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1.
EMBO Rep ; 24(12): e56997, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37975164

RESUMO

Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.


Assuntos
Proteínas de Drosophila , Proteínas de Membrana , Animais , Polaridade Celular/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
2.
JCI Insight ; 8(2)2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36692018

RESUMO

The G protein-coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor-associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Receptor Tipo 4 de Melanocortina , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/metabolismo , Cílios/metabolismo , Homeostase
3.
J Cell Sci ; 135(19)2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-36222105

RESUMO

Cilia sense and transduce sensory stimuli, homeostatic cues and developmental signals by orchestrating signaling reactions. Extracellular vesicles (EVs) that bud from the ciliary membrane have well-studied roles in the disposal of excess ciliary material, most dramatically exemplified by the shedding of micrometer-sized blocks by photoreceptors. Shedding of EVs by cilia also affords cells with a powerful means to shorten cilia. Finally, cilium-derived EVs may enable cell-cell communication in a variety of organisms, ranging from single-cell parasites and algae to nematodes and vertebrates. Mechanistic understanding of EV shedding by cilia is an active area of study, and future progress may open the door to testing the function of ciliary EV shedding in physiological contexts. In this Cell Science at a Glance and the accompanying poster, we discuss the molecular mechanisms that drive the shedding of ciliary material into the extracellular space, the consequences of shedding for the donor cell and the possible roles that ciliary EVs may have in cell non-autonomous contexts.


Assuntos
Cílios , Vesículas Extracelulares , Animais , Comunicação Celular , Cílios/fisiologia , Vesículas Citoplasmáticas , Transdução de Sinais
4.
Sci Rep ; 8(1): 12534, 2018 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120317

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

5.
Sci Rep ; 8(1): 2211, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29396404

RESUMO

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in signal transduction. Cilia are anchored inside the cell through basal bodies (BBs), modified centrioles also acting as microtubule-organization centers. Photoreceptors (PRs) are sensory neurons, whose primary cilium forms a highly specialized compartment called the outer segment (OS) responsible for sensing incoming light. Thus, ciliopathies often present with retinal degeneration. Mutations in KIAA0586/TALPID3 (TA3) cause Joubert syndrome, in which 30% of affected individuals develop retinal involvement. To elucidate the function of TALPID3 in PRs, we studied talpid3 zebrafish mutants and identified a progressive retinal degeneration phenotype. The majority of PRs lack OS development due to defects in BB positioning and docking at the apical cell surface. Intracellular accumulation of the photopigment opsin leads to PR cell death of moderate severity. Electroretinograms demonstrate severe visual impairement. A small subset of PRs display normally docked BBs and extended OSs through rescue by maternally-deposited Talpid3. While localization of the small GTPase Rab8a, which plays an important role in BB docking, appears unaffected in talpid3-/- PRs, overexpression of constitutively active Rab8a rescues OS formation, indicating that the role of Ta3 in early ciliogenesis lies upstream of Rab8a activation in PRs.


Assuntos
Ciliopatias/patologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas Mutantes/metabolismo , Biogênese de Organelas , Células Fotorreceptoras/patologia , Degeneração Retiniana/patologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Eletrorretinografia , Proteínas Mutantes/genética , Opsinas/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
6.
PLoS Genet ; 13(12): e1007150, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29281629

RESUMO

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming ciliary-directed vesicles, through organization of the vesicle fusion machinery at the periciliary membrane.


Assuntos
Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Transporte Biológico , Movimento Celular , Cílios/genética , Cílios/metabolismo , Humanos , Membranas/metabolismo , Opsinas/genética , Opsinas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Transporte Proteico , Peixe-Zebra , Proteínas rab de Ligação ao GTP/genética
7.
J Vis Exp ; (129)2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29155784

RESUMO

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.


Assuntos
Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodos , Retina/diagnóstico por imagem , Animais , Retina/patologia , Peixe-Zebra
8.
PLoS Genet ; 9(7): e1003596, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23874211

RESUMO

Chemical defense is one of the most important traits, which endow insects the ability to conquer a most diverse set of ecological environments. Chemical secretions are used for defense against anything from vertebrate or invertebrate predators to prokaryotic or eukaryotic parasites or food competitors. Tenebrionid beetles are especially prolific in this category, producing several varieties of substituted benzoquinone compounds. In order to get a better understanding of the genetic and molecular basis of defensive secretions, we performed RNA sequencing in a newly emerging insect model, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae). To detect genes that are highly and specifically expressed in the odoriferous gland tissues that secret defensive chemical compounds, we compared them to a control tissue, the anterior abdomen. 511 genes were identified in different subtraction groups. Of these, 77 genes were functionally analyzed by RNA interference (RNAi) to recognize induced gland alterations morphologically or changes in gland volatiles by gas chromatography-mass spectrometry. 29 genes (38%) presented strong visible phenotypes, while 67 genes (87%) showed alterations of at least one gland content. Three of these genes showing quinone-less (ql) phenotypes - Tcas-ql VTGl; Tcas-ql ARSB; Tcas-ql MRP - were isolated, molecularly characterized, their expression identified in both types of the secretory glandular cells, and their function determined by quantification of all main components after RNAi. In addition, microbe inhibition assays revealed that a quinone-free status is unable to impede bacterial or fungal growth. Phylogenetic analyses of these three genes indicate that they have evolved independently and specifically for chemical defense in beetles.


Assuntos
Benzoquinonas/química , Besouros/química , Proteínas de Insetos/metabolismo , Glândulas Odoríferas/química , Animais , Benzoquinonas/metabolismo , Besouros/metabolismo , Besouros/fisiologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Insetos/genética , Filogenia , Interferência de RNA , Glândulas Odoríferas/metabolismo
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