RESUMO
Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. Here we show that in response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling.
Assuntos
Proteínas de Drosophila , Insulinas , Animais , Proliferação de Células , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Insulinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases , Células-Tronco/metabolismo , Traqueia/metabolismo , Transativadores/metabolismoRESUMO
The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 µm axially over an imaging volume of 3,000 × 800 × 300 µm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time.
Assuntos
Encéfalo , Peixe-Zebra , Animais , Encéfalo/diagnóstico por imagem , Drosophila , Desenvolvimento Embrionário , Microscopia de Fluorescência/métodosRESUMO
This protocol describes how to image time and spatially resolved time lapses of Drosophila air sac primordium (ASP) cytonemes in ex vivo cultures of wing imaginal discs. It describes how to manually measure the length of cytonemes using custom-made FIJI/ImageJ tools, and to analyze data using R/R-Studios pipeline. It can also be used for studies of cell division, organelle localization, and protein trafficking as well as other cellular materials that can be fluorescently tagged and imaged with minimal phototoxicity.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Discos Imaginais , Transdução de Sinais , Imagem com Lapso de TempoRESUMO
Morphogen signaling proteins disperse across tissues to activate signal transduction in target cells. We investigated dispersion of Hedgehog (Hh), Wnt homolog Wingless (Wg), and Bone morphogenic protein homolog Decapentaplegic (Dpp) in the Drosophila wing imaginal disc. We discovered that delivery of Hh, Wg, and Dpp to their respective targets is regulated. We found that <5% of Hh and <25% of Wg are taken up by disc cells and activate signaling. The amount of morphogen that is taken up and initiates signaling did not change when the level of morphogen expression was varied between 50 and 200% (Hh) or 50 and 350% (Wg). Similar properties were observed for Dpp. We analyzed an area of 150 µm×150 µm that includes Hh-responding cells of the disc as well as overlying tracheal cells and myoblasts that are also activated by disc-produced Hh. We found that the extent of signaling in the disc was unaffected by the presence or absence of the tracheal and myoblast cells, suggesting that the mechanism that disperses Hh specifies its destinations to particular cells, and that target cells do not take up Hh from a common pool.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Discos Imaginais/metabolismo , Transdução de Sinais , Proteína Wnt1/metabolismo , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Morfogênese , Asas de Animais/embriologia , Proteína Wnt1/genéticaRESUMO
Drosophila oocytes develop together with 15 sister germline nurse cells (NCs), which pass products to the oocyte through intercellular bridges. The NCs are completely eliminated during stages 12-14, but we discovered that at stage 10B, two specific NCs fuse with the oocyte and extrude their nuclei through a channel that opens in the anterior face of the oocyte. These nuclei extinguish in the ooplasm, leaving 2 enucleated and 13 nucleated NCs. At stage 11, the cell boundaries of the oocyte are mostly restored. Oocytes in egg chambers that fail to eliminate NC nuclei at stage 10B develop with abnormal morphology. These findings show that stage 10B NCs are distinguished by position and identity, and that NC elimination proceeds in two stages: first at stage 10B and later at stages 12-14.
