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1.
Science ; 373(6550)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210853

RESUMO

The mechanisms by which macrophages regulate energy storage remain poorly understood. We identify in a genetic screen a platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF)-family ortholog, Pvf3, that is produced by macrophages and is required for lipid storage in fat-body cells of Drosophila larvae. Genetic and pharmacological experiments indicate that the mouse Pvf3 ortholog PDGFcc, produced by adipose tissue-resident macrophages, controls lipid storage in adipocytes in a leptin receptor- and C-C chemokine receptor type 2-independent manner. PDGFcc production is regulated by diet and acts in a paracrine manner to control lipid storage in adipose tissues of newborn and adult mice. At the organismal level upon PDGFcc blockade, excess lipids are redirected toward thermogenesis in brown fat. These data identify a macrophage-dependent mechanism, conducive to the design of pharmacological interventions, that controls energy storage in metazoans.


Assuntos
Adipócitos/imunologia , Dieta Hiperlipídica , Proteínas de Drosophila/metabolismo , Metabolismo Energético , Linfocinas/metabolismo , Macrófagos/imunologia , Obesidade/imunologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Termogênese , Tecido Adiposo Marrom/imunologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Hemócitos/imunologia , Fígado/imunologia , Linfocinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Derivado de Plaquetas/genética , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Nat Commun ; 12(1): 3778, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145251

RESUMO

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.


Assuntos
Adenosina/análogos & derivados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adenosina/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster , Células HeLa , Humanos , Metilação , Metiltransferases/genética , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética
3.
Nat Commun ; 12(1): 3730, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140480

RESUMO

Acid taste, evoked mainly by protons (H+), is a core taste modality for many organisms. The hedonic valence of acid taste is bidirectional: animals prefer slightly but avoid highly acidic foods. However, how animals discriminate low from high acidity remains poorly understood. To explore the taste perception of acid, we use the fruit fly as a model organism. We find that flies employ two competing taste sensory pathways to detect low and high acidity, and the relative degree of activation of each determines either attractive or aversive responses. Moreover, we establish one member of the fly Otopetrin family, Otopetrin-like a (OtopLa), as a proton channel dedicated to the gustatory detection of acid. OtopLa defines a unique subset of gustatory receptor neurons and is selectively required for attractive rather than aversive taste responses. Loss of otopla causes flies to reject normally attractive low-acid foods. Therefore, the identification of OtopLa as a low-acid sensor firmly supports our competition model of acid taste sensation. Altogether, we have discovered a binary acid-sensing mechanism that may be evolutionarily conserved between insects and mammals.


Assuntos
Ácidos/metabolismo , Vias Aferentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neurônios/metabolismo , Vias Aferentes/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eletrofisiologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Malatos/metabolismo , Microscopia Confocal , Mutação , Neurônios/fisiologia , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes , Paladar/fisiologia , Percepção Gustatória/fisiologia
4.
Nat Commun ; 12(1): 3291, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078905

RESUMO

The formation of hyperphosphorylated intracellular Tau tangles in the brain is a hallmark of Alzheimer's disease (AD). Tau hyperphosphorylation destabilizes microtubules, promoting neurodegeneration in AD patients. To identify suppressors of tau-mediated AD, we perform a screen using a microRNA (miR) library in Drosophila and identify the miR-9 family as suppressors of human tau overexpression phenotypes. CG11070, a miR-9a target gene, and its mammalian orthologue UBE4B, an E3/E4 ubiquitin ligase, alleviate eye neurodegeneration, synaptic bouton defects, and crawling phenotypes in Drosophila human tau overexpression models. Total and phosphorylated Tau levels also decrease upon CG11070 or UBE4B overexpression. In mammalian neuroblastoma cells, overexpression of UBE4B and STUB1, which encodes the E3 ligase CHIP, increases the ubiquitination and degradation of Tau. In the Tau-BiFC mouse model, UBE4B and STUB1 overexpression also increase oligomeric Tau degradation. Inhibitor assays of the autophagy and proteasome systems reveal that the autophagy-lysosome system is the major pathway for Tau degradation in this context. These results demonstrate that UBE4B, a miR-9 target gene, promotes autophagy-mediated Tau degradation together with STUB1, and is thus an innovative therapeutic approach for AD.


