The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues.

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane.

Publisher Correction: The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.

In the version of this article initially published, authors Sarah E. Wilkins, Charlotte D. Eaton, Martine I. Abboud and Maximiliano J. Katz were incorrectly included in the equal contributions footnote in the affiliations list. Footnote number seven linking to the equal contributions statement should be present only for Suzana Markolovic and Qinqin Zhuang, and the statement should read "These authors contributed equally: Suzana Markolovic, Qinqin Zhuang." The error has been corrected in the HTML and PDF versions of the article.

The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.

Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(II)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.

miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga.

Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses.

Musashi mediates translational repression of the Drosophila hypoxia inducible factor.

Adaptation to hypoxia depends on a conserved α/ß heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3' UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available.

Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth.

Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2α and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase.

OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation.

2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia-inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation.

Striking Oxygen Sensitivity of the Peptidylglycine α-Amidating Monooxygenase (PAM) in Neuroendocrine Cells.

Interactions between biological pathways and molecular oxygen require robust mechanisms for detecting and responding to changes in cellular oxygen availability, to support oxygen homeostasis. Peptidylglycine α-amidating monooxygenase (PAM) catalyzes a two-step reaction resulting in the C-terminal amidation of peptides, a process important for their stability and biological activity. Here we show that in human, mouse, and insect cells, peptide amidation is exquisitely sensitive to hypoxia. Different amidation events on chromogranin A, and on peptides processed from proopiomelanocortin, manifest similar striking sensitivity to hypoxia in a range of neuroendocrine cells, being progressively inhibited from mild (7% O2) to severe (1% O2) hypoxia. In developing Drosophila melanogaster larvae, FMRF amidation in thoracic ventral (Tv) neurons is strikingly suppressed by hypoxia. Our findings have thus defined a novel monooxygenase-based oxygen sensing mechanism that has the capacity to signal changes in oxygen availability to peptidergic pathways.

Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

Robustness of the hypoxic response: another job for miRNAs?

Living organisms are constantly exposed to environmental and genetic perturbations. Biological robustness enables these organisms to maintain their functional stability in the presence of external or internal changes. It has been proposed that microRNAs (miRNAs), small non-coding regulatory RNAs, contribute to robustness of gene regulatory networks. The hypoxic response is a major and well-characterized example of a cellular and systemic response to environmental stress that needs to be robust. miRNAs regulate the response to hypoxia, both at the level of the main transcription factor that mediates this response, the hypoxia-inducible factor (HIF), and at the level of one of the most important systemic outcomes of the response: angiogenesis. In this review, we will take the hypoxic response as a paradigm of miRNAs participating in circuits that provide robustness to biological responses.

Metabolic FRET sensors in intact organs: Applying spectral unmixing to acquire reliable signals.

In multicellular organisms, metabolic coordination across multiple tissues and cell types is essential to satisfy regionalized energetic requirements and respond coherently to changing environmental conditions. However, most metabolic assays require the destruction of the biological sample, with a concomitant loss of spatial information. Fluorescent metabolic sensors and probes are among the most user-friendly techniques for collecting metabolic information with spatial resolution. In a previous work, we have adapted to an animal system, Drosophila melanogaster, genetically encoded metabolic FRET-based sensors that had been previously developed in single-cell systems. These sensors provide semi-quantitative data on the stationary concentrations of key metabolites of the bioenergetic metabolism: lactate, pyruvate, and 2-oxoglutarate. The use of these sensors in intact organs required the development of an image processing method that minimizes the contribution of spatially complex autofluorescence patterns, that would obscure the FRET signals. In this article, we show step by step how to design FRET-based sensor experiments and how to process the fluorescence signal to obtain reliable FRET values.

Adaptation to hypoxia in Drosophila melanogaster requires autophagy.

