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1.
PLoS One ; 18(1): e0280652, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662713

RESUMO

Drosophila RhoGAP18B was identified as a negative regulator of small GTPase in the behavioral response to ethanol. However, the effect of RhoGAP18B on cell migration is unknown. Here, we report that RhoGAP18B regulates the migration of border cells in Drosophila ovary. The RhoGAP18B gene produces four transcripts and encodes three translation isoforms. We use different RNAi lines to knockdown each RhoGAP18B isoform, and find that knockdown of RhoGAP18B-PA, but not PC or PD isoform, blocks border cell migration. Knockdown of RhoGAP18B-PA disrupts the asymmetric distribution of F-actin in border cell cluster and increases F-actin level. Furthermore, RhoGAP18B-PA may act on Rac to regulate F-actin organization. Our data indicate that RhoGAP18B shows isoform-specific regulation of border cell migration.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Feminino , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Actinas/metabolismo , Movimento Celular/genética , Citoesqueleto de Actina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
2.
Cells ; 12(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36672258

RESUMO

Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. We found that a deletion of the 3'UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, we detected more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain. Importantly, deletion of the 3'UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3'UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Regiões 3' não Traduzidas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Memória de Longo Prazo/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Junções Íntimas/metabolismo
3.
Cells ; 12(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36672267

RESUMO

NUMB, a plasma membrane-associated protein originally described in Drosophila, is involved in determining cell function and fate during early stages of development. It is secreted asymmetrically in dividing cells, with one daughter cell inheriting NUMB and the other inheriting its antagonist, NOTCH. NUMB has been proposed as a polarizing agent and has multiple functions, including endocytosis and serving as an adaptor in various cellular pathways such as NOTCH, Hedgehog, and the P53-MDM2 axis. Due to its role in maintaining cellular homeostasis, it has been suggested that NUMB may be involved in various human pathologies such as cancer and Alzheimer's disease. Further research on NUMB could aid in understanding disease mechanisms and advancing the field of personalized medicine and the development of new therapies.


Assuntos
Proteínas de Drosophila , Animais , Humanos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores Notch/metabolismo , Drosophila/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Hormônios Juvenis/metabolismo
4.
Development ; 150(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36692218

RESUMO

The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Our previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Here, through the first genome-wide binding and expression profiles obtained for a FUBP family protein, we demonstrate that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFß signalling). We further demonstrate tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.


Assuntos
Proteínas de Drosophila , Drosophila , Proteínas de Ligação a RNA , Animais , Divisão Celular , Proliferação de Células , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ativação Transcricional
5.
Development ; 150(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36628974

RESUMO

Src kinases are important regulators of cell adhesion. Here, we have explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular (bAJ) and tricellular (tAJ) junctions in germband cells, and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and CRISPR-Cas9-induced germline mosaics. We find that, during cell intercalations, Src42A is required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knockdown. Furthermore, we show that Src42A acts in concert with Abl kinase, which has also been implicated in cell intercalations. Our data suggest that Src42A is involved in two related processes: in addition to establishing tension generated by the planar polarity of MyoII, it may also act as a signalling factor at tAJs to control E-cadherin residence time.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Junções Aderentes/metabolismo , Caderinas/genética , Caderinas/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Junções Intercelulares/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
6.
PLoS One ; 18(1): e0280529, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36689407