Assuntos
Doença de Alzheimer/genética , Proteínas de Drosophila/genética , MicroRNAs/genética , Ubiquitina-Proteína Ligases/genética , Proteínas tau/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Autofagia/genética , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Olho/metabolismo , Olho/patologia , Humanos , Lisossomos/metabolismo , Camundongos , MicroRNAs/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas tau/metabolismo
5.
Nucleic Acids Res ; 49(11): 6296-6314, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107019

RESUMO

Metal-induced genes are usually transcribed at relatively low levels under normal conditions and are rapidly activated by heavy metal stress. Many of these genes respond preferentially to specific metal-stressed conditions. However, the mechanism by which the general transcription machinery discriminates metal stress from normal conditions and the regulation of MTF-1-meditated metal discrimination are poorly characterized. Using a focused RNAi screening in Drosophila Schneider 2 (S2) cells, we identified a novel activator, the Drosophila gawky, of metal-responsive genes. Depletion of gawky has almost no effect on the basal transcription of the metallothionein (MT) genes, but impairs the metal-induced transcription by inducing the dissociation of MTF-1 from the MT promoters and the deficient nuclear import of MTF-1 under metal-stressed conditions. This suggests that gawky serves as a 'checkpoint' for metal stress and metal-induced transcription. In fact, regular mRNAs are converted into gawky-controlled transcripts if expressed under the control of a metal-responsive promoter, suggesting that whether transcription undergoes gawky-mediated regulation is encrypted therein. Additionally, lack of gawky eliminates the DNA binding bias of MTF-1 and the transcription preference of metal-specific genes. This suggests a combinatorial control of metal discrimination by gawky, MTF-1, and MTF-1 binding sites.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Metais/toxicidade , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cobre/toxicidade , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Metalotioneína/genética , Regiões Promotoras Genéticas , Interferência de RNA , Splicing de RNA , Estresse Fisiológico/genética
6.
Nat Commun ; 12(1): 3845, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158506

RESUMO

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.


Assuntos
Axônios/metabolismo , Quinase 1 do Ponto de Checagem/genética , Canais Iônicos/genética , Regeneração Nervosa/genética , Animais , Animais Geneticamente Modificados , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Canais Iônicos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética
7.
Nat Commun ; 12(1): 3328, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099654

RESUMO

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of Drosophila larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ecdisona/farmacologia , Epiderme/metabolismo , Morfogênese/efeitos dos fármacos , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ecdisona/genética , Células Epidérmicas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Larva/metabolismo , Metamorfose Biológica , Morfogênese/genética , Receptores Acoplados a Proteínas G/genética , Relaxina/metabolismo
8.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064595

RESUMO

Experimental studies of Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular and cellular processes in metazoans at large. Since the publication of their genomes, functional genomic investigations have identified genes that are essential or non-essential for survival in each species. Recently, a range of features linked to gene essentiality have been inferred using a machine learning (ML)-based approach, allowing essentiality predictions within a species. Nevertheless, predictions between species are still elusive. Here, we undertake a comprehensive study using ML to discover and validate features of essential genes common to both C. elegans and D. melanogaster. We demonstrate that the cross-species prediction of gene essentiality is possible using a subset of features linked to nucleotide/protein sequences, protein orthology and subcellular localisation, single-cell RNA-seq, and histone methylation markers. Complementary analyses showed that essential genes are enriched for transcription and translation functions and are preferentially located away from heterochromatin regions of C. elegans and D. melanogaster chromosomes. The present work should enable the cross-prediction of essential genes between model and non-model metazoans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Evolução Molecular , Genes Essenciais , Aprendizado de Máquina , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ontologia Genética , Genômica
9.
Nat Cell Biol ; 23(5): 497-510, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33972730

