RESUMO
All animals must coordinate growth rate and timing of maturation to reach the appropriate final size. Here, we describe hobbit, a novel and conserved gene identified in a forward genetic screen for Drosophila animals with small body size. hobbit is highly conserved throughout eukaryotes, but its function remains unknown. We demonstrate that hobbit mutant animals have systemic growth defects because they fail to secrete insulin. Other regulated secretion events also fail in hobbit mutant animals, including mucin-like 'glue' protein secretion from the larval salivary glands. hobbit mutant salivary glands produce glue-containing secretory granules that are reduced in size. Importantly, secretory granules in hobbit mutant cells lack essential membrane fusion machinery required for exocytosis, including Synaptotagmin 1 and the SNARE SNAP-24. These membrane fusion proteins instead accumulate inside enlarged late endosomes. Surprisingly, however, the Hobbit protein localizes to the endoplasmic reticulum. Our results suggest that Hobbit regulates a novel step in intracellular trafficking of membrane fusion proteins. Our studies also suggest that genetic control of body size, as a measure of insulin secretion, is a sensitive functional readout of the secretory machinery.
Assuntos
Tamanho Corporal/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Insulina/metabolismo , Proteínas de Fusão de Membrana/metabolismo , Glândulas Salivares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas do Grude Salivar de Drosophila/genética , Proteínas do Grude Salivar de Drosophila/metabolismo , Secreção de Insulina , Tamanho do Órgão/genética , Transporte Proteico/genética , Vesículas Secretórias/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Sinaptotagmina I/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: At the very end of the larval stage Drosophila expectorate a glue secreted by their salivary glands to attach themselves to a substrate while pupariating. The glue is a mixture of apparently unrelated proteins, some of which are highly glycosylated and possess internal repeats. Because species adhere to distinct substrates (i.e. leaves, wood, rotten fruits), glue genes are expected to evolve rapidly. RESULTS: We used available genome sequences and PCR-sequencing of regions of interest to investigate the glue genes in 20 Drosophila species. We discovered a new gene in addition to the seven glue genes annotated in D. melanogaster. We also identified a phase 1 intron at a conserved position present in five of the eight glue genes of D. melanogaster, suggesting a common origin for those glue genes. A slightly significant rate of gene turnover was inferred. Both the number of repeats and the repeat sequence were found to diverge rapidly, even between closely related species. We also detected high repeat number variation at the intrapopulation level in D. melanogaster. CONCLUSION: Most conspicuous signs of accelerated evolution are found in the repeat regions of several glue genes.
Assuntos
Drosophila/genética , Evolução Molecular , Genes de Insetos , Proteínas do Grude Salivar de Drosophila/genética , Glândulas Salivares/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon sem Sentido/genética , Funções Verossimilhança , Família Multigênica , Nucleotídeos/genética , Sequências Repetitivas de Ácido Nucleico/genética , Especificidade da EspécieRESUMO
Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. We manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. We monitored expression of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, we found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. We suggest that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Ecdisterona/metabolismo , Proteínas do Grude Salivar de Drosophila/metabolismo , Estruturas Animais/efeitos dos fármacos , Estruturas Animais/metabolismo , Animais , Proteínas de Fluorescência Verde/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Mifepristona/farmacologia , Modelos Biológicos , Progesterona/análogos & derivados , Transdução de Sinais , Fatores de Tempo , TransgenesRESUMO
Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Secretórias/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Endossomos/metabolismo , Proteínas do Grude Salivar de Drosophila/metabolismo , Complexo de Golgi/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Transporte Proteico , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Mosquitoes transmit Plasmodium and certain arboviruses during blood feeding, when they are injected along with saliva. Mosquito saliva interferes with the host's hemostasis and inflammation response and influences the transmission success of some pathogens. One family of mosquito salivary gland proteins, named SGS, is composed of large bacterial-type proteins that in Aedes aegypti were implicated as receptors for Plasmodium on the basal salivary gland surface. Here, we characterize the biology of two SGSs in the malaria mosquito, Anopheles gambiae, and demonstrate their involvement in blood feeding. Western blots and RT-PCR showed that Sgs4 and Sgs5 are produced exclusively in female salivary glands, that expression increases with age and after blood feeding, and that protein levels fluctuate in a circadian manner. Immunohistochemistry showed that SGSs are present in the acinar cells of the distal lateral lobes and in the salivary ducts of the proximal lobes. SDS-PAGE, Western blots, bite blots, and immunization via mosquito bites showed that SGSs are highly immunogenic and form major components of mosquito saliva. Last, Western and bioinformatic analyses suggest that SGSs are secreted via a non-classical pathway that involves cleavage into a 300-kDa soluble fragment and a smaller membrane-bound fragment. Combined, these data strongly suggest that SGSs play an important role in blood feeding. Together with their role in malaria transmission, we propose that SGSs could be used as markers of human exposure to mosquito bites and in the development of disease control strategies.
