearance of actin occurred in the very first cell; therefore, the emergence of microfilaments precedes that of the eukaryotic cytoskeleton. The discovery of nuclear actin opened new perspective on the field, suggesting that the nuclear activities of actin reflect the functions of primordial actin-like proteins. In this paper, we review the recent literature to explore the evolutionary origin of nuclear actin. We conclude that both ancient and eukaryotic features of the actin world can be detected in the nucleus today, which supports the idea that the cytoskeleton attained significant eukaryotic innovations before the tandem evolution of the cytoskeleton and nucleus occurred.
Asunto(s)
Actinas/metabolismo , Núcleo Celular/metabolismo , Eucariontes/metabolismo , HumanosRESUMEN
Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.
Asunto(s)
Actinas/metabolismo , Nucléolo Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Actinas/genética , Animales , Nucléolo Celular/genética , Humanos , Proteínas de Microfilamentos/genéticaRESUMEN
Ezrin-Radixin-Moesin proteins are highly conserved, actin-binding cytoskeletal proteins that play an essential role in microvilli formation, T-cell activation, and tumor metastasis by linking actin filaments to the plasma membrane. Recent studies demonstrated that the only Ezrin-Radixin-Moesin protein of Drosophila melanogaster, Moesin, is involved in mitotic spindle function through stabilizing cell shape and microtubules at the cell cortex. We previously observed that Moesin localizes to the mitotic spindle; hence, we tested for the biological significance of this surprising localization and investigated whether it plays a direct role in spindle function. To separate the cortical and spindle functions of Moesin during mitosis we combined cell biological and genetic methods. We used early Drosophila embryos, in which mitosis occurs in the absence of a cell cortex, and found in vivo evidence for the direct requirement of Moesin in mitotic spindle assembly and function. We also found that the accumulation of Moesin precedes the construction of the microtubule spindle, and the fusiform structure formed by Moesin persists even after the microtubules have disassembled.
Asunto(s)
Proteínas de la Membrana/metabolismo , Huso Acromático/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Ciclo Celular/fisiología , Forma de la Célula/fisiología , Citoplasma/metabolismo , Drosophila melanogaster , Proteínas de la Membrana/genética , Microtúbulos/metabolismo , Mitosis/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil-DNA glycosylase and dUTPase. Lack of the major uracil-DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200-2,000 uracil/million bases, quantified using a novel real-time PCR-based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil-DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil-DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil-DNA in this evolutionary clade.
Asunto(s)
ADN/genética , Drosophila melanogaster/genética , Larva/genética , Pirofosfatasas , Uracilo , Animales , Línea Celular , ADN/química , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Genoma de los Insectos , Inestabilidad Genómica , Células HeLa , Humanos , Larva/crecimiento & desarrollo , Pirofosfatasas/genética , Interferencia de ARN , Uracilo/química , Uracilo/metabolismo , Uracilo/farmacología , Uracil-ADN Glicosidasa/genéticaRESUMEN
The FERM domain is a conserved and widespread protein module that appeared in the common ancestor of amoebae, fungi, and animals, and is therefore now found in a wide variety of species. The primary function of the FERM domain is localizing to the plasma membrane through binding lipids and proteins of the membrane; thus, for a long time, FERM domain-containing proteins (FDCPs) were considered exclusively cytoskeletal. Although their role in the cytoplasm has been extensively studied, the recent discovery of the presence and importance of cytoskeletal proteins in the nucleus suggests that FDCPs might also play an important role in nuclear function. In this review, we collected data on their nuclear localization, transport, and possible functions, which are still scattered throughout the literature, with special regard to the role of the FERM domain in these processes. With this, we would like to draw attention to the exciting, new dimension of the role of FDCPs, their nuclear activity, which could be an interesting novel direction for future research.
Asunto(s)
Proteínas del Citoesqueleto , Dominios FERM , Animales , Estructura Terciaria de Proteína , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismoRESUMEN
The ERM protein family, which consists of three closely related proteins in vertebrates, ezrin, radixin, and moesin (ERM), is an ancient and important group of cytoplasmic actin-binding and organizing proteins. With their FERM domain, ERMs bind various transmembrane proteins and anchor them to the actin cortex through their C-terminal F-actin binding domain, thus they are major regulators of actin dynamics in the cell. ERMs participate in many fundamental cellular processes, such as phagocytosis, microvilli formation, T-cell activation and tumor metastasis. We have previously shown that, besides its cytoplasmic activities, the single ERM protein of Drosophila melanogaster, moesin, is also present in the cell nucleus, where it participates in gene expression and mRNA export. Here we study the mechanism by which moesin enters the nucleus. We show that the nuclear import of moesin is an NLS-mediated, active process. The nuclear localization sequence of the moesin protein is an evolutionarily highly conserved, conventional bipartite motif located on the surface of the FERM domain. Our experiments also reveal that the nuclear import of moesin does not require PIP2 binding or protein activation, and occurs in monomeric form. We propose, that the balance between the phosphorylated and non-phosphorylated protein pools determines the degree of nuclear import of moesin.
