RESUMO
The optimization of brain circuit connectivity based on initial environmental input occurs during critical periods characterized by sensory experience-dependent, temporally restricted, and transiently reversible synapse elimination. This precise, targeted synaptic pruning mechanism is mediated by glial phagocytosis. Serotonin signaling has prominent, foundational roles in the brain, but functions in glia, or in experience-dependent brain circuit synaptic connectivity remodeling, have been relatively unknown. Here, we discover that serotonergic signaling between glia is essential for olfactory experience-dependent synaptic glomerulus pruning restricted to a well-defined Drosophila critical period. We find that experience-dependent serotonin signaling is restricted to the critical period, with both (1) serotonin production and (2) 5-HT2A receptors specifically in glia, but not neurons, absolutely required for targeted synaptic glomerulus pruning. We discover that glial 5-HT2A receptor signaling limits the experience-dependent synaptic connectivity pruning in the critical period and that conditional reexpression of 5-HT2A receptors within adult glia reestablishes "critical period-like" experience-dependent synaptic glomerulus pruning at maturity. These results reveal an essential requirement for glial serotonergic signaling mediated by 5-HT2A receptors for experience-dependent synapse elimination.
Assuntos
Neuroglia , Receptor 5-HT2A de Serotonina , Serotonina , Transdução de Sinais , Sinapses , Animais , Neuroglia/metabolismo , Sinapses/metabolismo , Sinapses/fisiologia , Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Plasticidade Neuronal/fisiologia , Drosophila melanogaster/metabolismo , Drosophila/metabolismoRESUMO
High dietary sugar (HDS), a contemporary dietary concern due to excessive intake of added sugars and carbohydrates, escalates the risk of metabolic disorders and concomitant cancers. However, the molecular mechanisms underlying HDS-induced cancer progression are not completely understood. We found that phosphoenolpyruvate carboxykinase 1 (PEPCK1), a pivotal enzyme in gluconeogenesis, is paradoxically upregulated in tumors by HDS, but not by normal dietary sugar (NDS), during tumor progression. Targeted knockdown of pepck1, but not pepck2, specifically in tumor tissue in Drosophila in vivo, not only attenuates HDS-induced tumor growth but also significantly improves the survival of Ras/Src tumor-bearing animals fed HDS. Interestingly, HP1a-mediated heterochromatin interacts directly with the pepck1 gene and downregulates pepck1 gene expression in wild-type Drosophila. Mechanistically, we demonstrated that, under HDS conditions, pepck1 knockdown reduces both wingless and TOR signaling, decreases evasion of apoptosis, reduces genome instability, and suppresses glucose uptake and trehalose levels in tumor cells in vivo. Moreover, rational pharmacological inhibition of PEPCK1, using hydrazinium sulfate, greatly improves the survival of tumor-bearing animals with pepck1 knockdown under HDS. This study is the first to show that elevated levels of dietary sugar induce aberrant upregulation of PEPCK1, which promotes tumor progression through altered cell signaling, evasion of apoptosis, genome instability, and reprogramming of carbohydrate metabolism. These findings contribute to our understanding of the complex relationship between diet and cancer at the molecular, cellular, and organismal levels and reveal PEPCK1 as a potential target for the prevention and treatment of cancers associated with metabolic disorders.
Assuntos
Progressão da Doença , Proteínas de Drosophila , Regulação para Cima , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Humanos , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/genética , Apoptose/genética , Transdução de Sinais , Proteína Wnt1/metabolismo , Proteína Wnt1/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Glucose/metabolismo , Instabilidade Genômica , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Linhagem Celular Tumoral , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Regulação Neoplásica da Expressão Gênica , Trealose/metabolismo , Carboidratos da Dieta/efeitos adversos , Drosophila/metabolismoRESUMO
Inflammatory bowel diseases (IBDs) are immune chronic diseases characterized by recurrent episodes, resulting in continuous intestinal barrier damage and intestinal microbiota dysbiosis. Safe strategies aimed at stabilizing and reducing IBDs recurrence have been vigorously pursued. Here, we constructed a recurrent intestinal injury Drosophila model and found that vitamin B12 (VB12), an essential co-factor for organism physiological functions, could effectively protect the intestine and reduce dextran sulfate sodium-induced intestinal barrier disruption. VB12 also alleviated microbial dysbiosis in the Drosophila model and inhibited the growth of gram-negative bacteria. We demonstrated that VB12 could mitigate intestinal damage by activating the hypoxia-inducible factor-1 signaling pathway in injured conditions, which was achieved by regulating the intestinal oxidation. In addition, we also validated the protective effect of VB12 in a murine acute colitis model. In summary, we offer new insights and implications for the potential supportive role of VB12 in the management of recurrent IBDs flare-ups.