Assuntos
Linhagem da Célula/genética , Células Germinativas/crescimento & desenvolvimento , Oócitos/crescimento & desenvolvimento , Oogênese/genética , Animais , Núcleo Celular/genética , Citoplasma/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células Germinativas/citologia , Oócitos/citologiaRESUMO
Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, we used high-pressure freezing and EM to visualize cytoneme structures, paths, contents, and contacts. We observed cytonemes previously seen by CM in the Drosophila wing imaginal disc system, including disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and identified cytonemes extending into invaginations of target cells, and cytonemes connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between repeating wide (206 ± 51.8 nm) and thin (55.9 ± 16.2 nm) segments. Actin, ribosomes, and membranous compartments are present throughout; rough ER and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Actinas/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Mioblastos/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais/fisiologia , Asas de Animais/metabolismoRESUMO
Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. Here, we report that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. We identify the C. elegans ortholog TMEM-39 from an unbiased RNAi screen and show that deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. We propose that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Caenorhabditis elegans/metabolismo , Colágeno/metabolismo , Drosophila/metabolismo , Estresse do Retículo Endoplasmático/genética , Proteínas de Membrana/metabolismo , Animais , Autofagia/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Corpo Adiposo/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Ligação Proteica , Transporte Proteico/genética , Interferência de RNA , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Hedgehog (Hh) is an evolutionarily conserved signaling protein that has essential roles in animal development and homeostasis. We investigated Hh signaling in the region of the Drosophila wing imaginal disc that produces Hh and is near the tracheal air sac primordium (ASP) and myoblasts. Hh distributes in concentration gradients in the anterior compartment of the wing disc, ASP and myoblasts, and activates genes in each tissue. Some targets of Hh signal transduction are common to the disc, ASP and myoblasts, whereas others are tissue-specific. Signaling in the three tissues is cytoneme-mediated and cytoneme-dependent. Some ASP cells project cytonemes that receive both Hh and Branchless (Bnl), and some targets regulated by Hh signaling in the ASP are also dependent on Bnl signal transduction. We conclude that the single source of Hh in the wing disc regulates cell type-specific responses in three discreet target tissues.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Discos Imaginais/metabolismo , Transdução de Sinais , Asas de Animais/embriologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/genética , Discos Imaginais/citologia , Asas de Animais/citologiaRESUMO
Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Colágeno/metabolismo , Espaço Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Drosophila/metabolismo , Estresse do Retículo Endoplasmático , Evolução Molecular , Genoma , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/química , Filogenia , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Interferência de RNA , Proteínas de Transporte Vesicular/metabolismoRESUMO
Communication between neoplastic cells and cells of their microenvironment is critical to cancer progression. To investigate the role of cytoneme-mediated signaling as a mechanism for distributing growth factor signaling proteins between tumor and tumor-associated cells, we analyzed EGFR and RET Drosophila tumor models and tested several genetic loss-of-function conditions that impair cytoneme-mediated signaling. Neuroglian, capricious, Irk2, SCAR, and diaphanous are genes that cytonemes require during normal development. Neuroglian and Capricious are cell adhesion proteins, Irk2 is a potassium channel, and SCAR and Diaphanous are actin-binding proteins, and the only process to which they are known to contribute jointly is cytoneme-mediated signaling. We observed that diminished function of any one of these genes suppressed tumor growth and increased organism survival. We also noted that EGFR-expressing tumor discs have abnormally extensive tracheation (respiratory tubes) and ectopically express Branchless (Bnl, a FGF) and FGFR. Bnl is a known inducer of tracheation that signals by a cytoneme-mediated process in other contexts, and we determined that exogenous over-expression of dominant negative FGFR suppressed tumor growth. Our results are consistent with the idea that cytonemes move signaling proteins between tumor and stromal cells and that cytoneme-mediated signaling is required for tumor growth and malignancy.
Assuntos
Carcinogênese/metabolismo , Transformação Celular Neoplásica/metabolismo , Pseudópodes/fisiologia , Animais , Carcinogênese/genética , Estruturas da Membrana Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Receptores ErbB/metabolismo , Discos Imaginais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Animais , Metástase Neoplásica/genética , Neoplasias/metabolismo , Receptores de Peptídeos de Invertebrados/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Asas de Animais/crescimento & desenvolvimentoRESUMO
A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila. Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.
Assuntos
Apoptose , Genes Reporter/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Imagem Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Conformação Proteica em Folha betaRESUMO
We investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII. Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by l-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.