RESUMEN
Transposons are genetic elements that use various mechanisms of transposition to move around the genome, thus posing a risk to genomic integrity. Repression of transposable elements (TEs) involves the complex PIWI pathway and several proteins associated with heterochromatinization. All players of TE repression are indispensable for proper reproductive fitness, as loss-of-function mutations in these genes result primarily in sterility and impaired reproductive development. When investigating the function of novel genes with similar phenotypes, elevated transposon expression in reproductive tissues can be a marker for involvement in the aforementioned processes. Here, we present a protocol for investigating TE levels in adult Drosophila ovaries, from dissection to data analysis.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Femenino , Drosophila/genética , Drosophila/metabolismo , Ovario/metabolismo , Elementos Transponibles de ADN/genética , ARN Interferente Pequeño/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMEN
Much evidence supports the presence of cytoskeletal elements in the nucleus; however, the exact functions of these proteins in the nucleus are still uncertain. Of the cytoskeletal proteins, the activity and biological significance of nuclear actin has been the most extensively researched. It is now clear that actin performs essential tasks both in the cytoplasm and the nucleus, and that the dynamic balance between the large cytoplasmic and the significantly smaller nuclear actin pools is maintained by robust transport mechanisms. Therefore, the compartment-specific manipulation or investigation of actin has been an enormous challenge. Here, we present a protocol for the detection of actin in isolated nuclear protein fractions from Drosophila ovaries.
Asunto(s)
Actinas , Proteínas Nucleares , Animales , Femenino , Actinas/metabolismo , Proteínas Nucleares/metabolismo , Ovario/metabolismo , Drosophila/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismoRESUMEN
Actin, as an ancient and fundamental protein, participates in various cytoplasmic as well as nuclear functions in eukaryotic cells. Based on its manifold tasks in the nucleus, it is a reasonable assumption that the nuclear presence of actin is essential for the cell, and consequently, its nuclear localization is ensured by a robust system. However, today only a single nuclear import and a single nuclear export pathway is known which maintain the dynamic balance between cytoplasmic and nuclear actin pools. In our work, we tested the robustness of the nuclear import of actin, and investigated whether the perturbations of nuclear localization affect the viability of the whole organism. For this aim, we generated a genetic system in Drosophila, in which we rescued the lethal phenotype of the null mutation of the Actin5C gene with transgenes that express different derivatives of actin, including a Nuclear Export Signal (NES)-tagged isoform which ensures forced nuclear export of the protein. We also disrupted the SUMOylation site of actin, suggested earlier to be responsible for nuclear retention, and eliminated the activity of the single nuclear import factor dedicated to actin. We found that, individually, none of the above mentioned manipulations led to a notable reduction in nuclear actin levels and thus, fully rescued lethality. However, the NES tagging of actin, together with the knock out of its importin, significantly reduced the amount of nuclear actin and induced lethality, confirming that the presence of actin in the nucleus is essential, and thereby, over-secured. Supporting this, we identified novel nuclear importins specific to actin, which sheds light on the mechanism behind the robustness of nuclear localization of actin, and supports the idea of essentiality of its nuclear functions.
RESUMEN
Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the in vivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression of protein chaperones requires the nuclear functions of Moesin. DATABASE: GEO accession number: GSE155778.
Asunto(s)
Proteínas de la Membrana/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Núcleo Celular/metabolismo , Drosophila , Regulación de la Expresión Génica/genética , Proteínas de la Membrana/genéticaRESUMEN
The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.
Asunto(s)
Proteínas de Drosophila/inmunología , Drosophila/inmunología , Hemocitos/inmunología , Fagocitosis , Receptores Inmunológicos/inmunología , Secuencias de Aminoácidos , Animales , Drosophila/citología , Drosophila/microbiología , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Escherichia coli/inmunología , Hemocitos/citología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Staphylococcus aureus/inmunologíaRESUMEN
The dorsoventral axis of the Drosophila embryo is patterned by a gradient of bone morphogenetic protein (BMP) ligands. In a process requiring at least three additional extracellular proteins, a broad domain of weak signaling forms and then abruptly sharpens into a narrow dorsal midline peak. Using experimental and computational approaches, we investigate how the interactions of a multiprotein network create the unusual shape and dynamics of formation of this gradient. Starting from observations suggesting that receptor-mediated BMP degradation is an important driving force in gradient dynamics, we develop a general model that is capable of capturing both subtle aspects of gradient behavior and a level of robustness that agrees with in vivo results.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica/fisiología , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/genética , Procesamiento Automatizado de Datos , Embrión no Mamífero , Técnica del Anticuerpo Fluorescente/métodos , Genotipo , Hibridación in Situ/métodos , Redes Neurales de la Computación , Factores de TiempoRESUMEN
Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.
