RESUMEN
One of a major function of programmed cell death (apoptosis) is the removal of cells which suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes which, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS-promoters at both ends which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS-promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, seven genes were identified which suppressed the p53-induced apoptosis. In four mutants, the suppression effect resulted from single genes activated by one UAS-promoter (Pka-R2, Rga, crol, Spt5). In the other three (Orct2, Polr2M, stg), deleting either UAS-promoter eliminated the suppression effect. In qPCR experiments we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eucaryotic genomes there are co-expressed gene clusters. Three of the DEP insertion mutants are included and two are in close vicinity of separate co-expressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.
RESUMEN
A central function of the Drosophila salivary glands (SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late-larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally-regulated programmed cell death (PCD) of the larval salivary glands, which culminates 16 h after puparium formation (APF). We demonstrate here that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions--glue secretion during pupariation--they remain dynamic and physiologically active up until the execution phase of PCD. We have used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68-2, vha36-1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7-8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early- to mid-prepupal period.
Asunto(s)
Ecdisona/metabolismo , Endosomas/metabolismo , Glándulas Salivales/metabolismo , Vacuolas/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ecdisona/genética , Endosomas/genética , Pupa , Glándulas Salivales/citología , Vacuolas/genéticaRESUMEN
In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal ß-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity.
Asunto(s)
Glándulas Apocrinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Glándulas Salivales/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Animales , Glándulas Apocrinas/ultraestructura , ADN/metabolismo , Colorantes Fluorescentes/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Biosíntesis de Proteínas , Pupa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Glándulas Salivales/ultraestructura , Fracciones Subcelulares/metabolismo , Transcripción GenéticaRESUMEN
The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA(+)-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Animales , Antibacterianos/farmacología , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimus/farmacologíaRESUMEN
The multifunctional factors Imp-α and Imp-ß are involved in nuclear protein import, mitotic spindle dynamics, and nuclear membrane formation. Furthermore, each of the three members of the Imp-α family exerts distinct tasks during development. In Drosophila melanogaster, the imp-α2 gene is critical during oogenesis for ring canal assembly; specific mutations, which allow oogenesis to proceed normally, were found to block early embryonic mitosis. Here, we show that imp-α2 and imp-ß genetically interact during early embryonic development, and we characterize the pattern of defects affecting mitosis in embryos laid by heterozygous imp-α2(D14) and imp-ß(KetRE34) females. Embryonic development is arrested in these embryos but is unaffected in combinations between imp-ß(KetRE34) and null mutations in imp-α1 or imp-α3. Furthermore, the imp-α2(D14)/imp-ß(KetRE34) interaction could only be rescued by an imp-α2 transgene, albeit not imp-α1 or imp-α3, showing the exclusive imp-α2 function with imp-ß. Use of transgenes carrying modifications in the major Imp-α2 domains showed the critical requirement of the nuclear localization signal binding (NLSB) site in this process. In the mutant embryos, we found metaphase-arrested mitoses made of enlarged spindles, suggesting an unrestrained activity of factors promoting spindle assembly. In accordance with this, we found that Imp-ß(KetRE34) and Imp-ß(KetD) bind a high level of RanGTP/GDP, and a deletion decreasing RanGTP level suppresses the imp-ß(KetRE34) phenotype. These data suggest that a fine balance among Imp-α2, Imp-ß, RanGTP, and the NLS cargos is critical for mitotic progression during early embryonic development.