Assuntos
Sulfato de Dextrana , Modelos Animais de Doenças , Microbioma Gastrointestinal , Fator 1 Induzível por Hipóxia , Mucosa Intestinal , Transdução de Sinais , Vitamina B 12 , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Vitamina B 12/farmacologia , Vitamina B 12/metabolismo , Camundongos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Transdução de Sinais/efeitos dos fármacos , Sulfato de Dextrana/toxicidade , Fator 1 Induzível por Hipóxia/metabolismo , Colite/metabolismo , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Colite/tratamento farmacológico , Disbiose/microbiologia , Disbiose/metabolismo , Camundongos Endogâmicos C57BL , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Drosophila/metabolismoRESUMO
Proteotoxic stress impairs cellular homeostasis and underlies the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, the loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in the tissues of ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.
Assuntos
Complexo de Endopeptidases do Proteassoma , Proteostase , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Humanos , Drosophila/metabolismo , Autofagia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Neurônios/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genéticaRESUMO
Stem cell niche is critical for regulating the behavior of stem cells. Drosophila neural stem cells (Neuroblasts, NBs) are encased by glial niche cells closely, but it still remains unclear whether glial niche cells can regulate the self-renewal and differentiation of NBs. Here, we show that ferritin produced by glia, cooperates with Zip13 to transport iron into NBs for the energy production, which is essential to the self-renewal and proliferation of NBs. The knockdown of glial ferritin encoding genes causes energy shortage in NBs via downregulating aconitase activity and NAD+ level, which leads to the low proliferation and premature differentiation of NBs mediated by Prospero entering nuclei. More importantly, ferritin is a potential target for tumor suppression. In addition, the level of glial ferritin production is affected by the status of NBs, establishing a bicellular iron homeostasis. In this study, we demonstrate that glial cells are indispensable to maintain the self-renewal of NBs, unveiling a novel role of the NB glial niche during brain development.
Iron is an essential nutrient for almost all living organisms. For example, iron contributes to the replication of DNA, the generation of energy inside cells, and the transport of oxygen around the body. Iron deficiency is the most common of all nutrient deficiencies, affecting over 40% of children worldwide. This can lead to anemia and also impair how the brain and nervous system develop, potentially resulting in long-lasting cognitive damage, even after the deficiency has been treated. It is poorly understood how iron contributes to the development of the brain and nervous system. In particular, whether and how it supports nerve stem cells (or NSCs for short) which give rise to the various neural types in the mature brain. To investigate, Ma et al. experimentally reduced the levels of ferritin (a protein which stores iron) in the developing brains of fruit fly larvae. This reduction in ferritin led to lower numbers of NSCs and a smaller brain. Unexpectedly, this effect was largest when ferritin levels were reduced in glial cells which support and send signals to NSCs, rather than in the stem cells themselves. Ma et al. then used fluorescence microscopy to confirm that glial cells make and contain a lot of ferritin which can be transported to NSCs. Adding iron supplements to the diet of flies lacking ferritin did not lead to normal numbers of stem cells in the brains of the developing fruit flies, whereas adding compounds that reduce the amount of iron led to lower numbers of stem cells. Together, this suggests that ferritin transports iron from glial cells to the NSCs. Without ferritin and iron, the NSCs could not produce enough energy to divide and make new stem cells. This caused the NSCs to lose the characteristics of stem cells and prematurely turn into other types of neurons or glial cells. Together, these findings show that when iron cannot move from glial cells to NSCs this leads to defects in brain development. Future experiments will have to test whether a similar transport of iron from supporting cells to NSCs also occurs in the developing brains of mammals, and whether this mechanism applies to stem cells in other parts of the body.