Assuntos
Anopheles/imunologia , Anopheles/metabolismo , Proteínas do Grude Salivar de Drosophila/metabolismo , Fatores Imunológicos/metabolismo , Saliva/imunologia , Saliva/metabolismo , Glândulas Salivares/metabolismo , Animais , Biologia Computacional , Proteínas de Drosophila , Feminino , Regulação da Expressão Gênica , Proteínas do Grude Salivar de Drosophila/genética , Proteínas do Grude Salivar de Drosophila/imunologia , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/imunologia , Proteólise , Fatores de TempoRESUMO
The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Ecdisterona/fisiologia , Metamorfose Biológica/fisiologia , Receptores de Esteroides/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/fisiologia , Dimerização , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Grude Salivar de Drosophila/genética , Proteínas do Grude Salivar de Drosophila/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Metamorfose Biológica/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transgenes/fisiologiaRESUMO
Glue protein as secretion from fruit fly larva plays a significant role in metamorphosis as cementing material for pupation sites. However, the biochemical composition of this macromolecule remains obscure. This study takes the advantage of high-resolution proteomic analysis to unveil the protein compositions. A glue protein group is identified as chitin-binding motifs by bioinformatic analysis. Computational modeling analysis of representative proteins illustrates the binding site between protein and chitin. A biosynthetic approach is used to fabricate a glue protein by a modified Escherichia coli recombinant system. The as-biosynthesized biomimetic glue protein is applied as an extracellular matrix to investigate its biocompatibility and functionality. It is found that the purified recombinant protein shows enhanced performance to cellular viability. This finding provides a potential biomacromolecule candidate as an extracellular matrix for cell culture.
Assuntos
Biomimética , Regulação da Expressão Gênica , Proteínas do Grude Salivar de Drosophila/farmacologia , Fígado/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Quitina/química , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas do Grude Salivar de Drosophila/biossíntese , Proteínas do Grude Salivar de Drosophila/química , Humanos , Fígado/efeitos dos fármacos , Modelos Moleculares , Filogenia , Proteômica , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Participation of RAS, RAF, and mitogen-activated protein kinase (MAPK) in learning and memory has been demonstrated in a number of studies, but the molecular events requisite for cascade activation and regulation have not been explored. We demonstrate that the adapter protein DRK (downstream of receptor kinase) which is essential for signaling to RAS in developmental contexts, is preferentially distributed in the adult mushroom bodies, centers for olfactory learning and memory. We demonstrate that drk mutant heterozygotes exhibit deficits in olfactory learning and memory, apparent under limited training conditions, but are not impaired in sensory responses requisite for the association of the stimuli, or brain neuroanatomy. Furthermore, we demonstrate that the protein is required acutely within mushroom body neurons to mediate efficient learning, a process that requires RAF activation. Importantly, 90 min memory remained impaired, even after differential training yielding equivalent learning in animals with compromised DRK levels and controls and did not require RAF. Sustained MAPK activation is compromised in drk mutants and surprisingly is negatively regulated by constitutive RAF activity. The data establish a role for DRK in Drosophila behavioral neuroplasticity and suggest a dual role for the protein, first in RAF activation-dependent learning and additionally in RAF-inhibition dependent sustained MAPK activation essential for memory formation or stability.
