dMyc-dependent upregulation of CD98 amino acid transporters is required for Drosophila brain tumor growth

Funding Information: We would like to thank Tiago Baptista and Hugo Silva for experimental help. We thank Florence Janody and Rita Teodoro for critical reading of the manuscript. We thank Juergen Knoblich for the Miranda antibody. We thank the cytometry and fly facilities at NOVA Medical School for technical support and CONGENTO: consortium for genetically tractable organisms (LISBOA-01-0145-FEDER-022170); Bloomington Drosophila Stock Center (NIH P40OD018537) and Vienna Drosophila Resource Center (VDRC) [52 ], for the stocks used in this study; the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Funding Information: Open access funding provided by FCT|FCCN (b-on). This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2017-STG-GA 759853-StemCellHabitat); by Wellcome Trust and Howard Hughes Medical Institute (HHMI-208581/Z/17/Z-Metabolic Reg SC fate); EMBO Installation grant (H2020-EMBO-3311/2017/G2017) and by Fundação para a Ciência e Tecnologia (IF/01265/2014/CP1252/CT0004, EXPL/BIA-BID/1394/2021 and 2020.05639.BD to A.R.R). This work was supported by iNOVA4Health—UIDB/04462/2020 and UIDP/04462/2020, and by the Associated Laboratory LS4FUTURE (LA/P/0087/2020), two programs financially supported by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior. Publisher Copyright: © 2023, The Author(s).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 (bratIR) 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.publishersversionpublishe

Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis

Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper our ability to test this hypothesis. We used loss- and gain-of-function approaches to examine the role of Diaphanous i

Exploring the Roles of Diaphanous and Enabled Activity in Shaping the Balance between Filopodia and Lamellipodia

During migration cell protrusions power cell extension and sample the environment. Different cells produce different protrusions, from keratocytes dominated by lamellipodia, to growth cones combining filopodia and lamellipodia, to dendritic spines. One key challenge is to determine how the toolkit of actin regulators are coordinated to generate these diverse protrusive arrays. Here we use Drosophila leading-edge (LE) cells to explore how Diaphanous (Dia)-related formins and Ena/VASP proteins cooperate in this process. We first dissect the Dia regulatory region, revealing novel roles for the GTPase-binding and FH3 domains in cortical localization, filopodial initiation, and lengthening. Second, we provide evidence that activating Dia mobilizes Ena from storage places near the LE to act at the LE. Further, Dia and Ena coIP and can recruit one another to new locations, suggesting cooperation is key to their mechanisms of action. Third, we directly explore the functional relationship between Dia and Ena, varying their levels and activity separately in the same cell type. Surprisingly, although each is sufficient to induce filopodia, together they induce lamellipodia. Our data suggest they work together in a complex and nonadditive way, with the ratio between active Dia and Ena being one factor that modulates the balance between filopodia and lamellipodia

Serine hydroxymethyl transferase is required for optic lobe neuroepithelia development in Drosophila