Assuntos
Proteínas de Drosophila , Ferritinas , Ferro , Células-Tronco Neurais , Neuroglia , Animais , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Ferro/metabolismo , Ferritinas/metabolismo , Ferritinas/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila/metabolismo , Proliferação de Células , Diferenciação Celular , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Autorrenovação CelularRESUMO
Spatially and temporally accurate termination of axon outgrowth, a process called axon termination, is required for efficient, precise nervous system construction and wiring. The mechanosensory neurons that sense low-threshold mechanical stimulation or gentle touch have proven exceptionally valuable for studying axon termination over the past 40â years. In this Review, we discuss progress made in deciphering the molecular and genetic mechanisms that govern axon termination in touch receptor neurons. Findings across model organisms, including Caenorhabditis elegans, Drosophila, zebrafish and mice, have revealed that complex signaling is required for termination with conserved principles and players beginning to surface. A key emerging theme is that axon termination is mediated by complex signaling networks that include ubiquitin ligase signaling hubs, kinase cascades, transcription factors, guidance/adhesion receptors and growth factors. Here, we begin a discussion about how these signaling networks could represent termination codes that trigger cessation of axon outgrowth in different species and types of mechanosensory neurons.
Assuntos
Axônios , Transdução de Sinais , Animais , Axônios/metabolismo , Axônios/fisiologia , Mecanorreceptores/metabolismo , Caenorhabditis elegans/metabolismo , Drosophila/metabolismoRESUMO
The number of neural stem cells reflects the total number of neurons in the mature brain. As neural stem cells arise from neuroepithelial cells, the neuroepithelial cell population must be expanded to secure a sufficient number of neural stem cells. However, molecular mechanisms that regulate timely differentiation from neuroepithelial to neural stem cells are largely unclear. Here, we show that TCF4/Daughterless is a key factor that determines the timing of the differentiation in Drosophila. The neuroepithelial cells initiated but never completed the differentiation in the absence of TCF4/Daughterless. We also found that TCF4/Daughterless binds to the Notch locus, suggesting that Notch is one of its downstream candidate genes. Consistently, Notch expression was ectopically induced in the absence of TCF4/Daughterless. Furthermore, ectopic activation of Notch signaling phenocopied loss of TCF4/Daughterless. Our findings demonstrate that TCF4/Daughterless directly inactivates Notch signaling pathway, resulting in completion of the differentiation from neuroepithelial cells into neural stem cells with optimal timing. Thus, the present results suggest that TCF4/Daughterless is essential for determining whether to move to the next state or stay in the current state in differentiating neuroepithelial cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Proteínas de Drosophila , Células-Tronco Neurais , Células Neuroepiteliais , Receptores Notch , Transdução de Sinais , Animais , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Receptores Notch/metabolismo , Receptores Notch/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Células Neuroepiteliais/metabolismo , Células Neuroepiteliais/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/citologia , Fatores de Tempo , Drosophila/metabolismoRESUMO
Most RNA-protein condensates are composed of heterogeneous immiscible phases. However, how this multiphase organization contributes to their biological functions remains largely unexplored. Drosophila germ granules, a class of RNA-protein condensates, are the site of mRNA storage and translational activation. Here, using super-resolution microscopy and single-molecule imaging approaches, we show that germ granules have a biphasic organization and that translation occurs in the outer phase and at the surface of the granules. The localization, directionality, and compaction of mRNAs within the granule depend on their translation status, translated mRNAs being enriched in the outer phase with their 5'end oriented towards the surface. Translation is strongly reduced when germ granule biphasic organization is lost. These findings reveal the intimate links between the architecture of RNA-protein condensates and the organization of their different functions, highlighting the functional compartmentalization of these condensates.