Assuntos
Aprendizagem por Associação/fisiologia , Proteínas de Drosophila/fisiologia , Memória de Curto Prazo/fisiologia , Condutos Olfatórios/fisiologia , Olfato/genética , Análise de Variância , Animais , Animais Geneticamente Modificados , Condicionamento Clássico/fisiologia , Drosophila , Proteínas de Drosophila/genética , Proteínas do Grude Salivar de Drosophila/genética , Deficiências da Aprendizagem/genética , Transtornos da Memória/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Corpos Pedunculados/citologia , Corpos Pedunculados/metabolismo , Mutação/fisiologia , Odorantes , RNA Interferente Pequeno/genéticaRESUMO
Autophagic-lysosomal degradation is essential for the maintenance of normal homeostasis in eukaryotic cells. Several types of such self-degradative and recycling pathways have been identified. From these, probably the least known autophagic process is crinophagy, during which unnecessary or obsolete secretory granules directly fuse with late endosomes/lysosomes as a means of rapid elimination of unused secretory material from the cytoplasm. This process was identified in 1966, but we are only beginning to understand the molecular mechanisms and regulation of crinophagy. In this review, we summarize the current examination methods and possible model systems, discuss the recently identified factors that are required for crinophagy, and give an overview of the potential medical relevance of this process.
Assuntos
Autofagia/fisiologia , Vesículas Secretórias/fisiologia , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Sistema Endócrino/fisiologia , Endossomos/fisiologia , Previsões , Genes Reporter , Proteínas do Grude Salivar de Drosophila/metabolismo , Humanos , Larva , Lisossomos/enzimologia , Lisossomos/fisiologia , Fusão de Membrana , Pupa , Glândulas Salivares/citologia , Glândulas Salivares/metabolismoRESUMO
Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.
Assuntos
Citoplasma/metabolismo , Dineínas/metabolismo , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Células Cultivadas , Embrião de Galinha , DNA , Complexo Dinactina , Dineínas/química , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Biblioteca Genômica , Proteínas do Grude Salivar de Drosophila/genética , Interfase , Proteínas dos Microtúbulos/genética , Mitose , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The larval salivary glands of Drosophila express the FOXA transcription factor Fork head (Fkh) before, but not after, puparium formation. Forced expression of Fkh in late prepupae prevents the programmed destruction of the tissue, which normally occurs in the early pupa. Using Affymetrix GeneChips, we analysed changes in gene expression brought about by Fkh when expressed shortly before the normal time of salivary gland death. Genes identified as responsive to Fkh include not only cell death genes, but also genes involved in autophagy, phospholipid metabolism and hormone-controlled signalling pathways. In addition, Fkh changed the expression of genes involved in glucose and fatty acid metabolism that are known to be target genes of the FOXAs in vertebrates. Premature loss of fkh induced by RNAi and gain of Fkh by ectopic expression at earlier times of development confirmed that genes identified in the microarray study are under normal developmental control by Fkh. These genes include Eip63F-1, which is expressed in both salivary glands and Malpighian tubules, suggesting that Fkh controls common aspects of the secretory function of the two organs. Eip63F-1 is one of many genes controlled by the steroid hormone 20-hydroxyecdysone that appear to be co-regulated by Fkh.
Assuntos
Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Nucleares/fisiologia , Glândulas Salivares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Northern Blotting , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Fatores de Transcrição Forkhead , Proteínas do Grude Salivar de Drosophila/biossíntese , Proteínas do Grude Salivar de Drosophila/genética , Proteínas do Grude Salivar de Drosophila/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfolipídeos/metabolismo , RNA/química , RNA/genética , Interferência de RNA , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. We find that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.