RESUMO

The Drosophila trachea, as the functional equivalent of mammalian blood vessels, senses hypoxia and oxygenates the body. Here, we show that the adult intestinal tracheae are dynamic and respond to enteric infection, oxidative agents and tumours with increased terminal branching. Increased tracheation is necessary for efficient damage-induced intestinal stem cell (ISC)-mediated regeneration and is sufficient to drive ISC proliferation in undamaged intestines. Gut damage or tumours induce HIF-1α (Sima in Drosophila), which stimulates tracheole branching via the FGF (Branchless (Bnl))-FGFR (Breathless (Btl)) signalling cascade. Bnl-Btl signalling is required in the intestinal epithelium and the trachea for efficient damage-induced tracheal remodelling and ISC proliferation. Chemical or Pseudomonas-generated reactive oxygen species directly affect the trachea and are necessary for branching and intestinal regeneration. Similarly, tracheole branching and the resulting increase in oxygenation are essential for intestinal tumour growth. We have identified a mechanism of tracheal-intestinal tissue communication, whereby damage and tumours induce neo-tracheogenesis in Drosophila, a process reminiscent of cancer-induced neoangiogenesis in mammals.


Assuntos
Transformação Celular Neoplásica/metabolismo , Hipóxia/metabolismo , Oxigênio/metabolismo , Regeneração/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/genética
10.
Neuron ; 109(12): 1979-1995.e6, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34015253

RESUMO

Nutrient sensors allow animals to identify foods rich in specific nutrients. The Drosophila nutrient sensor, diuretic hormone 44 (DH44) neurons, helps the fly to detect nutritive sugar. This sensor becomes operational during starvation; however, the mechanisms by which DH44 neurons or other nutrient sensors are regulated remain unclear. Here, we identified two satiety signals that inhibit DH44 neurons: (1) Piezo-mediated stomach/crop stretch after food ingestion and (2) Neuromedin/Hugin neurosecretory neurons in the ventral nerve cord (VNC) activated by an increase in the internal glucose level. A subset of Piezo+ neurons that express DH44 neuropeptide project to the crop. We found that DH44 neuronal activity and food intake were stimulated following a knockdown of piezo in DH44 neurons or silencing of Hugin neurons in the VNC, even in fed flies. Together, we propose that these two qualitatively distinct peripheral signals work in concert to regulate the DH44 nutrient sensor during the fed state.


Assuntos
Proteínas de Drosophila/metabolismo , Trato Gastrointestinal/fisiologia , Glucose/metabolismo , Canais Iônicos/metabolismo , Inibição Neural/fisiologia , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Resposta de Saciedade/fisiologia , Animais , Drosophila , Drosophila melanogaster , Comportamento Alimentar/fisiologia , Trato Gastrointestinal/inervação , Hormônios de Inseto , Mecanotransdução Celular/fisiologia , Neurônios/fisiologia , Estômago/inervação , Estômago/fisiologia
11.
J Cell Sci ; 134(9)2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33973638

RESUMO

The synaptic cleft manifests enriched glycosylation, with structured glycans coordinating signaling between presynaptic and postsynaptic cells. Glycosylated signaling ligands orchestrating communication are tightly regulated by secreted glycan-binding lectins. Using the Drosophila neuromuscular junction (NMJ) as a model glutamatergic synapse, we identify a new Ca2+-binding (C-type) lectin, Lectin-galC1 (LGC1), which modulates presynaptic function and neurotransmission strength. We find that LGC1 is enriched in motoneuron presynaptic boutons and secreted into the NMJ extracellular synaptomatrix. We show that LGC1 limits locomotor peristalsis and coordinated movement speed, with a specific requirement for synaptic function, but not NMJ architecture. LGC1 controls neurotransmission strength by limiting presynaptic active zone (AZ) and postsynaptic glutamate receptor (GluR) aligned synapse number, reducing both spontaneous and stimulation-evoked synaptic vesicle (SV) release, and capping SV cycling rate. During high-frequency stimulation (HFS), mutants have faster synaptic depression and impaired recovery while replenishing depleted SV pools. Although LGC1 removal increases the number of glutamatergic synapses, we find that LGC1-null mutants exhibit decreased SV density within presynaptic boutons, particularly SV pools at presynaptic active zones. Thus, LGC1 regulates NMJ neurotransmission to modulate coordinated movement.


Assuntos
Proteínas de Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Lectinas Tipo C , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo
12.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34028543

RESUMO

In metazoans, tissue growth and patterning is partly controlled by the Hedgehog (Hh) morphogen. Using immuno-electron microscopy on Drosophila wing imaginal discs, we identified a cellular structure, the Hherisomes, which contain the majority of intracellular Hh. Hherisomes are recycling tubular endosomes, and their formation is specifically boosted by overexpression of Hh. Expression of Rab11, a small GTPase involved in recycling endosomes, boosts the size of Hherisomes and their Hh concentration. Conversely, increased expression of the transporter Dispatched, a regulator of Hh secretion, leads to their clearance. We show that increasing Hh density in Hherisomes through Rab11 overexpression enhances both the level of Hh signaling and disc pouch growth, whereas Dispatched overexpression decreases high-level Hh signaling and growth. We propose that, upon secretion, a pool of Hh triggers low-level signaling, whereas a second pool of Hh is endocytosed and recycled through Hherisomes to stimulate high-level signaling and disc pouch growth. Altogether, our data indicate that Hherisomes are required to sustain physiological Hh activity necessary for patterning and tissue growth in the wing disc.


Assuntos
Proteínas de Drosophila , Proteínas Hedgehog , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endossomos/genética , Endossomos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Asas de Animais
13.
Nat Commun ; 12(1): 3175, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039988

RESUMO

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.


Assuntos
Envelhecimento/fisiologia , Evolução Biológica , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vertebrados/fisiologia , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Edição de Genes , Técnicas de Introdução de Genes , Masculino , Camundongos , Modelos Animais , Oxirredução , Filogenia , Aptidão Física/fisiologia , Mutação Puntual
14.
Nat Commun ; 12(1): 3231, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050142

RESUMO

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.


Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação Alostérica/genética , Animais , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma Fúngico , Histonas/genética , Histonas/isolamento & purificação , Humanos , Larva/genética , Larva/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
15.
Nat Commun ; 12(1): 3232, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050140

RESUMO

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the 'ruler' that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.


Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Epigênese Genética , Genoma Fúngico/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/isolamento & purificação , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/genética , Histonas/metabolismo , Larva/genética , Larva/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas dos Microfilamentos/metabolismo , Mutagênese , Nucleossomos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequenciamento Completo do Genoma
16.
Nat Commun ; 12(1): 2892, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001903

RESUMO

Flying insects have invaded all the aerial space on Earth and this astonishing radiation could not have been possible without a remarkable morphological diversification of their flight appendages. Here, we show that characteristic spatial expression profiles and levels of the Hox genes Antennapedia (Antp) and Ultrabithorax (Ubx) underlie the formation of two different flight organs in the fruit fly Drosophila melanogaster. We further demonstrate that flight appendage morphology is dependent on specific Hox doses. Interestingly, we find that wing morphology from evolutionary distant four-winged insect species is also associated with a differential expression of Antp and Ubx. We propose that variation in the spatial expression profile and dosage of Hox proteins is a major determinant of flight appendage diversification in Drosophila and possibly in other insect species during evolution.


Assuntos
Proteína do Homeodomínio de Antennapedia/genética , Proteínas de Drosophila/genética , Voo Animal , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Proteína do Homeodomínio de Antennapedia/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dosagem de Genes , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Asas de Animais/anatomia & histologia , Asas de Animais/metabolismo
17.
Methods Mol Biol ; 2281: 241-263, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847963

RESUMO

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of biological samples with a nanometric resolution. Determination of the physical properties of biological molecules at a single-molecule level is achieved through topographic analysis of the sample adsorbed on a flat and smooth surface. AFM has been widely used for the structural analysis of nucleic acid-protein interactions, providing insights on binding specificity and stoichiometry of proteins forming complexes with DNA substrates. Analysis of single-stranded DNA-binding proteins by AFM requires specific single-stranded/double-stranded hybrid DNA molecules as substrates for protein binding. In this chapter we describe the protocol for AFM characterization of binding properties of Drosophila telomeric protein Ver using DNA constructs that mimic the structure of chromosome ends. We provide details on the methodology used, including the procedures for the generation of DNA substrates, the preparation of samples for AFM visualization, and the data analysis of AFM images. The presented procedure can be adapted for the structural studies of any single-stranded DNA-binding protein.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , DNA de Cadeia Simples/química , Drosophila melanogaster/genética , Microscopia de Força Atômica , Ligação Proteica , Imagem Individual de Molécula , Telômero/genética , Telômero/metabolismo
18.
Neuron ; 109(11): 1836-1847.e5, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-33915110