Macroautophagy/autophagy, a mechanism of degradation of intracellular material required to sustain cellular homeostasis, is exacerbated under stress conditions like nutrient deprivation, protein aggregation, organelle senescence, pathogen invasion, and hypoxia, among others. Detailed in vivo description of autophagic responses triggered by hypoxia is limited. We have characterized the autophagic response induced by hypoxia in Drosophila melanogaster. We found that this process is essential for Drosophila adaptation and survival because larvae with impaired autophagy are hypersensitive to low oxygen levels. Hypoxia triggers a bona fide autophagic response, as evaluated by several autophagy markers including Atg8, LysoTracker, Lamp1, Pi3K59F/Vps34 activity, transcriptional induction of Atg genes, as well as by transmission electron microscopy. Autophagy occurs in waves of autophagosome formation and maturation as hypoxia exposure is prolonged. Hypoxia-triggered autophagy is induced cell autonomously, and different tissues are sensitive to hypoxic treatments. We found that hypoxia-induced autophagy depends on the basic autophagy machinery but not on the hypoxia master regulator sima/HIF1A. Overall, our studies lay the foundation for using D. melanogaster as a model system for studying autophagy under hypoxic conditions, which, in combination with the potency of genetic manipulations available in this organism, provides a platform for studying the involvement of autophagy in hypoxia-associated pathologies and developmentally regulated processes.Abbreviations: Atg: autophagy-related; FYVE: zinc finger domain from Fab1 (yeast ortholog of PIKfyve); GFP: green fluorescent protein; HIF: hypoxia-inducible factor; hsf: heat shock factor; Hx: hypoxia; mCh: mCherry; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; Rheb: Ras homolog enriched in brain; sima: similar; Stv: Starvation; TEM: transmission electron microscopy; Tor: target of rapamycin; UAS: upstream activating sequence; Vps: vacuolar protein sorting.

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway.

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex. SUMMARY: In this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.

Epigenetics: new questions on the response to hypoxia.

Reduction in oxygen levels below normal concentrations plays important roles in different normal and pathological conditions, such as development, tumorigenesis, chronic kidney disease and stroke. Organisms exposed to hypoxia trigger changes at both cellular and systemic levels to recover oxygen homeostasis. Most of these processes are mediated by Hypoxia Inducible Factors, HIFs, a family of transcription factors that directly induce the expression of several hundred genes in mammalian cells. Although different aspects of HIF regulation are well known, it is still unclear by which precise mechanism HIFs activate transcription of their target genes. Concomitantly, hypoxia provokes a dramatic decrease of general transcription that seems to rely in part on epigenetic changes through a poorly understood mechanism. In this review we discuss the current knowledge on chromatin changes involved in HIF dependent gene activation, as well as on other epigenetic changes, not necessarily linked to HIF that take place under hypoxic conditions.

The Latin American Society for Developmental Biology: a successful history.

The Latin American Society for Developmental Biology (LASDB) is one of the newest societies in this field. However, despite being new, this society already had a highly important impact on the advancement of Developmental Biology across Latin America and globally. From its conception, the society began with the establishment of courses and congresses at the frontiers of knowledge and with the participation of researchers from Latin American countries and other regions, creating an academic and fraternal environment. The first LASDB congress was held in 2003, and recently, in 2019, the LASDB celebrated its tenth meeting, besides the Pan-American congress organized in 2007. Since the creation of this society and throughout its consolidation, the LASDB has been fortunate in receiving the support of highly prominent Developmental Biology societies, with which it has established links and collaboration that have clearly promoted Development Biology not only in Latin America but also in other parts of the world. At this moment, the LASDB looks to the future to continue supporting science in Latin America as it has done up to the present.

Metabo-Devo: A metabolic perspective of development.