Assuntos
Autofagia/fisiologia , Drosophila melanogaster/embriologia , Proteínas do Grude Salivar de Drosophila/metabolismo , Lisossomos/metabolismo , Fusão de Membrana/fisiologia , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Classe III de Fosfatidilinositol 3-Quinases/genética , Proteínas de Drosophila/genética , Proteínas do Grude Salivar de Drosophila/genética , Proteínas Qa-SNARE/genética , Proteínas R-SNARE/genética , Proteínas SNARE/genética , Proteínas rab de Ligação ao GTP/genética , Proteína rab2 de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
The Sgs-3 gene of Drosophila melanogaster exhibits a tightly regulated pattern of expression governed by two functionally equivalent elements within 1 kb of the gene, each of which is sufficient to confer third-instar salivary gland-specific transcription. In this report we describe a detailed functional analysis of one of these, the proximal element. To determine the nucleotides responsible for specific expression, we have introduced mutations into the proximal element and then assessed the effects of each alteration on expression in the developing animal. We have identified six particularly important base pairs which are located in two regions separated by nonessential sequences. These base pairs, along with some surrounding sequence, are conserved within the upstream regions of the three glue genes at 68C. Nearly identical groups of base pairs can be found upstream of the other glue genes which have been cloned. This analysis has allowed us to derive a consensus sequence, which we believe contains binding sites for two different factors which interact to direct third-instar salivary gland-specific expression.
Assuntos
Drosophila melanogaster/genética , Genes , Proteínas do Grude Salivar de Drosophila/genética , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Deleção Cromossômica , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Mapeamento por Restrição , Glândulas Salivares/metabolismo , Moldes Genéticos , Transcrição GênicaRESUMO
The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between -211 and -43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from -133 to -48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region.
Assuntos
Drosophila melanogaster/genética , Genes , Proteínas do Grude Salivar de Drosophila/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Animais , Sequência de Bases , Clonagem Molecular , DNA Ribossômico/genética , Escherichia coli/genética , Expressão Gênica , Dados de Sequência Molecular , Plasmídeos , Homologia de Sequência do Ácido Nucleico , Transformação GenéticaRESUMO
Pig-1 and Sgs-4 are a pair of closely linked and divergently transcribed Drosophila melanogaster genes, which are both expressed in larval salivary glands but at different times during development. While Sgs-4 is expressed at high levels only at the end of the third instar, Pig-1 exhibits a major peak of expression during late second and early third instar. Thus, Pig-1 expression declines as Sgs-4 expression is induced. In this paper, we show that three adjacent elements located within the short region between these genes can account for the switch from Pig-1 to Sgs-4 expression. A 170-bp segment acts as an enhancer to direct Sgs-4 expression in late-third-instar salivary glands. A 64-bp sequence located just upstream from the enhancer can modify its temporal specificity so that it works throughout the third instar. Expression induced at mid-third instar by a combination of these two elements can be repressed by a negative regulatory sequence located still further upstream. We present evidence suggesting that the changing interactions between these regulatory elements and the Sgs-4 and Pig-1 promoters lead to the correct pattern of expression of the two genes.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genes de Insetos , Sequências Reguladoras de Ácido Nucleico , Proteínas e Peptídeos Salivares/genética , Fatores Etários , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Elementos Facilitadores Genéticos , Proteínas do Grude Salivar de Drosophila/genética , Larva , Pupa , RNA Mensageiro/genética , Glândulas Salivares/fisiologia , Transcrição GênicaRESUMO
The transcription of the Drosophila melanogaster 68C salivary gland glue gene Sgs-3 involves the interaction of a distal and a proximal regulatory region. These are marked in vivo by a specific chromatin structure which is established sequentially during development, starting early in embryogenesis. The distal region is characterized by a stage- and tissue-specific DNase I hypersensitive site. A stage- and tissue-specific factor, GEBF-I, binds in this region and is missing in 2B5 mutant larvae which lack Sgs-3 transcripts. This binding involves the simultaneous interaction with two distinct DNA sequences which induces conformational changes in the protein. Salivary glands acquire competence to respond to ecdysone in the mid-third larval instar, whereafter the hormone rapidly induces both the GEBF-I protein and Sgs-3 transcription.