Assuntos
Grânulos Citoplasmáticos , Proteínas de Drosophila , Drosophila melanogaster , Biossíntese de Proteínas , RNA Mensageiro , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Grânulos Citoplasmáticos/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Células Germinativas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Imagem Individual de Molécula , Drosophila/metabolismo , Drosophila/genética , Condensados Biomoleculares/metabolismoRESUMO
The initially homogeneous epithelium of the early Drosophila embryo differentiates into regional subpopulations with different behaviours and physical properties that are needed for morphogenesis. The factors at top of the genetic hierarchy that control these behaviours are known, but many of their targets are not. To understand how proteins work together to mediate differential cellular activities, we studied in an unbiased manner the proteomes and phosphoproteomes of the three main cell populations along the dorso-ventral axis during gastrulation using mutant embryos that represent the different populations. We detected 6111 protein groups and 6259 phosphosites of which 3398 and 3433 were differentially regulated, respectively. The changes in phosphosite abundance did not correlate with changes in host protein abundance, showing phosphorylation to be a regulatory step during gastrulation. Hierarchical clustering of protein groups and phosphosites identified clusters that contain known fate determinants such as Doc1, Sog, Snail, and Twist. The recovery of the appropriate known marker proteins in each of the different mutants we used validated the approach, but also revealed that two mutations that both interfere with the dorsal fate pathway, Toll10B and serpin27aex do this in very different manners. Diffused network analyses within each cluster point to microtubule components as one of the main groups of regulated proteins. Functional studies on the role of microtubules provide the proof of principle that microtubules have different functions in different domains along the DV axis of the embryo.
Assuntos
Proteínas de Drosophila , Fosfoproteínas , Proteoma , Animais , Proteoma/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Embrião não Mamífero/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Fosforilação , Gastrulação , Padronização Corporal/genéticaRESUMO
Signaling pathways activated by secreted Wnt ligands play an essential role in tissue development and the progression of diseases, like cancer. Secretion of the lipid-modified Wnt proteins is tightly regulated by a repertoire of intracellular factors. For instance, a membrane protein, Evi, interacts with the Wnt ligand in the ER, and it is essential for its further trafficking and release in the extracellular space. After dissociating from the Wnt, the Wnt-unbound Evi is recycled back to the ER via Golgi. However, where in this trafficking path Wnt proteins dissociate from Evi remains unclear. Here, we have used the Drosophila wing epithelium to trace the route of the Evi-Wg (Wnt homolog) complex leading up to their separation. In these polarized cells, Wg is first trafficked to the apical surface; however, the secretion of Wg is believed to occurs post-internalization via recycling. Our results show that the Evi-Wg complex is internalized from the apical surface and transported to the retromer-positive endosomes. Furthermore, using antibodies that specifically label the Wnt-unbound Evi, we show that Evi and Wg separation occurs post-internalization in the acidic endosomes. These results refine our understanding of the polarized trafficking of Wg and highlight the importance of Wg endocytosis in its secondary secretion.
Assuntos
Proteínas de Drosophila , Endossomos , Transporte Proteico , Proteína Wnt1 , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Asas de Animais/metabolismo , Proteína Wnt1/metabolismo , Proteína Wnt1/genéticaRESUMO
Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress continues to be made in our understanding of the pathways that control the apical (top) geometry of epithelial cells, we know comparatively little about those that control cell basal (bottom) geometry. To examine this, we used the Drosophila ommatidium, which is the basic visual unit of the compound eye. The ommatidium is shaped as a hexagonal prism, and generating this 3D structure requires ommatidial cells to adopt specific apical and basal polygonal geometries. Using this model system, we find that generating cell type-specific basal geometries starts with patterning of the basal extracellular matrix, whereby Laminin accumulates at discrete locations across the basal surface of the retina. We find the Dystroglycan receptor complex (DGC) is required for this patterning by promoting localized Laminin accumulation at the basal surface of cells. Moreover, our results reveal that localized accumulation of Laminin and the DGC are required for directing Integrin adhesion. This induces cell basal geometry remodeling by anchoring the basal surface of cells to the extracellular matrix at specific, Laminin-rich locations. We propose that patterning of a basal extracellular matrix by generating discrete Laminin domains can direct Integrin adhesion to induce cell shape remodeling in epithelial morphogenesis.
