RESUMO
The molecular mechanisms driving the conserved metazoan developmental shift referred to as the mid-blastula transition (MBT) remain mysterious. Typically, cleavage divisions give way to longer asynchronous cell cycles with the acquisition of a gap phase. In Drosophila, rapid synchronous nuclear divisions must pause at the MBT to allow the formation of a cellular blastoderm through a special form of cytokinesis termed cellularization. Drosophila Fragile X mental retardation protein (dFMRP; FMR1), a transcript-specific translational regulator, is required for cellularization. The role of FMRP has been most extensively studied in the nervous system because the loss of FMRP activity in neurons causes the misexpression of specific mRNAs required for synaptic plasticity, resulting in mental retardation and autism in humans. Here, we show that in the early embryo dFMRP associates specifically with Caprin, another transcript-specific translational regulator implicated in synaptic plasticity, and with eIF4G, a key regulator of translational initiation. dFMRP and Caprin collaborate to control the cell cycle at the MBT by directly mediating the normal repression of maternal Cyclin B mRNA and the activation of zygotic frühstart mRNA. These findings identify two new targets of dFMRP regulation and implicate conserved translational regulatory mechanisms in processes as diverse as learning, memory and early embryonic development.
Assuntos
Ciclo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Ciclina B/genética , Drosophila/citologia , Proteínas de Drosophila/genética , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ligação ProteicaRESUMO
Drosophila melanogaster cellularization is a dramatic form of cytokinesis in which a membrane furrow simultaneously encapsulates thousands of cortical nuclei of the syncytial embryo to generate a polarized cell layer. Formation of this cleavage furrow depends on Golgi-based secretion and microtubules. During cellularization, specific Golgi move along microtubules, first to sites of furrow formation and later to accumulate within the apical cytoplasm of the newly forming cells. Here we show that Golgi movements and furrow formation depend on cytoplasmic dynein. Furthermore, we demonstrate that Lava lamp (Lva), a golgin protein that is required for cellularization, specifically associates with dynein, dynactin, cytoplasmic linker protein-190 (CLIP-190) and Golgi spectrin, and is required for the dynein-dependent targeting of the secretory machinery. The Lva domains that bind these microtubule-dependent motility factors inhibit Golgi movement and cellularization in a live embryo injection assay. Our results provide new evidence that golgins promote dynein-based motility of Golgi membranes.
Assuntos
Citocinese/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Complexo Dinactina , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/fisiologia , Feminino , Complexo de Golgi/ultraestrutura , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Espectrina/metabolismoRESUMO
Fragile X mental retardation protein (FMRP) is an RNA-binding protein that is required for the translational regulation of specific target mRNAs. Loss of FMRP causes Fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. Understanding the basis for FXS has been limited because few in vivo targets of FMRP have been identified and mechanisms for how FMRP regulates physiological targets are unclear. We have previously demonstrated that Drosophila FMRP (dFMRP) is required in early embryos for cleavage furrow formation. In an effort to identify new targets of dFMRP-dependent regulation and new effectors of cleavage furrow formation, we used two-dimensional difference gel electrophoresis and mass spectrometry to identify proteins that are misexpressed in dfmr1 mutant embryos. Of the 28 proteins identified, we have identified three subunits of the Chaperonin containing TCP-1 (CCT) complex as new direct targets of dFMRP-dependent regulation. Furthermore, we found that the septin Peanut, a known effector of cleavage, is a likely conserved substrate of fly CCT and is mislocalized in both cct and in dfmr1 mutant embryos. Based on these results we propose that dFMRP-dependent regulation of CCT subunits is required for cleavage furrow formation and that at least one of its substrates is affected in dfmr1- embryos suggesting that dFMRP-dependent regulation of CCT contributes to the cleavage furrow formation phenotype.
