RESUMEN
Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. We found that beta-amyloid (Abeta42) initiated Abl kinase activity and that blockade of Abl kinase rescued both Drosophila and mammalian neuronal cells from cell death. We also found activated Abl kinase to be necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 was not observed in Abeta42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. Our genetic studies also showed that abl mutations repressed Abeta42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Abeta42 induced conversion of p35 to p25 in mammalian cells, it did not sufficiently induce Cdk5 activation when c-Abl kinase activity was suppressed. Therefore, we propose that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Abeta42-triggered neurodegeneration.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Drosophila melanogaster/enzimología , Regulación Enzimológica de la Expresión Génica , Degeneración Nerviosa/enzimología , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Animales , Línea Celular , Drosophila melanogaster/metabolismo , Activación Enzimática , Datos de Secuencia Molecular , Degeneración Nerviosa/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuronas/enzimología , Fragmentos de Péptidos/metabolismo , Fosforilación , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-abl/fisiología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The baculovirus expression vector system is considered to be a safe, powerful, but cell-lytic heterologous protein expression system in insect cells. We show here that there is a new baculovirus system for efficient gene transfer and expression using the popular and genetically well-understood Drosophila S2 cells. The recombinant baculovirus was constructed to carry an enhanced green fluorescent protein under the control of polyhedrin promoter as a fluorescent selection marker in the Sf21 cell line. Recombinant baculoviruses were then used to transduce S2 cells with target gene expression cassettes containing a Drosophila heat shock protein 70, an actin 5C, or a metallothionein promoter. Nearly 100% of the S2 cells showed evidence of gene expression after infection. The time course for the optimal protein expression peaked at 24 to 36 h postinfection, which is significantly earlier than a polyhedrin-driven protein expression in Sf21 cells. Importantly, S2 cells did not appear to be lysed after infection, and the protein expression levels are comparable to those of proteins under the control of polyhedrin promoter in several lepidopteran cell lines. Most surprisingly, S2 cells permit repetitive infections of multiple baculoviruses over time. These findings clearly suggest that this baculovirus-S2 system may effect the efficient gene transfer and expression system of the well-characterized Drosophila S2 cells.
Asunto(s)
Baculoviridae , Drosophila/virología , Vectores Genéticos , Sobreinfección , Transfección , Animales , Células Cultivadas , Regulación Viral de la Expresión Génica , Recombinación GenéticaRESUMEN
C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster, an invertebrate species supposed to be void of detectable m(5)CpG. We now report the genomic structure of a Drosophila gene, dMBD2/3, that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Delta. Interestingly, in vitro binding experiments showed that as was the case for vertebrate MBD proteins, dMBD2/3Delta could preferentially recognize m(5)CpG-containing DNA through its MBD. Furthermore, dMBD2/3Delta as well as one of its orthologs in mouse, MBD2b, could function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appeared to be dispensable for transcriptional repression by dMBD2/3Delta. Finally, dMBD2/3Delta also could repress transcription effectively in transfected Drosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions.
Asunto(s)
Islas de CpG , Metilación de ADN , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Genes de Insecto , Proteínas de Insectos/fisiología , Proteínas Represoras/fisiología , Transcripción Genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatina/metabolismo , Cromatina/ultraestructura , ADN/genética , ADN/metabolismo , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Evolución Molecular , Histona Desacetilasas/fisiología , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/fisiología , Especificidad de la EspecieRESUMEN
The blood-brain barrier (BBB) ensures brain function in vertebrates and insects by maintaining ionic integrity of the neuronal bathing fluid. Without this barrier, paralysis and death ensue. The structural analogs of the BBB are occlusive (pleated-sheet) septate and tight junctions between perineurial cells, glia and perineurial cells, and possibly between glia. Immature Diptera have such septate junctions (without tight junctions) while both junctional types are found in the imago. Genetic and molecular biology of these junctions are discussed, namely tight (occludin) and pleated-sheet septate (neurexin IV). A temporal succession of blood barriers form in immature Diptera. The first barrier forms in the peripheral nervous system where pleated-sheet septate junctions bond cells of the nascent (embryonic) chordotonal organs in early neurogenesis. At the end of embryonic life, the central nervous system is fully vested with a blood-brain barrier. A blood-eye barrier arises in early pupal life. Future prospects in blood-barrier research are discussed.