Assuntos
Forma Celular , Proteínas de Drosophila , Drosophila melanogaster , Distroglicanas , Matriz Extracelular , Integrinas , Laminina , Retina , Animais , Distroglicanas/metabolismo , Laminina/metabolismo , Integrinas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Matriz Extracelular/metabolismo , Retina/metabolismo , Retina/crescimento & desenvolvimento , Retina/citologia , Retina/embriologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Morfogênese , Adesão Celular , Drosophila/metabolismo , Drosophila/crescimento & desenvolvimentoRESUMO
Post-larval hematopoiesis in Drosophila largely depends upon the stockpile of progenitors present in the blood-forming organ/lymph gland of the larvae. During larval stages, the lymph gland progenitors gradually accumulate reactive oxygen species (ROS), which is essential to prime them for differentiation. Studies have shown that ROS triggers the activation of JNK (c-Jun Kinase), which upregulates fatty acid oxidation (FAO) to facilitate progenitor differentiation. Intriguingly, despite having ROS, the entire progenitor pool does not differentiate simultaneously in the late larval stages. Using expression analyses, genetic manipulation and pharmacological approaches, we found that the Drosophila NF-κB transcription factor Relish (Rel) shields the progenitor pool from the metabolic pathway that inducts them into the differentiation program by curtailing the activation of JNK. Although ROS serves as the metabolic signal for progenitor differentiation, the input from ROS is monitored by the developmental signal TAK1, which is regulated by Relish. This developmental circuit ensures that the stockpile of ROS-primed progenitors is not exhausted entirely. Our study sheds light on how, during development, integrating NF-κB-like factors with metabolic pathways seem crucial to regulating cell fate transition during development.
Assuntos
Diferenciação Celular , Proteínas de Drosophila , Hematopoese , Homeostase , Larva , NF-kappa B , Espécies Reativas de Oxigênio , Fatores de Transcrição , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Espécies Reativas de Oxigênio/metabolismo , NF-kappa B/metabolismo , NF-kappa B/genética , Diferenciação Celular/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Hematopoese/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Transdução de Sinais , Tecido Linfoide/metabolismo , Tecido Linfoide/crescimento & desenvolvimento , Células-Tronco/metabolismo , Células-Tronco/citologia , Drosophila/genética , Drosophila/metabolismo , Drosophila/crescimento & desenvolvimentoRESUMO
Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins.
Assuntos
Caenorhabditis elegans , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Optogenética , Proteínas RGS , Transdução de Sinais , Peixe-Zebra , Animais , Optogenética/métodos , Caenorhabditis elegans/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas RGS/metabolismo , Proteínas RGS/genética , Peixe-Zebra/embriologia , Neurônios/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Domínios Proteicos , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Drosophila/metabolismoRESUMO
Golgi-resident enzymes remain in place while their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3, acts to recruit enzymes into vesicles in part of the stack. Here, we used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi-resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.
Assuntos
Complexo de Golgi , Proteínas de Membrana , Complexo de Golgi/metabolismo , Humanos , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ligação Proteica , Transporte Proteico , Complexo I de Proteína do Envoltório/metabolismo , Complexo I de Proteína do Envoltório/genética , Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , GlicosilaçãoRESUMO
Testicular aging manifests as impaired spermatogenesis and morphological alterations in Drosophila. Nonetheless, the comprehensive molecular regulatory framework remains largely undisclosed. This investigation illustrates the impact of copper overload on testicular aging and underscores the interplay between copper overload and lncRNA. Copper overload triggers Cuproptosis through the mitochondrial TCA cycle, facilitating intracellular interactions with Ferroptosis, thereby governing testicular aging. Dysfunction of lncRNA:CR43306 also contributes to testicular aging in Drosophila, emphasizing the significance of lncRNA:CR43306 as a novel aging-associated lncRNA. Moreover, copper overload exacerbates spermatid differentiation defects mediated by lncRNA:CR43306 deficiency through oxidative stress, copper, and iron transport. Therapeutically, Ferrostatin-1 and Resveratrol emerge as potential remedies for addressing testicular aging. This study offers perspectives on the regulatory mechanisms involving copper overload and lncRNA:CR43306 deficiency in the context of testicular aging.