Funding Information: This work was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2017-STG-GA 759853-StemCellHabitat); Wellcome Trust and Howard Hughes Medical Institute (HHMI-208581/Z/17/ZMetabolicRegSCfate); EMBO Installation grant (H2020-EMBO-3311/2017/G2017) and Fundaçaõ para a Ciência e a Tecnologia (IF/01265/2014/CP1252/CT0004 and SFRH/BD/135262/2017 Eunice Silva); ‘Inova4Health – UIDB/04462/2020 and UIDP/04462/2020, and the Associated Laboratory LS4FUTURE (LA/P/0087/2020), Fundaçaõ para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior’. E.A.B.S. was partially funded by a Fundaçaõ para a Ciência e a Tecnologia doctoral fellowship under the PGCD-Graduate Program Science for Development (SFRH/BD/135262/ 2017). This research was funded in part by the Wellcome Trust [208581/Z/17/Z]. Open Access funding provided by the European Research Council. Deposited in PMC for immediate release. Publisher Copyright: © 2023. Published by The Company of Biologists Ltd.Cell fate and growth require one-carbon units for the biosynthesis of nucleotides, methylation reactions and redox homeostasis, provided by one-carbon metabolism. Consistently, defects in one-carbon metabolism lead to severe developmental defects, such as neural tube defects. However, the role of this pathway during brain development and in neural stem cell regulation is poorly understood. To better understand the role of one carbon metabolism we focused on the enzyme Serine hydroxymethyl transferase (Shmt), a key factor in the one-carbon cycle, during Drosophila brain development. We show that, although loss of Shmt does not cause obvious defects in the central brain, it leads to severe phenotypes in the optic lobe. The shmt mutants have smaller optic lobe neuroepithelia, partly justified by increased apoptosis. In addition, shmt mutant neuroepithelia have morphological defects, failing to form a lamina furrow, which likely explains the observed absence of lamina neurons. These findings show that one-carbon metabolism is crucial for the normal development of neuroepithelia, and consequently for the generation of neural progenitor cells and neurons. These results propose a mechanistic role for one-carbon during brain development.publishersversionpublishe

Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis

Funding Information: This work has been funded by National funds, through the Foundation for Science and Technology (FCT)\u2013project UIDB/50026/2020 (DOI 10.54499/UIDB/50026/2020), UIDP/50026/2020 (DOI 10.54499/UIDP/50026/2020) and LA/P/0050/2020 (DOI 10.54499/LA/P/0050/2020); by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI\u2013Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122); and by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also supported by the European Research Council (ERC) under the European Union\u2019s Horizon 2020 research and innovation programme (H2020-ERC-2017-STG-GA 759853-StemCellHabitat). Hugo Fernandes-Silva was supported by a doctoral fellowship (PD/BD/137655/2018) from FCT as part of the Inter-University Doctoral Programme in Ageing and Chronic Disease (PhDOC). Publisher Copyright: © 2024 by the authors.Lung branching morphogenesis relies on intricate epithelial–mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal–distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA’s pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.publishersversionpublishe

Rho1 regulates Drosophila adherens junctions independently of p120ctn

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. Here we examine the roles of these candidate AJ regulators durin

Enabled and Capping protein play important roles in shaping cell behavior during Drosophila oogenesis

During development, cells craft an impressive array of actin-based structures, mediating events as diverse as cytokinesis, apical constriction, and cell migration. One challenge is to determine how cells regulate actin assembly and disassembly to carry out these cell behaviors. During Drosophila oogenesis diverse cell behaviors are seen in the soma and germline. We used oogenesis to explore developmental roles of two important actin regulators: Enabled/VASP proteins and Capping protein. We found that Enabled plays an important role in cortical integrity of nurse cells, formation of robust bundled actin filaments in late nurse cells that facilitate nurse cell dumping, and migration of somatic border cells. During nurse cell dumping, Enabled localizes to barbed ends of the nurse cell actin filaments, suggesting its mechanism of action. We further pursued this mechanism using mutant Enabled proteins, each affecting one of its protein domains. These data suggest critical roles for the EVH2 domain and its tetramerization subdomain, while the EVH1 domain appears less critical. Enabled appears to be negatively regulated during oogenesis by Abelson kinase. We also explored the function of Capping protein. This revealed important roles in oocyte determination, nurse cell cortical integrity and nurse cell dumping, and support the idea that Capping protein and Enabled act antagonistically during dumping. Together these data reveal places these actin regulators shape oogenesis

Opposing effects of ecdysone signaling regulate neuroblast proliferation to ensure coordination of brain and organism development