Assuntos
Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Proteínas do Grude Salivar de Drosophila/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores Etários , Animais , Sequência de Bases , Cromatina/ultraestrutura , Mapeamento Cromossômico , Drosophila melanogaster/crescimento & desenvolvimento , Ecdisona/farmacologia , Regulação da Expressão Gênica , Genes , Dados de Sequência Molecular , Oligonucleotídeos/química , Ligação Proteica , Mapeamento por Restrição , Transcrição GênicaRESUMO
An actin filament coat promotes cargo expulsion from large exocytosing vesicles, but the mechanisms of coat formation and force generation have been poorly characterized. Elegant imaging studies of the Drosophila melanogaster salivary gland now reveal how actin and myosin are recruited, and show that myosin II forms a contractile 'cage' that facilitates exocytosis.
Assuntos
Actomiosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Exocitose , Proteínas do Grude Salivar de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , AnimaisRESUMO
Releasing content from large vesicles measuring several micrometres in diameter poses exceptional challenges to the secretory system. An actomyosin network commonly coats these vesicles, and is thought to provide the necessary force mediating efficient cargo release. Here we describe the spatial and temporal dynamics of the formation of this actomyosin coat around large vesicles and the resulting vesicle collapse, in live Drosophila melanogaster salivary glands. We identify the Formin family protein Diaphanous (Dia) as the main actin nucleator involved in generating this structure, and uncover Rho as an integrator of actin assembly and contractile machinery activation comprising this actomyosin network. High-resolution imaging reveals a unique cage-like organization of myosin II on the actin coat. This myosin arrangement requires branched-actin polymerization, and is critical for exerting a non-isotropic force, mediating efficient vesicle contraction.
Assuntos
Actomiosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Exocitose , Proteínas do Grude Salivar de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Forminas , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microscopia de Vídeo , Miosina Tipo II/metabolismo , Tamanho das Organelas , Glândulas Salivares/ultraestrutura , Vesículas Secretórias/ultraestrutura , Imagem com Lapso de Tempo , Quinases Associadas a rho/metabolismoRESUMO
DNA comprising the larval glue protein gene Lgp-3 of Drosophila virilis was isolated from a lambda genomic and a cDNA library. The transcription start site, two polyadenylation sites and the boundaries of the single intron were determined. An open reading frame encoding 379 amino acids was found. At the DNA level the presence of similar introns and three conserved sequence motifs in the proximal promoters suggest that the gene is related to those of the D. virilis lgp-1 and the D. melanogaster sgs-3, -7 and -8 glue proteins. Their common ancestry is also substantiated by the comparisons of the deduced amino acid sequences and the profiles of hydropathic indices, which reveal striking similarities of the N- and C-termini and of the central repeat domains, although the lengths and the primary structures of the proteins diverged considerably during 60 million years of separate evolution of the two Drosophila species.
Assuntos
Drosophila/genética , Proteínas do Grude Salivar de Drosophila/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Genes de Insetos , Proteínas do Grude Salivar de Drosophila/química , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
The X-linked Sgs-4 gene of Drosophila melanogaster encodes a salivary glue protein. Here we report the molecular characterization of a non-dosage compensated variant strain, named Karsnas, in which males accumulate only about half of the Sgs-4 polypeptide amount as do females. The results obtained show that significant nucleotide sequence alterations are accumulated within the Sgs-4 coding and 3' untranslated region of the variant strain, thus suggesting a possible role of these sequences in the Sgs-4 dosage compensation.