Assuntos
Envelhecimento , Cobre , Ferroptose , Estresse Oxidativo , RNA Longo não Codificante , Testículo , Animais , Masculino , RNA Longo não Codificante/genética , Testículo/metabolismo , Ferroptose/genética , Envelhecimento/genética , Envelhecimento/metabolismo , Cobre/metabolismo , Cobre/deficiência , Espermatogênese/genética , Ferro/metabolismo , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genéticaRESUMO
RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.
Assuntos
Citosol , Proteínas de Drosophila , Endocitose , Lisossomos , Interferência de RNA , RNA de Cadeia Dupla , Animais , RNA de Cadeia Dupla/metabolismo , Lisossomos/metabolismo , Citosol/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Linhagem Celular , Membranas Intracelulares/metabolismo , Permeabilidade , Drosophila/metabolismo , Drosophila/genéticaRESUMO
Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive vinegar fly that is a major threat to the small fruits industries globally. Insect capa genes encode multiple neuropeptides, including CAPA-periviscerokinin (CAPA-PVK) peptides, that are specifically known to cause diuresis or anti-diuresis in various organisms. Here we identified and characterized a corresponding G protein-coupled receptor (GPCR) of the D. suzukii CAPA-PVK peptides: CAPA receptor (CAPA-R). To better characterize the behavior of D. suzukii CAPA-R, we used insect cell-based functional expression assays to evaluate responses of CAPA-R against D. suzukii CAPA-PVKs, CAPA-PVKs from five species in Insecta, one species from Mollusca, modified CAPA-PVK peptides, and some PRXamide family peptides: pyrokinin (PK), diapause hormone (DH), and ecdysis-triggering hormone (ETH). Functional studies revealed that the D. suzukii CAPA-R is strongly activated by both of its own natural D. suzukii CAPA-PVKs, and interestingly, it was strongly activated by other CAPA-PVK peptides from Frankliniella occidentallis (Thysanoptera), Solenopsis invicta (Hymenoptera), Helicoverpa zea (Lepidoptera) and Plutella xylostella (Lepidoptera). However, D. suzukii CAPA-R was not activated by Mollusca CAPA-PVK or the other PRXamide peptides. Gene expression analyses showed that the CAPA-R was highly expressed in the Malpighian tubules and moderately in hindgut compared to other digestive organs or the rest of body, supporting diuretic/antidiuretic functionality. When compared across life stages of D. suzukii, expression of CAPA-R was approximately 1.5x greater in the third instar than the other stages and minimally detected in the eggs, 4-day old pupae and 3-day old adults. Our results functionally characterized the D. suzukii CAPA-R and a few short peptides were identified as potential biological targets to exploit the CAPA-R for D. suzukii management.
Assuntos
Proteínas de Drosophila , Drosophila , Neuropeptídeos , Animais , Feminino , Sequência de Aminoácidos , Drosophila/metabolismo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Trato Gastrointestinal/metabolismo , Hormônios de Inseto/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Pupa/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
In nephrotic syndrome, the podocyte filtration structures are damaged in a process called foot process effacement. This is mediated by the actin cytoskeleton; however, which actins are involved and how they interact with other filtration components, like the basement membrane, remains poorly understood. Here, we used the well-established Drosophila pericardial nephrocyte-the equivalent of podocytes in flies-knockdown models (RNAi) to study the interplay of the actin cytoskeleton (Act5C, Act57B, Act42A, and Act87E), alpha- and beta-integrin (basement membrane), and the slit diaphragm (Sns and Pyd). Knockdown of an actin gene led to variations of formation of actin stress fibers, the internalization of Sns, and a disrupted slit diaphragm cortical pattern. Notably, deficiency of Act5C, which resulted in complete absence of nephrocytes, could be partially mitigated by overexpressing Act42A or Act87E, suggesting at least partial functional redundancy. Integrin localized near the actin cytoskeleton as well as slit diaphragm components, but when the nephrocyte cytoskeleton or slit diaphragm was disrupted, this switched to colocalization, both at the surface and internalized in aggregates. Altogether, the data show that the interdependence of the slit diaphragm, actin cytoskeleton, and integrins is key to the structure and function of the Drosophila nephrocyte.