RESUMEN
At the onset of Drosophila metamorphosis the steroid hormone ecdysone induces a process leading to a rapid degeneration of the larval salivary glands (SGs). Ecdysone acts through the ecdysone receptor heterodimer, which activates primary response genes. In particular these genes include the Broad-Complex (BR-C) gene encoding a set of BTB/POZ-transcription factors, among which the Z1 isoform is critical for SG cell death. The timing of SG disappearance depends upon of p127 (l(2)gl) , a cytoskeletal tumor suppressor that interacts with nonmuscle myosin II heavy chain (nmMHC) encoded by the zipper (zip) gene. Reduced l(2)gl expression delays SG histolysis whereas over-expression accelerates this process without affecting larval and pupal development. However, the mechanism by which l(2)gl controls SG histolysis remains yet unknown. Here we analyze the regulation controlled by p127 (l(2)gl) and nmMHC in the cytoplasm on the association of BR-C Z1 with chromatin and remodeling factors, such as Rpd3, Sin3A, and Smrter. In wild-type SGs these factors bind to chromatin but in l(2)gl SGs they accumulate in the cytoplasm and the cortical nuclear zone (CNZ). Similar chromatin exclusion occurs in SGs of developmentally delayed zip (E(br)) /+ larvae or can be achieved by high levels of nmMHC synthesis. The present data show that p127 (l(2)gl) and nmMHC regulate the access of BR-C Z1, Rpd3, Sin3A, and Smrter to chromatin. As the interaction between p127 (l(2)gl) and nmMHC occurs in the cytoplasm, we propose that these nuclear factors are processed by p127 (l(2)gl) and then released from p127 (l(2)gl) by nmMHC to allow their binding to chromatin. This process may constitute a novel mechanism of gene regulation, which in the absence of p127 (l(2)gl) , or excessive amounts of nmMHC, could lead to a fixed configuration in the pattern of gene expression that prevents further progression of SG differentiation, and programmed cell death (PCD). Such a transcriptional block could play a critical role in the neoplastic transformation of l(2)gl tissues.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Histona Desacetilasa 1/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis , Proteínas del Citoesqueleto/genética , Citoesqueleto/metabolismo , Drosophila/enzimología , Drosophila/genética , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Ecdisona/metabolismo , Larva , Miosina Tipo II/metabolismo , Receptores de Esteroides/metabolismo , Glándulas Salivales/enzimología , Glándulas Salivales/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3RESUMEN
Gamete development requires a coordinated soma-germ line interaction that ensures renewal and differentiation of germline and somatic stem cells. The physical contact between the germline and somatic cell populations is crucial because it allows the exchange of diffusible signals among them. The tumor suppressor gene discs large (dlg) encodes a septate junction protein with functions in epithelial cell polarity, asymmetric neuroblast division and formation of neuromuscular junctions. Our recent work reveals a new role of dlg in the Drosophila testis, as mutations in dlg lead to testis defects and cell death. Dlg is required throughout spermatogenesis in the somatic lineage and its localization changes from a uniform distribution along the plasma membrane of somatic cells in the testis apex, to a restricted localization on the distally located somatic cell in growing cysts. The extensive defects in dlg testis underline the importance of the somatic cells in the establishment and maintenance of the male stem cell niche and somatic cell differentiation. Here, we discuss our latest findings on the role of dlg in the Drosophila testis, supporting the view that junction proteins are dynamic structures, which can provide guiding cues to recruit scaffold proteins or other signaling molecules.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/genética , Transducción de Señal , Testículo/citología , Proteínas Supresoras de Tumor/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Supervivencia Celular/genética , Drosophila/citología , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Masculino , Espermatogénesis/genética , Espermatozoides/citología , Espermatozoides/metabolismo , Células Madre/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Gonad development requires a coordinated soma-germline interaction that ensures renewal and differentiation of germline and somatic stem cells to ultimately produce mature gametes. The Drosophila tumour suppressor gene discs large (dlg) encodes a septate junction protein functioning during epithelial polarization, asymmetric neuroblast division, and formation of neuromuscular junctions. Here, we report the role of dlg in testis development and its critical function in