Funding Information: This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme ( H2020-ERC-2017-STG-GA 759853 -StemCellHabitat); by Wellcome Trust and Howard Hughes Medical Institute ( HHMI-208581/Z/17/Z -Metabolic Reg SC fate); EMBO Installation grant ( H2020-EMBO-3311/2017/G2017 ); by Fundação para a Ciência e Tecnologia ( PTDC/BIA-BID/0681/2021 IF/01265/2014/CP1252/CT0004 and PD/BD/136895/2018 to AO). Nova Medical School was funded by iNOVA4Health — UIDB/04462/2020 and UIDP/04462/2020 , and by the Associated Laboratory LS4FUTURE ( LA/P/0087/2020 ), two programs financially supported by Fundação para a Ciência e Tecnologia / Ministério da Ciência , Tecnologia e Ensino Superior . The Fly facility was funded by CONGENTO LISBOA-01-0145-FEDER- 022170 . Funding Information: This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (H2020-ERC-2017-STG-GA 759853-StemCellHabitat); by Wellcome Trust and Howard Hughes Medical Institute (HHMI-208581/Z/17/Z-Metabolic Reg SC fate); EMBO Installation grant (H2020-EMBO-3311/2017/G2017); by Fundação para a Ciência e Tecnologia (PTDC/BIA-BID/0681/2021 IF/01265/2014/CP1252/CT0004 and PD/BD/136895/2018 to AO). Nova Medical School was funded by iNOVA4Health—UIDB/04462/2020 and UIDP/04462/2020, and by the Associated Laboratory LS4FUTURE (LA/P/0087/2020), two programs financially supported by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior. The Fly facility was funded by CONGENTO LISBOA-01-0145-FEDER- 022170. Publisher Copyright: © 2023 The AuthorsGrowth regulation must be robust to ensure correct final size, but also adaptative to adjust to less favorable environmental conditions. Developmental coordination between whole-organism and the brain is particularly important, as the brain is a critical organ with little adaptability. Brain growth mainly depends on neural stem cell (NSC) proliferation to generate differentiated neural cells, it is however unclear how organism developmental progression is coordinated with NSCs. Here we demonstrate that the steroid hormone ecdysone plays a multi-step, stage specific role in regulating Drosophila NSCs, the neuroblasts. We used animals that are unable to synthesize ecdysone, to show that the developmental milestone called “critical weight peak”, the peak that informs the body has reached minimum viable weight to survive metamorphosis, acts a checkpoint necessary to set neuroblast cell cycle pace during larval neurogenesis. The peaks of ecdysone that occur post-critical weight are no longer required to maintain neuroblast division rate. We additionally show that in a second stage, at the onset of pupariation, ecdysone is instead required to trigger neuroblast's proliferation exit and consequently the end of neurogenesis. We demonstrate that, without this signal from ecdysone, neuroblasts lose their ability to exit proliferation. Interestingly, although these neuroblasts proliferate for a longer period, the number of differentiated neurons is smaller compared to wild-type brains, suggesting a role for ecdysone in neuron maintenance. Our study provides insights into how neural stem cells coordinate their division rate with the pace of body growth, identifying a novel coordination mechanism between animal development and NSC proliferation.publishersversionpublishe

Using Bcr-Abl to Examine Mechanisms by Which Abl Kinase Regulates Morphogenesis in Drosophila

Signaling by the nonreceptor tyrosine kinase Abelson (Abl) plays key roles in normal development, whereas its inappropriate activation helps trigger the development of several forms of leukemia. Abl is best known for its roles in axon guidance, but Abl and its relatives also help regulate embryonic morphogenesis in epithelial tissues. Here, we explore the role of regulation of Abl kinase activity during development. We first compare the subcellular localization of Abl protein and of active Abl, by using a phosphospecific antibody, providing a catalog of places where Abl is activated. Next, we explore the consequences for morphogenesis of overexpressing wild-type Abl or expressing the activated form found in leukemia, Bcr-Abl. We find dose-dependent effects of elevating Abl activity on morphogenetic movements such as head involution and dorsal closure, on cell shape changes, on cell protrusive behavior, and on the organization of the actin cytoskeleton. Most of the effects of Abl activation parallel those caused by reduction in function of its target Enabled. Abl activation leads to changes in Enabled phosphorylation and localization, suggesting a mechanism of action. These data provide new insight into how regulated Abl activity helps direct normal development and into possible biological functions of Bcr-Abl