Assuntos
Chaperonina com TCP-1/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sequência de Aminoácidos , Animais , Blástula/embriologia , Chaperonina com TCP-1/química , Chaperonina com TCP-1/genética , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Eletroforese em Gel Bidimensional , Embrião não Mamífero/metabolismo , Imunofluorescência , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. We report a saturating genetic screen of the Drosophila autosomal genome to identify functional partners of dFmr1. We recovered 19 mutations in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. We provide direct evidence for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. Our data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Drosophila/metabolismo , Genes Supressores de Tumor/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Western Blotting/métodos , Fracionamento Celular/métodos , Células Cultivadas , Clonagem Molecular/métodos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Drosophila , Olho/patologia , Olho/ultraestrutura , Proteína do X Frágil da Deficiência Intelectual , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica/métodos , Camundongos , Microscopia Eletrônica de Varredura/métodos , Mutagênese , Mutação , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/metabolismo , Retina/patologia , Retina/ultraestrutura , Frações Subcelulares/metabolismo , Sinapses/metabolismo , Fatores de TempoRESUMO
Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Exocitose/fisiologia , Olho/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Junções Aderentes/genética , Junções Aderentes/metabolismo , Junções Aderentes/ultraestrutura , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/ultraestrutura , Células Epiteliais/ultraestrutura , Olho/metabolismo , Olho/ultraestrutura , Feminino , Células Germinativas/metabolismo , Células Germinativas/ultraestrutura , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação/genética , Células Fotorreceptoras de Invertebrados/ultraestrutura , Transporte Proteico/fisiologia , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/isolamento & purificação , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The early Drosophila embryo undergoes two distinct membrane invagination events believed to be mechanistically related to cytokinesis: metaphase furrow formation and cellularization. Both involve actin cytoskeleton rearrangements, and both have myosin II at or near the forming furrow. Actin and myosin are thought to provide the force driving membrane invagination; however, membrane addition is also important. We have examined the role of myosin during these events in living embryos, with a fully functional myosin regulatory light-chain-GFP chimera. We find that furrow invagination during metaphase and cellularization occurs even when myosin activity has been experimentally perturbed. In contrast, the basal closure of the cellularization furrows and the first cytokinesis after cellularization are highly dependent on myosin. Strikingly, when ingression of the cellularization furrow is experimentally inhibited by colchicine treatment, basal closure still occurs at the appropriate time, suggesting that it is regulated independently of earlier cellularization events. We have also identified a previously unrecognized reservoir of particulate myosin that is recruited basally into the invaginating furrow in a microfilament-independent and microtubule-dependent manner. We suggest that cellularization can be divided into two distinct processes: furrow ingression, driven by microtubule mediated vesicle delivery, and basal closure, which is mediated by actin/myosin based constriction.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Drosophila melanogaster/embriologia , Fusão de Membrana/fisiologia , Miosina Tipo II/metabolismo , Animais , Divisão Celular/fisiologia , Colchicina/farmacologia , Citoesqueleto/efeitos dos fármacos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Fusão de Membrana/efeitos dos fármacos , Microscopia ConfocalRESUMO
We describe the maternal-effect and zygotic phenotypes of null mutations in the Drosophila gene for the epsilon-subunit of mitochondrial ATP synthase, stunted (sun). Loss of zygotic sun expression leads to a dramatic delay in the growth rate of first instar larvae and ultimately death. Embryos lacking maternally supplied sun (sun embryos) have a sixfold reduction in ATP synthase activity. Cellular analysis of sun embryos shows defects only after the nuclei have migrated to the cortex. During the cortical divisions the actin-based metaphase and cellularization furrows do not form properly, and the nuclei show abnormal spacing and division failures. The most striking abnormality is that nuclei and spindles form lines and clusters, instead of adopting a regular spacing. This is reflected in a failure to properly position neighboring nonsister centrosomes during the telophase-to-interphase transition of the cortical divisions. Our study is consistent with a role for Sun in mitochondrial ATP synthesis and suggests that reduced ATP levels selectively affect molecular motors. As Sun has been identified as the ligand for the Methuselah receptor that regulates aging, Sun may function both within and outside mitochondria.
Assuntos
Drosophila/embriologia , ATPases Mitocondriais Próton-Translocadoras/química , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Movimento Celular , Núcleo Celular/metabolismo , Centrossomo/ultraestrutura , Citoesqueleto/metabolismo , DNA/metabolismo , Drosophila/fisiologia , Feminino , Interfase , Ligantes , Masculino , Microscopia de Fluorescência , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/fisiologia , Modelos Genéticos , Modelos Moleculares , Proteínas Motores Moleculares , Dados de Sequência Molecular , Fenótipo , Homologia de Sequência de Aminoácidos , Fuso Acromático , TelófaseRESUMO
We conducted a prospective, inpatient fever study in malaria-endemic Papua, Indonesia to determine non-malaria fever etiologies. Investigations included malaria blood films, blood culture, paired serologic samples analysis for dengue, Japanese encephalitis, leptospirosis, scrub typhus, murine typhus, and spotted fever group rickettsia. During 1997-2000, 226 patients (127 males and 99 females) 1-80 years of age (median age = 25 years) were enrolled. Positive blood cultures (n = 34, 15%) were obtained for Salmonella Typhi (n = 13), Escherichia coli (n = 8), Streptococcus pneumoniae (n = 6), Staphylococcus aureus (n = 5), Streptococcus pyogenes (n = 1), and Klebsiella pneumoniae (n = 1). Twenty (8.8%) patients were positive for leptospirosis by polymerase chain reaction. Eighty (35.4%) of 226 patients had ≥ 1 positive serology, diagnostic for 15 rickettsial and 9 dengue cases. Acid-fast bacilli-positive sputum was obtained from three patients. Most common confirmed (81 of 226, 35.8%)/suspected diagnoses were typhoid fever (n = 41), pneumonia (n = 29), leptospirosis (n = 28), urinary tract infections (n = 20), rickettsioses (n = 19), dengue (n = 17), and meningitis/encephalitis (n = 15). There were 17 deaths, 7 (46.7%) were caused by meningitis/encephalitis. Multiple positive serologic results and few confirmed diagnoses indicate the need for improved diagnostics.