RESUMO

Much is known about environmental influences on metabolism and systemic insulin levels. Less is known about how those influences are translated into molecular mechanisms regulating insulin production. To better understand the molecular mechanisms we generated marked cells homozygous for a null mutation in the Drosophila TGF-ß signal transducer dSmad2 in unmated adult females. We then conducted side-by-side single cell comparisons of the pixel intensity of two Drosophila insulin-like peptides (dILP2 and dILP5) in dSmad2- mutant and wild type insulin producing cells (IPCs). The analysis revealed multiple features of dSmad2 regulation of dILPs. In addition, we discovered that dILP5 is expressed and regulated by dSmad2 in circadian pacemaker cells (CPCs). Outcomes of regulation by dSmad2 differ between dILP2 and dILP5 within IPCs and differ for dILP5 between IPCs and CPCs. Modes of dSmad2 regulation differ between dILP2 and dILP5. dSmad2 antagonism of dILP2 in IPCs is robust but dSmad2 regulation of dILP5 in IPCs and CPCs toggles between antagonism and agonism depending upon dSmad2 dosage. Companion studies of dILP2 and dILP5 in the IPCs of dCORL mutant (fussel in Flybase and SKOR in mammals) and upd2 mutant unmated adult females showed no significant difference from wild type. Taken together, the data suggest that dSmad2 regulates dILP2 and dILP5 via distinct mechanisms in IPCs (antagonist) and CPCs (agonist) and in unmated adult females that dSmad2 acts independently of dCORL and upd2.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Feminino , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Insulina/metabolismo , Peptídeos/metabolismo , Mamíferos/metabolismo
7.
PLoS Genet ; 19(1): e1010571, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36689473

RESUMO

Drosophila trachea is a premier model to study tube morphogenesis. After the formation of continuous tubes, tube maturation follows. Tracheal tube maturation starts with an apical secretion pulse that deposits extracellular matrix components to form a chitin-based apical luminal matrix (aECM). This aECM is then cleared and followed by the maturation of taenidial folds. Finally, air fills the tubes. Meanwhile, the cellular junctions are maintained to ensure tube integrity. Previous research has identified several key components (ER, Golgi, several endosomes) of protein trafficking pathways that regulate the secretion and clearance of aECM, and the maintenance of cellular junctions. The Osiris (Osi) gene family is located at the Triplo-lethal (Tpl) locus on chromosome 3R 83D4-E3 and exhibits dosage sensitivity. Here, we show that three Osi genes (Osi9, Osi15, Osi19), function redundantly to regulate adherens junction (AJ) maintenance, luminal clearance, taenidial fold formation, tube morphology, and air filling during tube maturation. The localization of Osi proteins in endosomes (Rab7-containing late endosomes, Rab11-containing recycling endosomes, Lamp-containing lysosomes) and the reduction of these endosomes in Osi mutants suggest the possible role of Osi genes in tube maturation through endosome-mediated trafficking. We analyzed tube maturation in zygotic rab11 and rab7 mutants, respectively, to determine whether endosome-mediated trafficking is required. Interestingly, similar tube maturation defects were observed in rab11 but not in rab7 mutants, suggesting the involvement of Rab11-mediated trafficking, but not Rab7-mediated trafficking, in this process. To investigate whether Osi genes regulate tube maturation primarily through the maintenance of Rab11-containing endosomes, we overexpressed rab11 in Osi mutant trachea. Surprisingly, no obvious rescue was observed. Thus, increasing endosome numbers is not sufficient to rescue tube maturation defects in Osi mutants. These results suggest that Osi genes regulate other aspects of endosome-mediated trafficking, or regulate an unknown mechanism that converges or acts in parallel with Rab11-mediated trafficking during tube maturation.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Traqueia/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Proteínas de Drosophila/genética
8.
Cell Mol Life Sci ; 80(1): 24, 2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36600084

RESUMO

At the onset of Drosophila metamorphosis, plenty of secretory glue granules are released from salivary gland cells and the glue is deposited on the ventral side of the forming (pre)pupa to attach it to a dry surface. Prior to this, a poorly understood maturation process takes place during which secretory granules gradually grow via homotypic fusions, and their contents are reorganized. Here we show that the small GTPase Rab26 localizes to immature (smaller, non-acidic) glue granules and its presence prevents vesicle acidification. Rab26 mutation accelerates the maturation, acidification and release of these secretory vesicles as well as the lysosomal breakdown (crinophagy) of residual, non-released glue granules. Strikingly, loss of Mon1, an activator of the late endosomal and lysosomal fusion factor Rab7, results in Rab26 remaining associated even with the large glue granules and a concomitant defect in glue release, similar to the effects of Rab26 overexpression. Our data thus identify Rab26 as a key regulator of secretory vesicle maturation that promotes early steps (vesicle growth) and inhibits later steps (lysosomal transport, acidification, content reorganization, release, and breakdown), which is counteracted by Mon1.