RESUMO

Mature behaviors emerge from neural circuits sculpted by genetic programs and spontaneous and evoked neural activity. However, how neural activity is refined to drive maturation of learned behavior remains poorly understood. Here, we explore how transient hormonal signaling coordinates a neural activity state transition and maturation of associative learning. We identify spontaneous, asynchronous activity in a Drosophila learning and memory brain region, the mushroom body. This activity declines significantly over the first week of adulthood. Moreover, this activity is generated cell-autonomously via Cacophony voltage-gated calcium channels in a single cell type, α'/ß' Kenyon cells. Juvenile hormone, a crucial developmental regulator, acts transiently in α'/ß' Kenyon cells during a young adult sensitive period to downregulate spontaneous activity and enable subsequent enhanced learning. Hormone signaling in young animals therefore controls a neural activity state transition and is required for improved associative learning, providing insight into the maturation of circuits and behavior.


Assuntos
Hormônios Juvenis/metabolismo , Aprendizagem , Corpos Pedunculados/metabolismo , Neurogênese , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Corpos Pedunculados/citologia , Corpos Pedunculados/crescimento & desenvolvimento , Corpos Pedunculados/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transmissão Sináptica
19.
G3 (Bethesda) ; 11(3)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33793746

RESUMO

In Drosophila raised in nutrient-rich conditions, female body size is approximately 30% larger than male body size due to an increased rate of growth and differential weight loss during the larval period. While the mechanisms that control this sex difference in body size remain incompletely understood, recent studies suggest that the insulin/insulin-like growth factor signaling pathway (IIS) plays a role in the sex-specific regulation of processes that influence body size during development. In larvae, IIS activity differs between the sexes, and there is evidence of sex-specific regulation of IIS ligands. Yet, we lack knowledge of how changes to IIS activity impact body size in each sex, as the majority of studies on IIS and body size use single- or mixed-sex groups of larvae and/or adult flies. The goal of our current study was to clarify the body size requirement for IIS activity in each sex. To achieve this goal, we used established genetic approaches to enhance, or inhibit, IIS activity, and quantified pupal size in males and females. Overall, genotypes that inhibited IIS activity caused a female-biased decrease in body size, whereas genotypes that augmented IIS activity caused a male-specific increase in body size. These data extend our current understanding of body size regulation by showing that most changes to IIS pathway activity have sex-biased effects, and highlights the importance of analyzing body size data according to sex.


Assuntos
Proteínas de Drosophila , Insulina , Transdução de Sinais , Somatomedinas , Animais , Tamanho Corporal , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Masculino
20.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925631

RESUMO

In Drosophila, endoplasmic reticulum (ER) stress activates the protein kinase R-like endoplasmic reticulum kinase (dPerk). dPerk can also be activated by defective mitochondria in fly models of Parkinson's disease caused by mutations in pink1 or parkin. The Perk branch of the unfolded protein response (UPR) has emerged as a major toxic process in neurodegenerative disorders causing a chronic reduction in vital proteins and neuronal death. In this study, we combined microarray analysis and quantitative proteomics analysis in adult flies overexpressing dPerk to investigate the relationship between the transcriptional and translational response to dPerk activation. We identified tribbles and Heat shock protein 22 as two novel Drosophila activating transcription factor 4 (dAtf4) regulated transcripts. Using a combined bioinformatics tool kit, we demonstrated that the activation of dPerk leads to translational repression of mitochondrial proteins associated with glutathione and nucleotide metabolism, calcium signalling and iron-sulphur cluster biosynthesis. Further efforts to enhance these translationally repressed dPerk targets might offer protection against Perk toxicity.


Assuntos
eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Biologia Computacional/métodos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismo , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologia
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