Assuntos
Sinalização do Cálcio , Proteínas de Drosophila/fisiologia , Glutamatos/fisiologia , Discos Imaginais/fisiologia , Receptores Ionotrópicos de Glutamato/fisiologia , Sinapses/fisiologia , Animais , Animais Geneticamente Modificados , Canais de Cálcio/fisiologia , Drosophila melanogaster/fisiologia , Imagem Óptica , Pseudópodes/fisiologia , Proteínas R-SNARE/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia , Sinaptotagmina I/fisiologia , Técnicas de Cultura de TecidosRESUMO
Evidence in many experimental systems supports the idea that non-uniform distributions of morphogen proteins encode positional information in developing tissues. There is also strong evidence that morphogen dispersal is mediated by cytonemes and that morphogen proteins transfer from producing to receiving cells at morphogenetic synapses that form at sites of cytoneme contacts. This essay considers some implications of this mechanism and its relevance to various contexts including large single cells such as the pre-cellular Drosophila embryo and the ciliate Stentor.
Assuntos
Cilióforos/metabolismo , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/embriologia , Morfogênese/fisiologia , Proteínas de Protozoários/metabolismo , Transdução de Sinais/fisiologia , Animais , Cilióforos/citologia , Drosophila melanogaster , Embrião não Mamífero/citologiaRESUMO
Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.
Assuntos
Imagem Óptica/métodos , Fosfotransferases/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Drosophila , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Fosforilação , Fosfotransferases/metabolismoRESUMO
Morphogen concentration gradients that extend across developmental fields form by dispersion from source cells. In the Drosophila wing disc, Hedgehog (Hh) produced by posterior compartment cells distributes in a concentration gradient to adjacent cells of the anterior compartment. We monitored Hh:GFP after pulsed expression, and analyzed the movement and colocalization of Hh, Patched (Ptc) and Smoothened (Smo) proteins tagged with GFP or mCherry and expressed at physiological levels from bacterial artificial chromosome transgenes. Hh:GFP moved to basal subcellular locations prior to release from posterior compartment cells that express it, and was taken up by basal cytonemes that extend to the source cells. Hh and Ptc were present in puncta that moved along the basal cytonemes and formed characteristic apical-basal distributions in the anterior compartment cells. The basal cytonemes required diaphanous, SCAR, Neuroglian and Synaptobrevin, and both the Hh gradient and Hh signaling declined under conditions in which the cytonemes were compromised. These findings show that in the wing disc, Hh distributions and signaling are dependent upon basal release and uptake, and on cytoneme-mediated movement. No evidence for apical dispersion was obtained.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Hedgehog/metabolismo , Discos Imaginais/metabolismo , Asas de Animais/metabolismo , Animais , Padronização Corporal , Compartimento Celular , Cromossomos Artificiais Bacterianos/genética , Proteínas de Fluorescência Verde/metabolismo , Transporte Proteico , Transdução de Sinais , TransgenesRESUMO
Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the fragile X mental retardation 1 (FMR1) gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. We previously found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and Drosophila models of FXS and that these molecular and cellular markers were correlated in patients with FXS. We report that larval locomotion is augmented in a Drosophila FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. Our study demonstrates that (i) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (ii) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics.
Assuntos
Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/fisiopatologia , Locomoção/fisiologia , Sinapses/patologia , Algoritmos , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Comportamento Animal/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Drosophila/efeitos dos fármacos , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Ensaios de Triagem em Larga Escala , Larva/efeitos dos fármacos , Larva/fisiologia , Quinases Lim/antagonistas & inibidores , Quinases Lim/genética , Quinases Lim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Bibliotecas de Moléculas Pequenas/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
During development, cells use specialized filopodia called cytonemes to deploy the signaling proteins that coordinate growth and direct morphogenesis. Cytonemes are dynamic structures that can extend long distances across tissues to either deliver or take up signaling proteins. Signaling proteins transfer between cells at the tips of cytonemes where specific contacts termed morphogenetic synapses form. This review summarizes our current understanding of the roles and functions of cytonemes, and it explores some of the conceptual issues relevant to the cytoneme mechanism of contact-dependent cell-cell signaling.