Assuntos
Drosophila , Vesículas Secretórias , Proteínas rab de Ligação ao GTP , Animais , Drosophila/metabolismo , Lisossomos/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas de Drosophila/metabolismo
9.
Cell Mol Life Sci ; 80(1): 30, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609617

RESUMO

Tumor cells have an increased demand for nutrients to sustain their growth, but how these increased metabolic needs are ensured or how this influences tumor formation and progression remains unclear. To unravel tumor metabolic dependencies, particularly from extracellular metabolites, we have analyzed the role of plasma membrane metabolic transporters in Drosophila brain tumors. Using a well-established neural stem cell-derived tumor model, caused by brat knockdown, we have found that 13 plasma membrane metabolic transporters, including amino acid, carbohydrate and monocarboxylate transporters, are upregulated in tumors and are required for tumor growth. We identified CD98hc and several of the light chains with which it can form heterodimeric amino acid transporters, as crucial players in brat RNAi (brat IR) tumor progression. Knockdown of these components of CD98 heterodimers caused a dramatic reduction in tumor growth. Our data also reveal that the oncogene dMyc is required and sufficient for the upregulation of CD98 transporter subunits in these tumors. Furthermore, tumor-upregulated dmyc and CD98 transporters orchestrate the overactivation of the growth-promoting signaling pathway TOR, forming a core growth regulatory network to support brat IR tumor progression. Our findings highlight the important link between oncogenes, metabolism, and signaling pathways in the regulation of tumor growth and allow for a better understanding of the mechanisms necessary for tumor progression.


Assuntos
Neoplasias Encefálicas , Proteínas de Drosophila , Animais , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Regulação para Cima , Proteína-1 Reguladora de Fusão/metabolismo
10.
PLoS Genet ; 19(1): e1010581, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36626385

RESUMO

Glial cells play a critical role in maintaining homeostatic ion concentration gradients. Salt-inducible kinase 3 (SIK3) regulates a gene expression program that controls K+ buffering in glia, and upregulation of this pathway suppresses seizure behavior in the eag, Shaker hyperexcitability mutant. Here we show that boosting the glial SIK3 K+ buffering pathway suppresses seizures in three additional molecularly diverse hyperexcitable mutants, highlighting the therapeutic potential of upregulating glial K+ buffering. We then explore additional mechanisms regulating glial K+ buffering. Fray, a transcriptional target of the SIK3 K+ buffering program, is a kinase that promotes K+ uptake by activating the Na+/K+/Cl- co-transporter, Ncc69. We show that the Wnk kinase phosphorylates Fray in Drosophila glia and that this activity is required to promote K+ buffering. This identifies Fray as a convergence point between the SIK3-dependent transcriptional program and Wnk-dependent post-translational regulation. Bypassing both regulatory mechanisms via overexpression of a constitutively active Fray in glia is sufficient to robustly suppress seizure behavior in multiple Drosophila models of hyperexcitability. Finally, we identify cortex glia as a critical cell type for regulation of seizure susceptibility, as boosting K+ buffering via expression of activated Fray exclusively in these cells is sufficient to suppress seizure behavior. These findings highlight Fray as a key convergence point for distinct K+ buffering regulatory mechanisms and cortex glia as an important locus for control of neuronal excitability.


Assuntos
Proteínas de Drosophila , Animais , Proteínas de Drosophila/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Drosophila/metabolismo , Convulsões/genética , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Proteínas Serina-Treonina Quinases/genética
11.
Dev Cell ; 58(1): 51-62.e4, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36626871

RESUMO

Developmental enhancers bind transcription factors and dictate patterns of gene expression during development. Their molecular evolution can underlie phenotypical evolution, but the contributions of the evolutionary pathways involved remain little understood. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most point mutations in developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native pattern. In contrast, random sequences, often acting as developmental enhancers, drove expression across a range of cell types; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently. Our findings suggest that the phenotypic landscapes of developmental enhancers are constrained by enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity of de novo elements is a primary source of phenotypic novelty.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Evolução Molecular , Fenótipo , Regulação da Expressão Gênica no Desenvolvimento
12.
Int J Mol Sci ; 24(2)2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36674733