Assuntos
Infecções Bacterianas/complicações , Febre/epidemiologia , Febre/etiologia , Viroses/complicações , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/complicações , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Bacteriemia/mortalidade , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/mortalidade , Infecções do Sistema Nervoso Central/complicações , Infecções do Sistema Nervoso Central/epidemiologia , Infecções do Sistema Nervoso Central/etiologia , Infecções do Sistema Nervoso Central/mortalidade , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Papua Nova Guiné/epidemiologia , Viroses/epidemiologia , Viroses/mortalidade , Viroses/virologia , Adulto JovemRESUMO
INTRODUCTIONThe Drosophila embryo is a superb source of proteins for studies aimed at characterizing a variety of cellular functions, including replication, transcription, translation, signal transduction, and the functions of the extracellular matrix and the cytoskeleton. The success of these studies requires the efficient production of gram quantities of embryos by large populations of adult flies. This protocol outlines a simple and efficient method for maintaining a population of Drosophila.
RESUMO
INTRODUCTIONThe Drosophila embryo is a superb source of proteins for studies aimed at characterizing a variety of cellular functions, including replication, transcription, translation, signal transduction, and the functions of the extracellular matrix and the cytoskeleton. The success of these studies is dependent upon the maintenance of an efficient laboratory fly facility. This article outlines the basic requirements for maintaining Drosophila in large or small laboratories.
RESUMO
A method for assembling Drosophila embryos in a microfluidic device was developed for studies of thermal perturbation of early embryonic development. Environmental perturbation is a complimentary method to injection of membrane-impermeable macromolecules for assaying genetic function and investigating robustness in complex biochemical networks. The development of a high throughput method for perturbing embryos would facilitate the isolation and mapping of signaling pathways. We immobilize Drosophila embryos inside a microfluidic device on minimal potential-energy wells created through surface modification, and thermally perturb these embryos using binary laminar flows of warm and cold solutions. We self-assemble embryos onto oil adhesive pads with an alcohol surfactant carrier fluid (detachment: 0.1 mL/min), and when the surfactant is removed, the embryo-oil adhesion increases to approximately 25 mL/min flow rates, which allows for high velocities required for sharp gradients of thermal binary flows. The microfluidic thermal profile was numerically characterized by simulation and experimentally characterized by fluorescence thermometry. The effects of thermal perturbation were observed to induce abnormal morphogenetic movements in live embryos by using time-lapse differential interference contrast (DIC) microscopy.
Assuntos
Drosophila/embriologia , Técnicas Analíticas Microfluídicas/métodos , Morfogênese , Animais , Calibragem , Simulação por Computador , Dimetilpolisiloxanos/química , Drosophila/citologia , Embrião não Mamífero , Desenho de Equipamento , Etanol/química , Metanol/química , Técnicas Analíticas Microfluídicas/instrumentação , Óleos/química , Polímeros/química , Tensão Superficial , Temperatura , Água/químicaRESUMO
During the cleavage stage of animal embryogenesis, cell numbers increase dramatically without growth, and a shift from maternal to zygotic genetic control occurs called the midblastula transition. Although these processes are fundamental to animal development, the molecular mechanisms controlling them are poorly understood. Here, we demonstrate that Drosophila fragile X mental retardation protein (dFMRP) is required for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the midblastula transition. dFMRP is observed to colocalize with the cytoplasmic RNP body components Maternal expression at 31B (ME31B) and Trailer Hitch (TRAL) in a punctate pattern throughout the cytoplasm of cleavage-stage embryos. Complementary biochemistry demonstrates that dFMRP does not associate with polyribosomes, consistent with their reported exclusion from many cytoplasmic RNP bodies. By using a conditional mutation in small bristles (sbr), which encodes an mRNA nuclear export factor, to disrupt the normal cytoplasmic accumulation of zygotic transcripts at the midblastula transition, we observe the formation of giant dFMRP/TRAL-associated structures, suggesting that dFMRP and TRAL dynamically regulate RNA metabolism at the midblastula transition. Furthermore, we show that dFMRP associates with endogenous tral mRNA and is required for normal TRAL protein expression and localization, revealing it as a previously undescribed target of dFMRP control. We also show genetically that tral itself is required for cleavage furrow formation. Together, these data suggest that in cleavage-stage Drosophila embryos, dFMRP affects protein expression by controlling the availability and/or competency of specific transcripts to be translated.