Asunto(s)
Insectos/fisiología , Animales , Barrera HematoencefálicaRESUMEN
Human and mouse Abelson interacting proteins (Abi) are SH3-domain containing proteins that bind to the proline-rich motifs of the Abelson protein tyrosine kinase. We report a new member of this gene family, a Drosophila Abi (dAbi) that is a substrate for Abl kinase and that co-immunoprecipitates with Abl if the Abi SH3 domain is intact. We have identified a new function for both dAbi and human Abi-2 (hAbi-2). Both proteins activate the kinase activity of Abl as assayed by phosphorylation of the Drosophila Enabled (Ena) protein. Removal of the dAbi SH3 domain eliminates dAbi's activation of Abl kinase activity. dAbi is an unstable protein in cells and is present at low steady state levels but its protein level is increased coincident with phosphorylation by Abl kinase. Expression of the antisense strand of dAbi reduces dAbi protein levels and abolishes activation of Abl kinase activity. Modulation of Abi protein levels may be an important mechanism for regulating the level of Abl kinase activity in the cell.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/fisiología , Proteínas del Citoesqueleto , Proteínas de Drosophila , Drosophila melanogaster/enzimología , Proteínas de Homeodominio/fisiología , Proteínas de Insectos/aislamiento & purificación , Proteínas Oncogénicas v-abl/fisiología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-abl/fisiología , Virus de la Leucemia Murina de Abelson/enzimología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Clonación Molecular , Secuencia de Consenso , Drosophila melanogaster/genética , Activación Enzimática , Evolución Molecular , Proteínas de Homeodominio/química , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/fisiología , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas v-abl/química , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/química , Proteínas Proto-Oncogénicas c-abl/química , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transfección , Dominios Homologos srcRESUMEN
We targeted expression of human/fly chimeric Bcr-Abl proteins to the developing central nervous system (CNS) and eye imaginal disc of Drosophila melanogaster. Neural expression of human/fly chimeric P210 Bcr-Abl or P185 Bcr-Abl rescued abl mutant flies from pupal lethality, indicating that P210 and P185 Bcr-Abl can substitute functionally for Drosophila Abl during axonogenesis. However, increased levels of neurally expressed P210 or P185 Bcr-Abl but not Drosophila Abl produced CNS defects and lethality. Expression of P210 or P185 in the eye imaginal disc produced a dominant rough eye phenotype that was dependent on dosage of the transgene. Drosophila Enabled, previously identified as a suppressor of the abl mutant phenotype and substrate for Drosophila Abl kinase, had markedly increased phosphotyrosine levels in Bcr-Abl expressing Drosophila, indicating that it is a substrate for Bcr-Abl as well. Drosophila, therefore, is a suitable model system to identify Bcr-Abl interactions important for signal transduction and oncogenesis.
Asunto(s)
Drosophila melanogaster/embriología , Proteínas de Fusión bcr-abl/metabolismo , Animales , Animales Modificados Genéticamente , Sistema Nervioso Central/embriología , Proteínas de Unión al ADN/metabolismo , Ojo/embriología , Proteínas de Fusión bcr-abl/genética , Expresión Génica , Genes abl , Humanos , Morfogénesis , Fosforilación , Especificidad por SustratoRESUMEN
Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.
Asunto(s)
Proteínas de Unión al ADN/genética , Drosophila/genética , Genes abl , Animales , Proteínas de Unión al ADN/metabolismo , Drosophila/metabolismo , Mutación , Fosforilación , Unión Proteica , Transducción de Señal/genética , Dominios Homologos src/genéticaRESUMEN
Chordotonal organs of Drosophila embryos have become models for studies of developmental biology and molecular genetics due to their consistent segmental placement and mutability. Our first goal was to find the origin and anatomical correlate of the blood-nerve barrier of this PNS proprioreceptor in wild type embryos. The concept of a blood-nerve barrier for the PNS of the Drosophila embryo is new, and the present data are the first in this regard. A second goal was to reveal the ultrastructure of these four-celled stretch receptors, focusing particularly on the 'core' of this organ: the bipolar neuron enclosed by a scolopale cell. These latter data have resulted in a graphic reconstruction of the chordotonal organ which reveals how the four consistent cells fit together. At Stage 13 we first observed a clearly recognizable scolopale cell with an enclosed neuron. Surprisingly, an operative blood-nerve barrier, comprised of occlusive pleated-sheet septate junctions, exists at this relatively early stage. A blood-brain barrier is not yet functioning in the CNS during this same stage, as the perineurium is not present until Stage 17. Cross-sectional views of a more mature chordotonal organ show that the neuron's inner segment has a 'tongue-in-groove' formation which fits the dendrite into the scolopale cell. Other newly discovered fine structural features are: hemidesmosomes linking individual scolopale rod bundles to the inner dendrite, and a cap cell matrix bonding with the tip of the ciliary dendrite. Functional aspects of these findings are discussed.
Asunto(s)
Drosophila/embriología , Drosophila/ultraestructura , Animales , Anticuerpos Monoclonales , Drosophila/anatomía & histología , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/ultraestructura , Hemocitos/ultraestructura , Inmunohistoquímica , Microscopía Electrónica , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Sistema Nervioso/química , Sistema Nervioso/ultraestructuraRESUMEN
The chordotonal (scolopidial) organ is a segmental stretch receptor (proprioceptor) and a four-celled organ of the peripheral nervous system of Drosophila. This organ has become a model in studies of embryogenesis involving molecular genetics and developmental biology. We determined how three glial cells and a bipolar neuron (the chordotonal organ) develop and assemble to become a sensitive stretch receptor. Our focus was on the scolopale cell-neuron association which is the core of this organ. The first anatomical appearance of these developing organs appears in Stage 12 embryos: a central fissure forms in the scolopale cell and two intracellular adherence loci redirect the cleft to wall off a cylindrical sector. This portion hollows out and the dendrite of a sensory neuron enters the cavity. Cytoplasmic walls of the cylinder then regress to leave a more prominent lymph space, within which is the cilary portion of the sensory dendrite. A cap (glial) cell then covers and connects the distal portion of the scolopale-neuron pair. Chordotonal organs are formed in about two hours and assembled throughout Stages 13 to 15. We have drawn schematics of these developmental phases with the resultant four-celled organ. Lanthanum tracer in the embryonic hemocoel never gained access to the neuron housed by the scolopale cell. Thus a blood-nerve barrier forms as early as Stage 13 in the peripheral nervous system. Paracellular clefts, sealed by occlusive septate junctions between accessory (glial) cells, are the probable basis for barrier properties.