somatic cyst cells (SCCs). In these cells dlg is primarily required for their survival and expansion, and contributes to spermatocyte cyst differentiation. Cell death primarily occurred in SCCs at the end of spermatogonial amplification at a time when Dlg becomes restricted in wild-type (wt) testes to the distal somatic cells capping the growing spermatocyte cysts. RNAi depletion of dlg transcripts in early SCCs fully prevented testis development, whereas depletion in late SCCs resulted in a breakdown of spermatocyte cyst structure and germ cell individualization. Specific dlg expression in SCCs resulted in developmental rescue of dlg mutant testes, whereas its expression in germ cells exerted no such effect. dlg overexpression in wt testes led to spermatocyte cyst expansion at the expense of spermatogonial cysts. Our data demonstrate that dlg is essentially required in SCCs for their survival, expansion, and differentiation, and for the encapsulation of the germline cells.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/citología , Espermatocitos/citología , Testículo/citología , Proteínas Supresoras de Tumor/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Células Germinativas/citología , Masculino , Mutación , Interferencia de ARN , Espermatogénesis/fisiología , Testículo/crecimiento & desarrollo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Although arginine modification has been implicated in a number of cellular processes, the in vivo requirement of protein arginine methyltransferases (PRMTs) in specific biological processes remain to be clarified. In this study we characterize the Drosophila PRMT Capsuléen, homologous to human PRMT5. During Drosophila oogenesis, catalytic activity of Capsuléen is necessary for both the assembly of the nuage surrounding nurse cell nuclei and the formation of the pole plasm at the posterior end of the oocyte. In particular, we show that the nuage and pole plasm localization of Tudor, an essential component for germ cell formation, are abolished in csul mutant germ cells. We identify the spliceosomal Sm proteins as in vivo substrates of Capsuléen and demonstrate that Capsuléen, together with its associated protein Valois, is essential for the synthesis of symmetric di-methylated arginyl residues in Sm proteins. Finally, we show that Tudor can be targeted to the nuage in the absence of Sm methylation by Capsuléen, indicating that Tudor localization and Sm methylation are separate processes. Our results thus reveal the role of a PRMT in protein localization in germ cells.
Asunto(s)
Drosophila/enzimología , Oocitos/crecimiento & desarrollo , Proteína-Arginina N-Metiltransferasas/fisiología , Proteínas de Unión al ARN/metabolismo , Empalmosomas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia Conservada , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero , Femenino , Genes de Insecto , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Metilación , Datos de Secuencia Molecular , Oocitos/citología , Oocitos/metabolismo , Oogénesis/fisiología , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , TransgenesRESUMEN
Null-mutation in Drosophila importin-alpha2, such as the deficiency imp-alpha2(D14), causes recessive female sterility with the formation of dumpless eggs. In imp-alpha2(D14) the transfer of nurse cell components to the oocyte is interrupted and the Kelch protein, an oligomeric ring canal actin organizer, is normally produced but fails to associate with the ring canals resulting in their occlusion. To define domains regulating Kelch deposition on ring canals we performed site-directed mutagenesis on protein binding domains and putative phosphorylation sites of Imp-alpha2. Phenotypic analysis of the mutant transgenes in imp-alpha2(D14) revealed that mutations affecting the Imp-beta binding-domain, the dimerization domain, and specific serine residues of putative phosphorylation sites led to a normal or nearly normal oogenesis but arrested early embryonic development, whereas mutations in the nuclear localization signal (NLS) and CAS/exportin binding domains resulted in ring canal occlusion and a drastic nuclear accumulation of the mutant proteins. Deletion of the Imp-beta binding domain also gave rise to a nuclear localization of the mutant protein, which partially retained its function in ring canal assembly. Thus, we propose that mutations in NLS and CAS binding domains affect the deposition of Kelch onto the ring canals and prevent the association of Imp-alpha2 with a negative regulator of Kelch function.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Drosophila/ultraestructura , Oogénesis , Ovario/ultraestructura , alfa Carioferinas/química , Actinas/metabolismo , Animales , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/análisis , Proteínas de Drosophila/genética , Cáscara de Huevo/citología , Femenino , Polarización de Fluorescencia , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/genética , Mitosis , Mutación , Óvulo/citología , Estructura Terciaria de Proteína , alfa Carioferinas/genética , alfa Carioferinas/metabolismoRESUMEN