Assuntos
Citoesqueleto de Actina , Proteínas de Drosophila , Integrinas , Podócitos , Animais , Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Integrinas/metabolismo , Podócitos/metabolismo , Drosophila melanogaster/metabolismo , Drosophila/metabolismo , Actinas/metabolismo , ImunoglobulinasRESUMO
The ellipsoid body (EB) of the insect brain performs pivotal functions in controlling navigation. Input and output of the EB is provided by multiple classes of R-neurons (now referred to as ER-neurons) and columnar neurons which interact with each other in a stereotypical and spatially highly ordered manner. The developmental mechanisms that control the connectivity and topography of EB neurons are largely unknown. One indispensable prerequisite to unravel these mechanisms is to document in detail the sequence of events that shape EB neurons during their development. In this study, we analyzed the development of the Drosophila EB. In addition to globally following the ER-neuron and columnar neuron (sub)classes in the spatial context of their changing environment we performed a single cell analysis using the multi-color flip out (MCFO) system to analyze the developmental trajectory of ER-neurons at different pupal stages, young adults (4d) and aged adults (â¼60d). We show that the EB develops as a merger of two distinct elements, a posterior and anterior EB primordium (prEBp and prEBa, respectively. ER-neurons belonging to different subclasses form growth cones and filopodia that associate with the prEBp and prEBa in a pattern that, from early pupal stages onward, foreshadows their mature structure. Filopodia of all ER-subclasses are initially much longer than the dendritic and terminal axonal branches they give rise to, and are pruned back during late pupal stages. Interestingly, extraneous branches, particularly significant in the dendritic domain, are a hallmark of ER-neuron structure in aged brains. Aging is also associated with a decline in synaptic connectivity from columnar neurons, as well as upregulation of presynaptic protein (Brp) in ER-neurons. Our findings advance the EB (and ER-neurons) as a favorable system to visualize and quantify the development and age-related decline of a complex neuronal circuitry.
Assuntos
Envelhecimento , Neurônios , Animais , Neurônios/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Encéfalo/metabolismo , Encéfalo/embriologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Pseudópodes/metabolismo , Pupa/metabolismo , Pupa/crescimento & desenvolvimento , Drosophila/metabolismo , Cones de Crescimento/metabolismoRESUMO
Obesity remains one of the largest health problems in the world, arising from the excess storage of triglycerides (TAGs). However, the full complement of genes that are important for regulating TAG storage is not known. The Glut1 gene encodes a Drosophila glucose transporter that has been identified as a potential obesity gene through genetic screening. Yet, the tissue-specific metabolic functions of Glut1 are not fully understood. Here, we characterized the role of Glut1 in the fly brain by decreasing neuronal Glut1 levels with RNAi and measuring glycogen and TAGs. Glut1RNAi flies had decreased TAG and glycogen levels, suggesting a nonautonomous role of Glut1 in the fly brain to regulate nutrient storage. A group of hormones that regulate metabolism and are expressed in the fly brain are Drosophila insulin-like peptides (Ilps) 2, 3, and 5. Interestingly, we observed blunted Ilp3 and Ilp5 expression in neuronal Glut1RNAi flies, suggesting Glut1 functions in insulin-producing neurons (IPCs) to regulate whole-organism TAG and glycogen storage. Consistent with this hypothesis, we also saw fewer TAGs and glycogens and decreased expression of Ilp3 and Ilp5 in flies with IPC-specific Glut1RNAi. Together, these data suggest Glut1 functions as a nutrient sensor in IPCs, controlling TAG and glycogen storage and regulating systemic energy homeostasis.