RESUMO

Skuld (skd) is a subunit of the Mediator complex subunit complex. In the heart, skd controls systemic obesity, is involved in systemic energy metabolism, and is closely linked to cardiac function and aging. However, it is unclear whether the effect of cardiac skd on cardiac energy metabolism affects cardiac function. We found that cardiac-specific knockdown of skd showed impaired cardiac function, metabolic impairment, and premature aging. Drosophila was subjected to an exercise and high-fat diet (HFD) intervention to explore the effects of exercise on cardiac skd expression and cardiac function in HFD Drosophila. We found that Hand-Gal4>skd RNAi (KC) Drosophila had impaired cardiac function, metabolic impairment, and premature aging. Regular exercise significantly improved cardiac function and metabolism and delayed aging in HFD KC Drosophila. Thus, our study found that the effect of skd on cardiac energy metabolism in the heart affected cardiac function. Exercise may counteract age-related cardiac dysfunction and metabolic disturbances caused by HFD and heart-specific knockdown of skd. Skd may be a potential therapeutic target for heart disease.


Assuntos
Senilidade Prematura , Proteínas de Drosophila , Cardiopatias , Animais , Camundongos , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Coração , Dieta Hiperlipídica/efeitos adversos , Camundongos Endogâmicos C57BL , Proteínas do Olho/metabolismo
13.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675288

RESUMO

Mutations in ATP13A2 (PARK9), an autophagy-related protein, cause Kufor-Rakeb syndrome, an autosomal recessive, juvenile-onset form of parkinsonism. α-Synuclein (α-syn) is a presynaptic neuronal protein that forms toxic aggregates in Parkinson's disease (PD). We studied α-syn aggregation and autophagic flux in ATP13A2-knockdown Drosophila expressing either wild-type (WT) or mutant α-syn. Dopaminergic (DA) neuron loss was studied by confocal microscopy. Sleep and circadian activity were evaluated in young and old flies using a Drosophila activity monitor. Thirty-day-old ATP13A2-RNAi A53T-α-syn flies had increased Triton-insoluble α-syn levels, compared to control A53T-α-syn flies without ATP13A2-RNAi. Whole-brain staining revealed significantly fewer dopaminergic (DA) neurons in the PPL2 cluster of 30-day-old ATP13A2-RNAi flies expressing WT-, A30P-, and A53T-α-syn than in that of controls. In ATP13A2-RNAi A53T-α-syn flies, autophagic flux was decreased, as indicated by increased accumulation of Ref(2)P, the Drosophila p62 homologue. ATP13A2 silencing decreased total locomotor activity in young, and enhanced sleep features, similar to PD (decreasing bout length), in old flies expressing A53T-α-syn. ATP13A2 silencing also altered the circadian locomotor activity of A30P- and A53T-α-syn flies. Thus, ATP13A2 may play a role in the autophagic degradation of A53T-α-syn.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Drosophila/genética , Drosophila/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mutação , Inativação Gênica
14.
PLoS One ; 18(1): e0274716, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36595500

RESUMO

The C2H2 zinc finger gene cucoid establishes anterior-posterior (AP) polarity in the early embryo of culicine mosquitoes. This gene is unrelated to genes that establish embryo polarity in other fly species (Diptera), such as the homeobox gene bicoid, which serves this function in the traditional model organism Drosophila melanogaster. The cucoid gene is a conserved single copy gene across lower dipterans but nothing is known about its function in other species, and its evolution in higher dipterans, including Drosophila, is unresolved. We found that cucoid is a member of the ZAD-containing C2H2 zinc finger (ZAD-ZNF) gene family and is orthologous to 27 of the 91 members of this family in D. melanogaster, including M1BP, ranshi, ouib, nom, zaf1, odj, Nnk, trem, Zif, and eighteen uncharacterized genes. Available knowledge of the functions of cucoid orthologs in Drosophila melanogaster suggest that the progenitor of this lineage specific expansion may have played a role in regulating chromatin. We also describe many aspects of the gene duplication history of cucoid in the brachyceran lineage of D. melanogaster, thereby providing a framework for predicting potential redundancies among these genes in D. melanogaster.