In an effort to identify the role of Rab11, a small GTP binding protein, during Drosophila differentiation, phenotypic manifestations associated with different alleles of Rab11 were studied. The phenotypes ranged from eye-defects, bristle abnormalities and sterility to lethality during various developmental stages. In this paper, our focus is targeted on eye defects caused by Rab11 mutations. A novel P-element insertion in the Rab11 locus, Rab11mo, displayed characteristic retinal anomalies, which could be reverted by P-element excision and expression of Rab11+ transgenes. During larval development, Rab11 is widely synthesized in photoreceptor cells and localizes to the rhabdomeres and lamina neuropil in adult eyes. Photoreceptors and associated bristles failed to be formed in homozygous clones generated in Rab11EP(3)3017 eyes. Decreased levels of Rab11 protein and increased cell death in Rab11mo third-instar larval eye-antennal discs suggest that the retinal defects originate during larval development. Our data indicate a requirement for Rab11 in ommatidial differentiation during Drosophila eye development.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Alelos , Animales , Animales Modificados Genéticamente , Muerte Celular , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Células Fotorreceptoras/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
Using the Capsuleen (Csul) methyltransferase as bait in the yeast two-hybrid system, we have identified a novel Drosophila protein containing multiple WD repeats and encoded by the valois (vsl) gene, which acts in pole plasm function. Vls is homologous to human MEP50, which forms a complex with the PRMT5 methyltransferase--the human homologue of Csul. We found that Vls localizes to the nuage in the nurse cells and to the pole plasm in the oocyte. Moreover vls is required for the synthesis and/or stability of Oskar and the localization of Tudor (Tud) in both the nuage and at the posterior pole of the oocyte. Furthermore, we show that Vls and a fragment of Tud interact directly in binding assay. As the PMRT5/MEP50 complex is involved in ribonucleoprotein complex assembly, we hypothesize that the Vls complex may play a similar function in assembling the nuage in nurse cells and the polar granules in the oocyte.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteína Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Femenino , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Mutación , Oogénesis/fisiología , Proteína Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila, and encodes two protein isoforms. Here, we report the pattern of Scrib1 synthesis in pole cells and embryonic gonads. We found that Scrib1 synthesis became strongly enhanced in pole cells at the time of gonad formation and was also detectable in cortical domains of gonadal mesodermal cells adjacent to pole cells. Scrib1 synthesis in mesodermal cells was independent of pole cells and occurred in agametic valois and capsuléen embryonic gonads. In contrast, Scrib1 synthesis in pole cells required contact with gonadal mesodermal cells as revealed by the absence of Scrib1 in wunen or tinman-zinc finger homeodomain-1 pseudo-gonads made only of aggregated pole cells.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Gónadas/embriología , Proteínas de la Membrana/genética , Animales , ARN Helicasas DEAD-box , Proteínas de Drosophila/biosíntesis , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica , Células Germinativas , Gónadas/metabolismo , Proteínas de la Membrana/biosíntesis , ARN Helicasas/metabolismoRESUMEN
We used ends-in gene targeting to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila melanogaster. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1(KO) knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes we examined conversion tracts in the recombination products. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. We conclude that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome.
Asunto(s)
Reparación del ADN/genética , Drosophila melanogaster/genética , Proteínas/genética , Animales , Proteínas de Ciclo Celular , Proteínas de Drosophila , Marcación de Gen , Genes Letales , Proteínas Nucleares , Proteína 1 de Ensamblaje de NucleosomasRESUMEN