In the last years, several reports have established the notion that metabolism is not just a housekeeping process, but instead an active effector of physiological changes. The idea that the metabolic status may rule a wide range of phenomena in cell biology is starting to be broadly accepted. Thus, current developmental biology has begun to describe different ways by which the metabolic profile of the cell and developmental programs of the organism can crosstalk. In this review, we discuss mechanisms by which metabolism impacts on processes governing development. We review the growing body of evidence that supports the notion that aerobic glycolysis is required in cells undergoing fast growth and high proliferation, similarly to the Warburg effect described in tumor cells. Glycolytic metabolism explains not only the higher ATP synthesis rate required for cell growth, but also the uncoupling between mitochondrial activity and bioenergetics needed to provide anabolism with sufficient precursors. We also discuss some recent studies, which show that in addition to its role in providing energy and carbon chains, the metabolic status of the cell can also influence epigenetic regulation of developmental processes. Although metabolic aspects of development are just starting to be explored, there is no doubt that ongoing research in this field will shape the future landscape of Developmental Biology.

Cloning of hif-1alpha and hif-2alpha and mRNA expression pattern during development in zebrafish.

Hypoxia-inducible factors (HIFs) regulate gene expression in response to hypoxia and in vertebrates they are known to participate in several developmental processes, including angiogenesis, vasculogenesis, heart and central nervous system development. Over the last decade, major progress in unraveling the molecular mechanisms that mediate regulation of HIF proteins by oxygen tension has been reported, but our knowledge on their developmental regulation during embryogenesis in model organisms is limited. Expression of hif-1alpha and hif-2alpha genes has been characterized during normal mouse development and they were found to be expressed from stages E7.5, later in E9.5 and E15.5 in several different tissues such as the brain, heart and blood vessels. However, there is no detailed temporal information on their expression at other embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe the cloning and detailed expression pattern of zebrafish hif-1alpha and hif-2alpha genes. Sequence analysis revealed that zebrafish Hif proteins are highly homologous to other vertebrate orthologues. Zebrafish hif-1alpha and hif-2alpha are both expressed throughout development in discrete territories in a dynamic pattern. Interestingly, in the notochord the expression of hif-1alpha is switched off, while hif-2alpha transcription is turned on, signifying that the two genes might have partially overlapping, although non-redundant functions in development. This is the first time that a detailed comparison of the expression of hif-1alpha and hif-2alpha is directly assessed in a vertebrate model system throughout development.

Cellular and developmental adaptations to hypoxia: a Drosophila perspective.

The fruit fly Drosophila melanogaster, a widely utilized genetic model, is highly resistant to oxygen starvation and is beginning to be used for studying physiological, developmental, and cellular adaptations to hypoxia. The Drosophila respiratory (tracheal) system has features in common with the mammalian circulatory system so that an angiogenesis-like response occurs upon exposure of Drosophila larvae to hypoxia. A hypoxia-responsive system homologous to mammalian hypoxia-inducible factor (HIF) has been described in the fruit fly, where Fatiga is a Drosophila oxygen-dependent HIF prolyl hydroxylase, and the basic helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins Sima and Tango are, respectively, the Drosophila homologues of mammalian HIF-alpha (alpha) and HIF-beta (beta). Tango is constitutively expressed regardless of oxygen tension and, like in mammalian cells, Sima is controlled at the level of protein degradation and subcellular localization. Sima is critically required for development in hypoxia, but, unlike mammalian model systems, it is dispensable for development in normoxia. In contrast, fatiga mutant alleles are all lethal; however, strikingly, viability to adulthood is restored in fatiga sima double mutants, although these double mutants are not entirely normal, suggesting that Fatiga has Sima-independent functions in fly development. Studies in cell culture and in vivo have revealed that Sima is activated by the insulin receptor (InR) and target-of-rapamycin (TOR) pathways. Paradoxically, Sima is a negative regulator of growth. This suggests that Sima is engaged in a negative feedback loop that limits growth upon stimulation of InR/TOR pathways.

Control of the hypoxic response in Drosophila melanogaster by the basic helix-loop-helix PAS protein similar.

In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using transcriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-alpha and HIF-beta homologues, respectively, and demonstrate a conserved mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.

Zonda is a novel early component of the autophagy pathway in Drosophila.

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation.