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1.
Development ; 151(10)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38738602

RESUMO

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.


Assuntos
Proteínas de Drosophila , Morfogênese , Proteínas do Tecido Nervoso , Neurópilo , Lobo Óptico de Animais não Mamíferos , Receptores de Superfície Celular , Semaforinas , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Semaforinas/metabolismo , Semaforinas/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Morfogênese/genética , Neurópilo/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Lobo Óptico de Animais não Mamíferos/embriologia , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/embriologia , Neurônios/metabolismo , Drosophila/metabolismo , Drosophila/embriologia , Mutação/genética
2.
bioRxiv ; 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38293108

RESUMO

The biconvex shape of the Drosophila corneal lens, which enables it to focus light onto the retina, arises by organized assembly of chitin and other apical extracellular matrix components. We show here that the Zona Pellucida domain-containing protein Dusky-like is essential for normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical surfaces of the corneal lens-secreting cells, and in its absence, these cells undergo apical constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other Zona Pellucida-domain proteins, Dumpy and Piopio, external to the developing corneal lens. Loss of either dusky-like or dumpy delays chitin accumulation and disrupts the outer surface of the corneal lens. Artificially inducing apical constriction with constitutively active Myosin light chain kinase is sufficient to similarly alter chitin deposition and corneal lens morphology. These results demonstrate the importance of cell shape for the morphogenesis of overlying apical extracellular matrix structures.

3.
bioRxiv ; 2023 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-37609142

RESUMO

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of Semaphorin function indicates that Semaphorin 1a, provided by cells that include Tm5 neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A does not disrupt the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles. Summary statement: The axon guidance molecule Plexin A has two functions in Drosophila medulla development; morphogenesis of the neuropil requires its cytoplasmic domain, but establishing synaptic layers through Semaphorin 1a does not.

4.
Development ; 149(7)2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35297965

RESUMO

The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. Here, we show that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Our transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisona , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas Repressoras/genética , Fatores de Transcrição/genética
5.
J Cell Biol ; 221(5)2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35258563

RESUMO

Contractile actomyosin and protrusive branched F-actin networks interact in a dynamic balance, repeatedly contracting and expanding apical cell contacts to organize the epithelium of the developing fly retina. Previously we showed that the immunoglobulin superfamily protein Sidekick (Sdk) contributes to contraction by recruiting the actin binding protein Polychaetoid (Pyd) to vertices. Here we show that as tension increases during contraction, Sdk progressively accumulates at vertices, where it toggles to recruit the WAVE regulatory complex (WRC) to promote actin branching and protrusion. Sdk alternately interacts with the WRC and Pyd using the same C-terminal motif. With increasing protrusion, levels of Sdk and the WRC decrease at vertices while levels of Pyd increase paving the way for another round of contraction. Thus, by virtue of dynamic association with vertices and interchangeable associations with contractile and protrusive effectors, Sdk is central to controlling the balance between contraction and expansion that shapes this epithelium.


Assuntos
Citoesqueleto de Actina , Actinas , Proteínas de Drosophila , Proteínas do Olho , Moléculas de Adesão de Célula Nervosa , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Epitélio/metabolismo , Proteínas do Olho/metabolismo , Morfogênese , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteínas de Junções Íntimas/metabolismo
6.
EMBO Rep ; 23(1): e53231, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34726300

RESUMO

The exon junction complex controls the translation, degradation, and localization of spliced mRNAs, and three of its core subunits also play a role in splicing. Here, we show that a fourth subunit, Barentsz, has distinct functions within and separate from the exon junction complex in Drosophila neuromuscular development. The distribution of mitochondria in larval muscles requires Barentsz as well as other exon junction complex subunits and is not rescued by a Barentsz transgene in which residues required for binding to the core subunit eIF4AIII are mutated. In contrast, interactions with the exon junction complex are not required for Barentsz to promote the growth of neuromuscular synapses. We find that the Activin ligand Dawdle shows reduced expression in barentsz mutants and acts downstream of Barentsz to control synapse growth. Both barentsz and dawdle are required in motor neurons, muscles, and glia for normal synapse growth, and exogenous Dawdle can rescue synapse growth in the absence of barentsz. These results identify a biological function for Barentsz that is independent of the exon junction complex.


Assuntos
Proteínas de Drosophila , Proteínas de Ligação a RNA , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Éxons/genética , Proteínas de Ligação a RNA/metabolismo , Sinapses/metabolismo
7.
Elife ; 102021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34003117

RESUMO

As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here, we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections.


New nerve cells in a developing organism face a difficult challenge: finding the right partners to connect with in order to form the complex neural networks characteristic of a fully formed brain. Each cell encounters many potential matches but it chooses to connect to only a few, partly based on the proteins that decorate the surface of both cells. Still, too many cell types exist for each to have its own unique protein label, suggesting that nerve cells may also use the amount of each protein to identify suitable partners. Douthit, Hairston et al. explored this possibility in developing fruit flies, focusing on how R7 photoreceptor cells ­ present in the eye to detect UV light ­ connect to nerve cells in a specific brain layer. It is easy to spot when the process goes awry, as the incorrect connections will be in a different layer. Experiments allowed Douthit, Hairston et al. to identify a protein baptized 'Lost and found' ­ 'Loaf' for short ­ which R7 photoreceptors use to find their partners. Removing Loaf from the photoreceptors prevented them from connecting with their normal partners. Surprisingly though, removing Loaf from both the eye and the brain solved this problem ­ the cells, once again, formed the right connections. This suggests that R7 photoreceptors identify their partners by looking for cells that have less Loaf than they do: removing Loaf only from the photoreceptors disrupts this balance, leaving the cells unable to find their match. Another unexpected discovery was that Loaf is not present on the surface of cells, but instead occupies internal structures involved in protein transport. It may therefore work indirectly by controlling the movement of proteins to the cell surface. These findings provide a new way of thinking about how nerve cells connect. In the future, this may help to understand the origins of conditions in which the brain is wired differently, such as schizophrenia and autism.


Assuntos
Axônios/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Sinapses/fisiologia , Animais , Axônios/classificação , Proteínas de Drosophila/metabolismo , Expressão Gênica , Células Fotorreceptoras de Invertebrados/metabolismo
8.
Dev Cell ; 54(1): 117-131.e5, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32516570

RESUMO

Developmental growth requires coordination between the growth rates of individual tissues and organs. Here, we examine how Drosophila neuromuscular synapses grow to match the size of their target muscles. We show that changes in muscle growth driven by autonomous modulation of insulin receptor signaling produce corresponding changes in synapse size, with each muscle affecting only its presynaptic motor neuron branches. This scaling growth is mechanistically distinct from synaptic plasticity driven by neuronal activity and requires increased postsynaptic differentiation induced by insulin receptor signaling in muscle. We identify the guanine-nucleotide exchange factor dPix as an effector of insulin receptor signaling. Alternatively spliced dPix isoforms that contain a specific exon are necessary and sufficient for postsynaptic differentiation and scaling growth, and their mRNA levels are regulated by insulin receptor signaling. These findings define a mechanism by which the same signaling pathway promotes both autonomous muscle growth and non-autonomous synapse growth.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Drosophila/metabolismo , Junção Neuromuscular/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Neurogênese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo
9.
Dev Cell ; 50(3): 313-326.e5, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31353315

RESUMO

Tricellular adherens junctions are points of high tension that are central to the rearrangement of epithelial cells. However, the molecular composition of these junctions is unknown, making it difficult to assess their role in morphogenesis. Here, we show that Sidekick, an immunoglobulin family cell adhesion protein, is highly enriched at tricellular adherens junctions in Drosophila. This localization is modulated by tension, and Sidekick is itself necessary to maintain normal levels of cell bond tension. Loss of Sidekick causes defects in cell and junctional rearrangements in actively remodeling epithelial tissues like the retina and tracheal system. The adaptor proteins Polychaetoid and Canoe are enriched at tricellular adherens junctions in a Sidekick-dependent manner; Sidekick functionally interacts with both proteins and directly binds to Polychaetoid. We suggest that Polychaetoid and Canoe link Sidekick to the actin cytoskeleton to enable tricellular adherens junctions to maintain or transmit cell bond tension during epithelial cell rearrangements.


Assuntos
Junções Aderentes/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Proteínas do Olho/genética , Moléculas de Adesão de Célula Nervosa/genética , Ligação Proteica , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo
10.
Cell Rep ; 26(2): 322-329.e3, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625314

RESUMO

Calcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. We show here that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/ß-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed us to define three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics.


Assuntos
Caderinas/metabolismo , Retículo Endoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Via de Sinalização Wnt , Animais , Apoptose , Proteínas do Domínio Armadillo/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Notch/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Fatores de Transcrição/metabolismo
11.
J Cell Sci ; 131(18)2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30158176

RESUMO

Mammalian members of the ErbB family, including the epidermal growth factor receptor (EGFR), can regulate transcription, DNA replication and repair through nuclear entry of either the full-length proteins or their cleaved cytoplasmic domains. In cancer cells, these nuclear functions contribute to tumor progression and drug resistance. Here, we examined whether the single Drosophila EGFR can also localize to the nucleus. A chimeric EGFR protein fused at its cytoplasmic C-terminus to DNA-binding and transcriptional activation domains strongly activated transcriptional reporters when overexpressed in cultured cells or in vivo However, this activity was independent of cleavage and endocytosis. Without an exogenous activation domain, EGFR fused to a DNA-binding domain did not activate or repress transcription. Addition of the same DNA-binding and transcriptional activation domains to the endogenous Egfr locus through genome editing led to no detectable reporter expression in wild-type or oncogenic contexts. These results show that, when expressed at physiological levels, the cytoplasmic domain of the Drosophila EGFR does not have access to the nucleus. Therefore, nuclear EGFR functions are likely to have evolved after vertebrates and invertebrates diverged.


Assuntos
Núcleo Celular/metabolismo , Drosophila/metabolismo , Receptores ErbB/metabolismo , Animais
12.
Development ; 145(3)2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29361567

RESUMO

The assembly of functional neuronal circuits requires growth cones to extend in defined directions and recognize the correct synaptic partners. Homophilic adhesion between vertebrate Sidekick proteins promotes synapse formation between retinal neurons involved in visual motion detection. We show here that Drosophila Sidekick accumulates in specific synaptic layers of the developing motion detection circuit and is necessary for normal optomotor behavior. Sidekick is required in photoreceptors, but not in their target lamina neurons, to promote the alignment of lamina neurons into columns and subsequent sorting of photoreceptor axons into synaptic modules based on their precise spatial orientation. Sidekick is also localized to the dendrites of the direction-selective T4 and T5 cells, and is expressed in some of their presynaptic partners. In contrast to its vertebrate homologs, Sidekick is not essential for T4 and T5 to direct their dendrites to the appropriate layers or to receive synaptic contacts. These results illustrate a conserved requirement for Sidekick proteins in establishing visual motion detection circuits that is achieved through distinct cellular mechanisms in Drosophila and vertebrates.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Proteínas do Olho/fisiologia , Percepção de Movimento/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Feminino , Genes de Insetos , Masculino , Mutação , Moléculas de Adesão de Célula Nervosa/genética , Células Fotorreceptoras de Invertebrados/citologia , Sinapses/metabolismo , Vias Visuais/citologia , Vias Visuais/crescimento & desenvolvimento , Vias Visuais/fisiologia
13.
Fly (Austin) ; 12(2): 118-126, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29355077

RESUMO

The COP9 signalosome inhibits the activity of Cullin-RING E3 ubiquitin ligases by removing Nedd8 modifications from their Cullin subunits. Neddylation renders these complexes catalytically active, but deneddylation is also necessary for them to exchange adaptor subunits and avoid auto-ubiquitination. Although deneddylation is thought to be the primary function of the COP9 signalosome, additional activities have been ascribed to some of its subunits. We recently showed that COP9 subunits protect the transcriptional repressor and tumor suppressor Capicua from two distinct modes of degradation. Deneddylation by the COP9 signalosome inactivates a Cullin 1 complex that ubiquitinates Capicua following its phosphorylation by MAP kinase in response to Epidermal Growth Factor Receptor signaling. The CSN1b subunit also stabilizes unphosphorylated Capicua to control its basal level, independently of the deneddylase function of the complex. Here we further examine the importance of deneddylation for COP9 functions in vivo. We use an uncleavable form of Nedd8 to show that preventing deneddylation does not reproduce the effects of loss of COP9. In contrast, in the presence of COP9, conjugation to uncleavable Nedd8 renders Cullins unable to promote the degradation of their substrates. Our results suggest that irreversible neddylation prolongs COP9 binding to and inhibition of Cullin-based ubiquitin ligases.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Proteínas Culina/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteína NEDD8/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Complexo do Signalossomo COP9/genética , Células Cultivadas , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteína NEDD8/genética , Proteólise , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
14.
PLoS Genet ; 14(1): e1007173, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29324767

RESUMO

Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, we compared the effects of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc. Glass activated overlapping but distinct sets of genes in these neuronal and non-neuronal contexts, including markers of photoreceptors, cone cells and pigment cells. Coexpression of other transcription factors such as Pax2, Eyes absent, Lozenge and Escargot enabled Glass to induce additional genes characteristic of the non-neuronal cell types. Cell type-specific glass mutations generated in cone or pigment cells using somatic CRISPR revealed autonomous developmental defects, and expressing Glass specifically in these cells partially rescued glass mutant phenotypes. These results indicate that Glass is a determinant of organ identity that acts in both neuronal and non-neuronal cells to promote their differentiation into functional components of the eye.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Olho/embriologia , Olho/metabolismo , Células-Tronco Neurais/fisiologia , Células-Tronco/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero , Olho/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética
15.
Development ; 144(14): 2673-2682, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28619822

RESUMO

The COP9 signalosome removes Nedd8 modifications from the Cullin subunits of ubiquitin ligase complexes, reducing their activity. Here, we show that mutations in the Drosophila COP9 signalosome subunit 1b (CSN1b) gene increase the activity of ubiquitin ligases that contain Cullin 1. Analysis of CSN1b mutant phenotypes revealed a requirement for the COP9 signalosome to prevent ectopic expression of Epidermal growth factor receptor (EGFR) target genes. It does so by protecting Capicua, a transcriptional repressor of EGFR target genes, from EGFR pathway-dependent ubiquitylation by a Cullin 1/SKP1-related A/Archipelago E3 ligase and subsequent proteasomal degradation. The CSN1b subunit also maintains basal Capicua levels by protecting it from a separate mechanism of degradation that is independent of EGFR signaling. As a suppressor of tumor growth and metastasis, Capicua may be an important target of the COP9 signalosome in cancer.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas HMGB/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Animais Geneticamente Modificados , Complexo do Signalossomo COP9 , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Receptores ErbB/genética , Receptores ErbB/metabolismo , Olho/crescimento & desenvolvimento , Olho/metabolismo , Feminino , Genes de Insetos , Proteínas HMGB/genética , Sistema de Sinalização das MAP Quinases , Masculino , Modelos Biológicos , Complexos Multiproteicos/genética , Mutação , Peptídeo Hidrolases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteólise , Receptores de Peptídeos de Invertebrados/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Proteínas Repressoras/genética , Ubiquitinação , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
16.
Development ; 142(8): 1480-91, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25790850

RESUMO

Endocytic trafficking of signaling receptors is an important mechanism for limiting signal duration. Components of the Endosomal Sorting Complexes Required for Transport (ESCRT), which target ubiquitylated receptors to intra-lumenal vesicles (ILVs) of multivesicular bodies, are thought to terminate signaling by the epidermal growth factor receptor (EGFR) and direct it for lysosomal degradation. In a genetic screen for mutations that affect Drosophila eye development, we identified an allele of Vacuolar protein sorting 4 (Vps4), which encodes an AAA ATPase that interacts with the ESCRT-III complex to drive the final step of ILV formation. Photoreceptors are largely absent from Vps4 mutant clones in the eye disc, and even when cell death is genetically prevented, the mutant R8 photoreceptors that develop fail to recruit surrounding cells to differentiate as R1-R7 photoreceptors. This recruitment requires EGFR signaling, suggesting that loss of Vps4 disrupts the EGFR pathway. In imaginal disc cells mutant for Vps4, EGFR and other receptors accumulate in endosomes and EGFR target genes are not expressed; epistasis experiments place the function of Vps4 at the level of the receptor. Surprisingly, Vps4 is required for EGFR signaling even in the absence of Shibire, the Dynamin that internalizes EGFR from the plasma membrane. In ovarian follicle cells, in contrast, Vps4 does not affect EGFR signaling, although it is still essential for receptor degradation. Taken together, these findings indicate that Vps4 can promote EGFR activity through an endocytosis-independent mechanism.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Receptores ErbB/metabolismo , Adenosina Trifosfatases/genética , Animais , Drosophila , Proteínas de Drosophila/genética , Dinaminas/genética , Dinaminas/metabolismo , Endocitose/genética , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Receptores ErbB/genética , Masculino , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
17.
Mol Cell Biol ; 34(14): 2710-20, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24820420

RESUMO

Human MED26 was originally purified in the cofactor required for the Sp1 activation complex (CRSP) as a 70-kDa component named CRSP70. This polypeptide was specific to metazoans and the "small" form of the Mediator complex. We report here that a Drosophila melanogaster homologue of MED26 similarly interacts with other components of the core Drosophila Mediator complex but not with the kinase module and is recruited to genes upon activation. Using a null allele of Med26, we show that Med26 is required for organismal viability but not for cell proliferation or survival. Clones lacking Med26 in the wing disc lead to loss of the adult wing margin and reduced expression of genes involved in wing margin formation. Surprisingly, when polytene chromosomes from the salivary gland were examined using antibodies to Med26, it was apparent that a fraction of the protein was associated with the chromocenter, which contains pericentric heterochromatin. This staining colocalizes with heterochromatin protein 1 (HP1). Immunoprecipitation experiments show that Med26 interacts with HP1. The interaction is mediated through the chromoshadow domain of HP1 and through the conserved motif in the carboxy terminus of the Med26 protein. This work is the first characterization of the metazoan-specific Mediator subunit in an animal model.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Complexo Mediador/genética , Complexo Mediador/metabolismo , Sequência de Aminoácidos , Animais , Proliferação de Células , Sobrevivência Celular , Sequência Conservada , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Heterocromatina/metabolismo , Humanos , Cromossomos Politênicos , Glândulas Salivares/metabolismo , Asas de Animais/crescimento & desenvolvimento
18.
Wiley Interdiscip Rev Dev Biol ; 2(4): 545-57, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24014422

RESUMO

Drosophila eye development has been extensively studied, due to the ease of genetic screens for mutations disrupting this process. The eye imaginal disc is specified during embryonic and larval development by the Pax6 homolog Eyeless and a network of downstream transcription factors. Expression of these factors is regulated by signaling molecules and also indirectly by growth of the eye disc. Differentiation of photoreceptor clusters initiates in the third larval instar at the posterior of the eye disc and progresses anteriorly, driven by the secreted protein Hedgehog. Within each cluster, the combined activities of Hedgehog signaling and Notch-mediated lateral inhibition induce and refine the expression of the transcription factor Atonal, which specifies the founding R8 photoreceptor of each ommatidium. Seven additional photoreceptors, followed by cone and pigment cells, are successively recruited by the signaling molecules Spitz, Delta, and Bride of sevenless. Combinations of these signals and of intrinsic transcription factors give each ommatidial cell its specific identity. During the pupal stages, rhodopsins are expressed, and the photoreceptors and accessory cells take on their final positions and morphologies to form the adult retina. Over the past few decades, the genetic analysis of this small number of cell types arranged in a repetitive structure has allowed a remarkably detailed understanding of the basic mechanisms controlling cell differentiation and morphological rearrangement.