Asunto(s)
Drosophila/embriología , Neuroglía/fisiología , Neuronas/fisiología , Animales , Microscopía Electrónica , Neuroglía/ultraestructura , Neuronas/ultraestructuraRESUMEN
Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, disabled, reveal that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase.
Asunto(s)
Axones/fisiología , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto , Proteínas del Tejido Nervioso/genética , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Western Blotting , Cartilla de ADN , Drosophila melanogaster/enzimología , Drosophila melanogaster/fisiología , Genes Dominantes , Genes Letales , Genotipo , Heterocigoto , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis , Proteínas del Tejido Nervioso/biosíntesis , Fenómenos Fisiológicos del Sistema Nervioso , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-abl/genéticaRESUMEN
Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identified alleles of only one gene, enabled (ena). We report that the ena protein contains proline-rich motifs and binds to Abl and Src SH3 domains, ena is also a substrate for the Abl kinase; tyrosine phosphorylation of ena is increased when it is coexpressed in cells with human or Drosophila Abl and endogenous ena tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos have defects in axonal architecture. We conclude that a critical function of Drosophila Abl is to phosphorylate and negatively regulate ena protein during neural development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Drosophila/enzimología , Genes Supresores/genética , Genes abl/genética , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila/embriología , Drosophila/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Genes de Insecto/genética , Humanos , Masculino , Datos de Secuencia Molecular , Sistema Nervioso/química , Sistema Nervioso/embriología , Fosforilación , Fosfotirosina , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Especificidad por Sustrato , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
The blood-brain barrier ensures brain function in vertebrates and in some invertebrates by maintaining ionic integrity of the extraneuronal bathing fluid. Recent studies have demonstrated that anionic sites on the luminal surface of vascular endothelial cells collaborate with tight junctions to effect this barrier in vertebrates. We characterize these two analogous barrier factors for the first time on Drosophila larva by an electron-dense tracer and cationic gold labeling. Ionic lanthanum entered into but not through the extracellular channels between perineurial cells. Tracer is ultimately excluded from neurons in the ventral ganglion mainly by an extensive series of (pleated sheet) septate junctions between perineurial cells. Continuous junctions, a variant of the septate junction, were not as efficient as the pleated sheet variety in blocking tracer. An anionic domain now is demonstrated in Drosophila central nervous system through the use of cationic colloidal gold in LR White embedment. Anionic domains are specifically stationed in the neural lamella and not noted in the other cell levels of the blood-brain interface. It is proposed that in the central nervous system of the Drosophila larva the array of septate junctions between perineurial cells is the physical barrier, while the anionic domains in neural lamella are a "charge-selective barrier" for cations. All of these results are discussed relative to analogous characteristics of the vertebrate blood-brain barrier.
Asunto(s)
Membrana Celular/fisiología , Drosophila melanogaster/fisiología , Sistema Nervioso/ultraestructura , Neuronas/ultraestructura , Animales , Barrera Hematoencefálica , Membrana Celular/ultraestructura , Drosophila melanogaster/ultraestructura , Ganglios de Invertebrados/fisiología , Ganglios de Invertebrados/ultraestructura , Larva , Microscopía Electrónica , Modelos Biológicos , Fenómenos Fisiológicos del Sistema Nervioso , Neuroglía/ultraestructura , VertebradosRESUMEN
Using scanning and transmission electron microscopy, we studied basic ultrastructure, membrane specializations, and blood-brain barrier properties of the ventral ganglion and abdominal nerves of the last (third) instar larva of a dipteran fly, Delia platura. Both ganglion and nerves are covered with a non-cellular neural lamella. A monolayer of flattened perineurial cells lies beneath the neural lamella. Perineurial cells contain stores of metabolites and nutrients and these cells extensively interdigitate with one another. An extensive extracellular series of channels pervades perineurial cells. Glial cells beneath the perineurium envelope but do not entwine axons. In a minority of cases, adjacent axons in nerve and neuropil appear to be contiguous without glial intervention. Extensive (pleated) septate junctions with triangular septa are present between perineurial cells. Hemidesmosomes, half desmosomes (a first report for invertebrates), and desmosomes were also observed. Although no tight junctions were discovered, an effective blood-brain barrier exists, and tracer (ionic lanthanum) in no case reached neuronal surfaces. Extracellular tracer halted within the extensive septate junctions between perineurial cells. We postulate that in the absence of tight junctions the functional blood-brain barrier is effected by the septate junctions in the central nervous system of the Delia larva.