Asunto(s)
Sitios de Unión , Secuencia Conservada , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Humanos , Fosfoproteínas Fosfatasas/genética , Unión Proteica , Relación Estructura-ActividadRESUMEN
Previously, a novel cell type, the multinucleated giant hemocyte (MGH) was identified in the ananassae subgroup of Drosophilidae. These cells share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation. We were able to show that MGHs also differentiate in Zaprionus indianus, an invasive species belonging to the vittiger subgroup of the family, highly resistant to a large number of parasitoid wasp species. We have classified the MGHs of Z. indianusas giant hemocytes belonging to a class of cells which also include elongated blood cells carrying a single nucleus and anuclear structures. They are involved in encapsulating parasites, originate from the lymph gland, can develop by cell fusion, and generally carry many nuclei, while possessing an elaborated system of canals and sinuses, resulting in a spongiform appearance. Their nuclei are all transcriptionally active and show accretion of genetic material. Multinucleation and accumulation of the genetic material in the giant hemocytes represents a two-stage amplification of the genome, while their spongy ultrastructure substantially increases the contact surface with the extracellular space. These features may furnish the giant hemocytes with a considerable metabolic advantage, hence contributing to the mechanism of the effective immune response.
Asunto(s)
Drosophilidae/inmunología , Genoma de los Insectos , Células Gigantes/inmunología , Hemocitos/inmunología , Inmunidad Celular , Animales , Drosophilidae/genéticaRESUMEN
Autophagy ensures the turnover of cytoplasm and requires the coordinated action of Atg proteins, some of which also have moonlighting functions in higher eukaryotes. Here we show that the transmembrane protein Atg9 is required for female fertility, and its loss leads to defects in actin cytoskeleton organization in the ovary and enhances filopodia formation in neurons in Drosophila. Atg9 localizes to the plasma membrane anchor points of actin cables and is also important for the integrity of the cortical actin network. Of note, such phenotypes are not seen in other Atg mutants, suggesting that these are independent of autophagy defects. Mechanistically, we identify the known actin regulators profilin and Ena/VASP as novel binding partners of Atg9 based on microscopy, biochemical, and genetic interactions. Accordingly, the localization of both profilin and Ena depends on Atg9. Taken together, our data identify a new and unexpected role for Atg9 in actin cytoskeleton regulation.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Profilinas/metabolismo , Alelos , Animales , Autofagia , Proteínas Relacionadas con la Autofagia/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Femenino , Fertilidad , Proteínas de la Membrana/genética , Mutación/genética , Neuronas/metabolismo , Unión Proteica , Transporte de Proteínas , Seudópodos/metabolismo , TransgenesRESUMEN
Hemocytes, cellular elements of the innate immune system in insects, play a crucial role in the cellular and humoral immune response. Although a significant amount of information has been collected on their differentiation and function, our understanding of hemocyte development is far from complete. Their characterisation is mostly based on morphological criteria. However, molecular markers were recently identified, defining functional subsets by the aid of monoclonal antibodies. Isolated subsets of hemocytes, in sufficient quantity and purity could help to analyse their development in vitro and also to further define their molecular characteristics. Here we describe an antibody-based rosetting technique for the physical separation of Drosophila hemocyte sub-populations. We have applied anti-hemocyte antibodies coupled to sheep red blood cells for separation. The method relies on the formation of rosettes between hemocytes and sheep erythrocytes, sensitised with discriminative anti-hemocyte monoclonal antibodies. Using this method the rosetting and non-rosetting hemocytes can be separated from each other by gradient centrifugation. Rosette-forming cells from the pellet and non-rosetting cells from the interface can be isolated in high recovery. The method can be used for functional and molecular characterisation of hemocyte sub-populations. The procedure is sensitive, reproducible and easy to perform.
Asunto(s)
Separación Celular/métodos , Drosophila melanogaster/inmunología , Eritrocitos/inmunología , Hemocitos/citología , Formación de Roseta/métodos , Animales , Anticuerpos Monoclonales/inmunología , Eritrocitos/citología , Técnica del Anticuerpo Fluorescente Indirecta , Hemocitos/inmunología , Inmunofenotipificación , Fagocitosis , OvinosRESUMEN
In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general.