The destruction of obsolete larval tissues at the onset of insect metamorphosis is a complex process triggered by the steroid hormone ecdysone. Among the genes required for the implementation of salivary gland (SG) degeneration the reduced bristles on palpus (rbp) gene of the Broad-Complex (BR-C) locus plays a critical role. This gene encodes the BR-C Z1 transcription factor and its expression is directly regulated by ecdysone through the ecdysone receptor (EcR/Usp). The BR-C locus encodes four major protein isoforms, including BR-C Z1, Z2, Z3, and Z4. With the exceptions of mutations in BR-C Z1 all mutations affecting the other BR-C isoforms produce pupal lethality. To gain insight into the function of the different BR-C isoforms on the process of SG degeneration, we used transgenes expressing each of the four major BR-C isoform proteins. This study revealed that, depending upon the period of expression relative to the major peak of ecdysone production, BR-C Z1, Z2, and Z4 first inhibited and then stimulated the process of SG degeneration. In contrast, BR-C Z3 exerted all time points an inhibition on SG degeneration.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila/metabolismo , Glándulas Salivales/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Animales , Western Blotting , Muerte Celular , Ecdisona/metabolismo , Inmunohistoquímica , Isoformas de Proteínas , Factores de Tiempo , TransgenesRESUMEN
Importin-alpha proteins do not only mediate the nuclear import of karyophilic proteins but also regulate spindle assembly during mitosis and the assembly of ring canals during Drosophila oogenesis. Three importin-alpha genes are present in the genome of Drosophila. To gain further insights into their function we analysed their expression during spermatogenesis by using antibodies raised against each of the three Importin-alpha proteins identified in Drosophila, namely, Imp-alpha1, -alpha2, and -alpha3. We found that each Imp-alpha is expressed during a specific and limited period of spermatogenesis. Strong expression of Imp-alpha2 takes place in spermatogonial cells, persists in spermatocytes, and lasts up to the completion of meiosis. In growing spermatocytes, the intracellular localisation of Imp-alpha2 appears to be dependent upon the rate of cell growth. In pupal testes Imp-alpha2 is essentially present in the spermatocyte nucleus but is localised in the cytoplasm of spermatocytes from adult testes. Both Imp-alpha1 and -alpha3 expression initiates at the beginning of meiosis and ends during spermatid differentiation. Imp-alpha1 expression extends up to the onset of the elongation phase, whereas that of Imp-alpha3 persists up to the completion of nuclear condensation when the spermatids become individualised. During meiosis Imp-alpha1 and -alpha3 are dispersed in the karyoplasm where they are partially associated with the nuclear spindle, albeit not with the asters. At telophase they aggregate around the chromatin. During sperm head differentiation, both Imp-alpha1 and -alpha3 are nuclear. These data indicate that each Imp-alpha protein carries during Drosophila spermatogenesis distinct, albeit overlapping, functions that may involve nuclear import of proteins, microtubule organisation, and other yet unknown processes.
Asunto(s)
Drosophila/metabolismo , Espermatogénesis , Testículo/metabolismo , alfa Carioferinas/biosíntesis , Animales , Diferenciación Celular , División Celular , Núcleo Celular/metabolismo , Clonación Molecular , Femenino , Fertilidad/genética , Inmunohistoquímica , Masculino , Meiosis , Microscopía Confocal , Mitosis , Modelos Moleculares , Mutación , Fenotipo , Espermatocitos/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
The interstitial deletion D14 affecting the importin-alpha 2 gene of Drosophila, or imp-alpha 2(D14), causes recessive female sterility characterized by a block of nurse cell-oocyte transport during oogenesis. In wild-type egg chambers, the Imp-alpha 2 protein is uniformly distributed in the nurse cell cytoplasm with a moderate accumulation along the oocyte cortex. Cytochalasin D treatment of wild-type egg chambers disrupts the in vivo association of Imp-alpha 2 with F-actin and results in its release from the oocyte cortex and its transfer into nurse cell nuclei. Binding assay shows that the interaction of Imp-alpha 2 with F-actin, albeit not monomeric actin, requires the occurrence of NLS peptides. Phenotypic analysis of imp-alpha 2(D14) ovaries reveals that the block of nurse cell-oocyte transport results from the occlusion of the ring canals that constitute cytoplasmic bridges between the nurse cells and the oocyte. Immunohistochemistry shows that, although the Imp-alpha2 protein cannot be detected on the ring canals, the Kelch protein, a known ring canal component, fails to bind to ring canals in imp-alpha 2(D14) egg chambers. Since loss-of-function mutations of kelch results in a similar dumpless phenotype, we propose that the Imp-alpha 2 protein plays a critical role in Kelch function by regulating its deposition on ring canals during their assembly.