Assuntos
Drosophila/citologia , Neurogênese , Células Fotorreceptoras de Invertebrados/metabolismo , Animais , Linhagem da Célula , Drosophila/embriologia , Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
J Cell Sci ; 126(Pt 19): 4469-78, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23902690

RESUMO

Epidermal growth factor receptor (EGFR) ligands undergo a complex series of processing events during their maturation to active signaling proteins. Like its mammalian homologs, the predominant Drosophila EGFR ligand Spitz is produced as a transmembrane pro-protein. In the secretory pathway, Spitz is cleaved within its transmembrane domain to release the extracellular signaling domain. This domain is modified with an N-terminal palmitate group that tethers it to the plasma membrane. We found that the pro-protein can reach the cell surface in the absence of proteolysis, but that it fails to activate the EGFR. To address why the transmembrane pro-protein is inactive, whereas membrane association through the palmitate group promotes activity, we generated a panel of chimeric constructs containing the Spitz extracellular region fused to exogenous transmembrane proteins. Although the orientation of the EGF domain and its distance from the plasma membrane varies in these chimeras, they are all active in vivo. Thus, tethering Spitz to the membrane via a transmembrane domain at either terminus does not prevent activity. Conversely, removing the N-terminal palmitate group from the C-terminally tethered pro-protein does not render it active. Furthermore, we show that the Spitz transmembrane pro-protein can activate the EGFR in a tissue culture assay, indicating that its failure to signal in vivo is not due to structural features. In polarized imaginal disc cells, unprocessed Spitz pro-protein localizes to apical puncta, whereas the active chimeric Spitz constructs are basolaterally localized. Taken together, our data support the model that localized trafficking of the pro-protein restricts its ability to activate the receptor in polarized tissues.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Ligantes , Proteínas de Membrana/genética , Transdução de Sinais , Transfecção
20.
Genetics ; 190(2): 601-16, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22095083

RESUMO

The Wnt and Hedgehog signaling pathways are essential for normal development and are misregulated in cancer. The casein kinase family of serine/threonine kinases regulates both pathways at multiple levels. However, it has been difficult to determine whether individual members of this family have distinct functions in vivo, due to their overlapping substrate specificities. In Drosophila melanogaster, photoreceptor differentiation is induced by Hedgehog and inhibited by Wingless, providing a sensitive system in which to identify regulators of each pathway. We used a mosaic genetic screen in the Drosophila eye to identify mutations in genes on the X chromosome required for signal transduction. We recovered mutations affecting the transcriptional regulator CREB binding protein, the small GTPase dynamin, the cytoskeletal regulator Actin-related protein 2, and the protein kinase Casein kinase 1α. Consistent with its reported function in the ß-Catenin degradation complex, Casein Kinase 1α mutant cells accumulate ß-Catenin and ectopically induce Wingless target genes. In contrast to previous studies based on RNA interference, we could not detect any effect of the same Casein Kinase 1α mutation on Hedgehog signaling. We thus propose that Casein kinase 1α is essential to allow ß-Catenin degradation and prevent inappropriate Wingless signaling, but its effects on the Hedgehog pathway are redundant with other Casein kinase 1 family members.


Assuntos
Caseína Quinase Ialfa/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Mutação , Células Fotorreceptoras de Invertebrados/citologia , Transdução de Sinais , Proteína Wnt1/metabolismo , Proteína 2 Relacionada a Actina/genética , Animais , Proteína de Ligação a CREB/metabolismo , Caseína Quinase Ialfa/genética , Diferenciação Celular/genética , Dinaminas/genética , Dinaminas/metabolismo , Receptores ErbB/metabolismo , Feminino , Genes Ligados ao Cromossomo X , Proteínas Hedgehog/metabolismo , Masculino , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Notch/metabolismo
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