Assuntos
Culicidae , Proteínas de Drosophila , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Culicidae/metabolismo , Dedos de Zinco/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/genética
15.
Elife ; 122023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36647607

RESUMO

Nutrition in early life has profound effects on an organism, altering processes such as organogenesis. However, little is known about how specific nutrients affect neuronal development. Dendrites of class IV dendritic arborization neurons in Drosophila larvae become more complex when the larvae are reared on a low-yeast diet compared to a high-yeast diet. Our systematic search for key nutrients revealed that the neurons increase their dendritic terminal densities in response to a combined deficiency in vitamins, metal ions, and cholesterol. The deficiency of these nutrients upregulates Wingless in a closely located tissue, body wall muscle. Muscle-derived Wingless activates Akt in the neurons through the receptor tyrosine kinase Ror, which promotes the dendrite branching. In larval muscles, the expression of wingless is regulated not only in this key nutrient-dependent manner, but also by the JAK/STAT signaling pathway. Additionally, the low-yeast diet blunts neuronal light responsiveness and light avoidance behavior, which may help larvae optimize their survival strategies under low-nutritional conditions. Together, our studies illustrate how the availability of specific nutrients affects neuronal development through inter-organ signaling.


Assuntos
Dendritos , Proteínas de Drosophila , Animais , Dendritos/fisiologia , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios/fisiologia , Nutrientes , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
16.
Development ; 150(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36504086

RESUMO

Evolutionarily conserved insulin/insulin-like growth factor (IGF) signaling (IIS) correlates nutrient levels to metabolism and growth, thereby playing crucial roles in development and adult fitness. In the fruit fly Drosophila, ImpL2, an ortholog of IGFBP7, binds to and inhibits the function of Drosophila insulin-like peptides. In this study, we isolated a temperature-sensitive mutation in the insulin receptor (InR) gene as a spontaneous revertant in ImpL2 null mutants. The p.Y902C missense mutation is located at the functionally conserved amino acid residue of the first fibronectin type III domain of InR. The hypomorphic InR mutant animals showed a temperature-dependent reduction in IIS and body size. The mutant animals also exhibited metabolic defects, such as increased triglyceride and carbohydrate levels. Metabolomic analysis further revealed that defects in InR caused dysregulation of amino acid and ribonucleotide metabolism. We also observed that InR mutant females produced tiny irregular-shaped embryos with reduced fecundity. In summary, this novel allele of InR is a valuable tool for the Drosophila genetic model of insulin resistance and type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Proteínas de Drosophila , Animais , Feminino , Drosophila/metabolismo , Receptor de Insulina/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mutação de Sentido Incorreto/genética , Mutação/genética , Insulina/genética , Insulina/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo
17.
Methods Mol Biol ; 2557: 635-644, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512242

RESUMO

Neurons are polarized cells whose polarity and morphology rely on the robust localization of cellular organelles and cargo to axons or dendrites. Developing neurons require an active secretory pathway, which includes the endoplasmic reticulum and Golgi apparatus, to supply membrane and proteins to growing dendrites and axons. In some neurons, a subset of the Golgi called Golgi "outposts" localize to dendrites and contribute to local secretory networks. The movement and positioning of Golgi outposts have been correlated with dendrite branch growth and stabilization as the dendritic arbor is established. Live imaging is essential to capture the dynamic nature of these organelles. Here we outline a protocol to image and quantify Golgi outposts in peripheral sensory neurons in live, intact Drosophila larvae.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Dendritos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Células Receptoras Sensoriais/metabolismo
18.
Biol Open ; 12(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36524613

RESUMO

Stem cells cycle between periods of quiescence and proliferation to promote tissue health. In Drosophila ovaries, quiescence to proliferation transitions of follicle stem cells (FSCs) are exquisitely feeding-dependent. Here, we demonstrate feeding-dependent induction of follicle cell differentiation markers, eyes absent (Eya) and castor (Cas) in FSCs, a patterning process that does not depend on proliferation induction. Instead, FSCs extend micron-scale cytoplasmic projections that dictate Eya-Cas patterning. We identify still life and sickie as necessary and sufficient for FSC projection growth and Eya-Cas induction. Our results suggest that sequential, interdependent events establish long-term differentiation patterns in follicle cell precursors, independently of FSC proliferation induction.