Asunto(s)
Proteínas de Drosophila , Drosophila/fisiología , Carioferinas/fisiología , Proteínas de Microfilamentos , Oogénesis/fisiología , Actinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proteínas Portadoras/fisiología , Femenino , Infertilidad , MasculinoRESUMEN
The Drosophila malate dehydrogenase, or malic enzyme (ME) encoded by the Men gene, is a non-mitochondrial enzyme recovered in the cytosolic fraction. By using mutation in the Men gene and deficiencies uncovering this locus, we could show that the ME activity recovered in cytosolic fractions originates exclusively from the Men gene located at map position 87D-1 on the right arm of the 3rd chromosome. We found that juvenile hormone (JH) can induce ME activity by two mechanisms. One mechanism corresponds to a direct effect of JH on the enzyme, whose activity was enhanced by a twofold factor in the absence of transcription and translation. This enhancement can be noticed 1 h after JH treatment and lasts for approx 3-4 h. The other mechanism involves the transcription of the MEN gene. In the absence of inhibitors the induction of ME activity by JH is increased by a three to fourfold factor and extends over a period of 10-16 h. Since induction of ME activity by JH and JH analogs displayed a dose-response curve, specific for each tested component, we concluded that the hormonal action could be mediated through a receptor. The use of two temperature sensitive mutations deficient in the production of ecdysteroid, ecd1 and su(f)ts67g revealed that ME response to JH requires the presence of a minimal level of the steroid hormone ecdysone, showing a complex hormonal regulatory circuit in the execution of the JH response.
Asunto(s)
Drosophila melanogaster/enzimología , Malato Deshidrogenasa/biosíntesis , Sesquiterpenos/farmacología , Amanitinas/farmacología , Animales , Dactinomicina/farmacología , Ecdisteroides/farmacología , Inducción Enzimática , Larva/efectos de los fármacos , Larva/enzimología , Malato Deshidrogenasa/metabolismo , Metopreno/farmacología , Puromicina/farmacología , Sesquiterpenos/agonistas , Sesquiterpenos/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
The Drosophila gene dMBD2/3 encodes a protein with significant homologies to the mammalian methyl-DNA binding proteins MBD2 and MBD3. These proteins are essential components of chromatin complexes involved in epigenetic gene regulation. Because the available in vitro data on dMBD2/3 are conflicting we have started an in vivo characterization of dMBD2/3. We detected expression of two isoforms specifically during embryonic development. Staining of whole embryos combined with high-resolution confocal microscopy revealed a highly regulated spatial distribution. During the syncytial blastoderm stage, dMBD2/3 formed speckles that localized to the cytoplasm. Shortly after, during the cellular blastoderm stage, the protein entered the nucleus and formed bright foci that associated with DNA. This rapid transition coincided with the activation of the embryonic genome. A similar observation was made during activation of the spermatocyte genome as dMBD2/3 formed distinct foci associated with the activated Y chromosome. Our results indicate that dMBD2/3 forms specialized nuclear compartments to keep certain genes epigenetically silenced during genome activation.
Asunto(s)
Cromosomas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Animales , Drosophila/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Immunoblotting , Masculino , Microscopía Confocal , Unión Proteica , Espermatocitos/metabolismo , Cromosoma Y/metabolismoRESUMEN
The Drosophila gene for snRNP SmD3 (SmD3) is contained in reverse orientation within the first intron of the Ornithine Decarboxylase Antizyme (AZ) gene. Previous studies show that two closely linked P elements cause the gutfeeling phenotype characterized by embryonic lethality and aberrant neuronal and muscle cell differentiation. However, the exact nature of the gene(s) affected in the gutfeeling phenotype remained unknown. This study shows that a series of P inserts located within the 5'-untranslated region (5'-UTR) of SmD3 or its promoter affects only the expression of SmD3. Our analysis reveals that the gutfeeling phenotype associated with P elements inserted in the 5'-UTR of SmD3 results from amorphic or strongly hypomorphic mutations. In contrast, P inserts in the SmD3 promoter region reduce the expression of SmD3 without abolishing it and produce larval lethality with overgrown imaginal discs, brain hemispheres, and hematopoietic organs. The lethality of these mutations could be rescued by an SmD3+ transgene. Finally, inactivation of AZ was obtained by complementing with SmD3+ the deficiency Df(2R)guf(lex47) that uncovers both SmD3 and AZ. Interestingly, AZ inactivation causes a new phenotype characterized by late larval lethality and atrophy of the brain, imaginal discs, hematopoietic organs, and salivary glands.