Assuntos
Proteínas de Drosophila , Ovário , Animais , Feminino , Ovário/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Divisão Celular , Diferenciação Celular
19.
Insect Biochem Mol Biol ; 152: 103888, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36493962

RESUMO

Phosphorylation is a key post-translational modification in regulating autophagy in yeast and mammalians, yet it is not fully illustrated in invertebrates such as insects. ULK1/Atg1 is a functionally conserved serine/threonine protein kinase involved in autophagosome initiation. As a result of alternative splicing, Atg1 in the silkworm, Bombyx mori, is present as three mRNA isoforms, with BmAtg1c showing the highest expression levels. Here, we found that BmAtg1c mRNA expression, BmAtg1c protein expression and phosphorylation, and autophagy simultaneously peaked in the fat body during larval-pupal metamorphosis. Importantly, two BmAtg1c phosphorylation sites were identified at Ser269 and Ser270, which were activated by BmAMPK, the major energy-sensing kinase, upon stimulation with 20-hydroxyecdysone and starvation; additionally, these Atg1 phosphorylation sites are evolutionarily conserved in insects. The two BmAMPK-activated phosphorylation sites in BmAtg1c were found to be required for BmAMPK-induced autophagy. Moreover, the two corresponding DmAtg1 phosphorylation sites in the fruit fly, Drosophila melanogaster, are functionally conserved for autophagy induction. In conclusion, AMPK-activated Atg1 phosphorylation is indispensable for autophagy induction and evolutionarily conserved in insects, shedding light on how various groups of organisms differentially regulate ULK1/Atg1 phosphorylation for autophagy induction.


Assuntos
Proteínas Quinases Ativadas por AMP , Proteínas de Drosophila , Animais , Fosforilação , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Drosophila/metabolismo , Autofagia/genética , Mamíferos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas de Drosophila/metabolismo
20.
Insect Biochem Mol Biol ; 152: 103892, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36493963

RESUMO

High-fat diets (HFDs) are often used to study metabolic disorders using different animal models. However, the underlying cellular mechanisms pertaining to the concurrent loss of metabolic homeostasis characteristics of these disorders are still unclear mainly because the effects of such diets are also dependent on the time frame of the experiments. Here, we used the fruit fly, Drosophila melanogaster, to investigate the metabolic dynamic effects following 0, 2, 4, 7 and 9 days of an exposure to a HFD (standard diet supplemented with 20% w/v coconut oil, rich in 12:0 and 14:0) by combining NMR metabolomics and GC-FID fatty acid profiling. Our results show that after 2 days, the ingested 12:0 and 14:0 fatty acids are used for both lipogenesis and fatty acid oxidation. After 4 days, metabolites from several different pathways are highly modulated in response to the HFD, and an accumulation of 12:0 is also observed, suggesting that the balance of lipid, amino acid and carbohydrate metabolism is profoundly perturbed at this specific time point. Following a longer exposure to the HFD (and notably after 9 days), an accumulation of many metabolites is observed indicating a clear dysfunction of the metabolic system. Overall, our study highlights the relevance of the Drosophila model to study metabolic disorders and the importance of the duration of the exposure to a HFD to study the dynamics of the fundamental mechanisms that control metabolism following exposure to dietary fats. This knowledge is crucial to understand the development and progression of metabolic diseases.


Assuntos
Dieta Hiperlipídica , Doenças Metabólicas , Animais , Ácidos Graxos/metabolismo , Drosophila melanogaster/metabolismo , Metabolismo dos Lipídeos , Metaboloma , Drosophila/metabolismo
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