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1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35050379

RESUMO

During endosome maturation, neutral sphingomyelinase 2 (nSMase2, encoded by SMPD3) is involved in budding of intraluminal vesicles (ILVs) into late endosomes or multivesicular bodies (MVBs). Fusion of these with the plasma membrane results in secretion of exosomes or small extracellular vesicles (sEVs). Here, we report that nSMase2 activity controls sEV secretion through modulation of vacuolar H+-ATPase (V-ATPase) activity. Specifically, we show that nSMase2 inhibition induces V-ATPase complex assembly that drives MVB lumen acidification and consequently reduces sEV secretion. Conversely, we further demonstrate that stimulating nSMase2 activity with the inflammatory cytokine TNFα (also known as TNF) decreases acidification and increases sEV secretion. Thus, we find that nSMase2 activity affects MVB membrane lipid composition to counteract V-ATPase-mediated endosome acidification, thereby shifting MVB fate towards sEV secretion. This article has an associated First Person interview with the first author of the paper.


Assuntos
Exossomos , ATPases Vacuolares Próton-Translocadoras , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Corpos Multivesiculares/metabolismo , Transporte Proteico , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
2.
Nat Methods ; 18(9): 1013-1026, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34446922

RESUMO

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.


Assuntos
Vesículas Extracelulares , Microscopia/métodos , Animais , Corantes/química , Epitopos , Vesículas Extracelulares/química , Vesículas Extracelulares/patologia , Vesículas Extracelulares/fisiologia , Corantes Fluorescentes/química , Humanos
3.
Development ; 147(15)2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665246

RESUMO

Development and tissue homeostasis rely on the tight regulation of morphogen secretion. In the Drosophila wing imaginal disc epithelium, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory endosomal transport. Although Wg release appears to occur from the apical and basal cell sides, its exact post-endocytic fate and the functional relevance of polarized endosomal Wg trafficking are poorly understood. Here, we identify the kinesin-3 family member Klp98A as the master regulator of intracellular Wg transport after apical endocytosis. In the absence of Klp98A, functional mature endosomes accumulate in the apical cytosol, and endosome transport to the basal cytosol is perturbed. Despite the resulting Wg mislocalization, Wg signal transduction occurs normally. We conclude that transcytosis-independent routes for Wg trafficking exist and demonstrate that Wg can be recycled apically via Rab4-recycling endosomes in the absence of Klp98A.


Assuntos
Proteínas de Drosophila/metabolismo , Endocitose , Endossomos , Cinesinas/metabolismo , Transdução de Sinais , Proteína Wnt1/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Cinesinas/genética , Transporte Proteico , Proteína Wnt1/genética
4.
Development ; 147(15)2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32611603

RESUMO

Morphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. Here, we have investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, we show that most Ykt6 is present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, we propose a molecular mechanism by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels.


Assuntos
Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Proteínas R-SNARE/metabolismo , Asas de Animais/embriologia , Proteínas Wnt/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Endossomos/genética , Epitélio/embriologia , Proteínas R-SNARE/genética , Proteínas Wnt/genética
5.
J Biol Chem ; 295(26): 8759-8774, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32381507

RESUMO

The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD1-10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/ß-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.


Assuntos
Receptores Frizzled/metabolismo , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , Animais , Receptores Frizzled/análise , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Microscopia Confocal/métodos , Mapas de Interação de Proteínas , Transporte Proteico , Proteína Wnt3A/análise , Xenopus
6.
Handb Exp Pharmacol ; 269: 29-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34505202

RESUMO

WNT signaling is a key developmental pathway in tissue organization. A recent focus of research is the secretion of WNT proteins from source cells. Research over the past decade on how WNTs are produced and released into the extracellular space has unravelled very specific control mechanisms in the early secretory pathway, specialized trafficking routes, and redundant forms of packaging for delivery to target cells. In this review I discuss the findings that WNT proteins have been found on extracellular vesicles (EVs) such as exosomes and possible functional implications. There is an ongoing debate in the WNT signaling field whether EV are relevant in vivo and can fulfill specific functions, also fueled by the general preconception of EV secretion as cellular garbage disposal. As part of the EV research community, I want to give an overview of what we know and don't know about WNT secretion on EVs and offer a more unifying model that can explain current discrepancies in observations regarding WNT secretion.


Assuntos
Exossomos , Vesículas Extracelulares , Ligantes , Proteínas Wnt , Via de Sinalização Wnt
7.
Mol Cell Proteomics ; 16(6): 998-1008, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28396511

RESUMO

In prostate cancer and other malignancies sensitive and robust biomarkers are lacking or have relevant limitations. Prostate specific antigen (PSA), the only biomarker widely used in prostate cancer, is suffering from low specificity. Exosomes offer new perspectives in the discovery of blood-based biomarkers. Here we present a proof-of principle study for a proteomics-based identification pipeline, implementing existing data sources, to exemplarily identify exosome-based biomarker candidates in prostate cancer.Exosomes from malignant PC3 and benign PNT1A cells and from FBS-containing medium were isolated using sequential ultracentrifugation. Exosome and control samples were analyzed on an LTQ-Orbitrap XL mass spectrometer. Proteomic data is available via ProteomeXchange with identifier PXD003651. We developed a scoring scheme to rank 64 proteins exclusively found in PC3 exosomes, integrating data from four public databases and published mass spectrometry data sets. Among the top candidates, we focused on the tight junction protein claudin 3. Retests under serum-free conditions using immunoblotting and immunogold labeling confirmed the presence of claudin 3 on PC3 exosomes. Claudin 3 levels were determined in the blood plasma of patients with localized (n = 58; 42 with Gleason score 6-7, 16 with Gleason score ≥8) and metastatic prostate cancer (n = 11) compared with patients with benign prostatic hyperplasia (n = 15) and healthy individuals (n = 15) using ELISA, without prior laborious exosome isolation. ANOVA showed different CLDN3 plasma levels in these groups (p = 0.004). CLDN3 levels were higher in patients with Gleason ≥8 tumors compared with patients with benign prostatic hyperplasia (p = 0.012) and Gleason 6-7 tumors (p = 0.029). In patients with localized tumors CLDN3 levels predicted a Gleason score ≥ 8 (AUC = 0.705; p = 0.016) and did not correlate with serum PSA.By using the described workflow claudin 3 was identified and validated as a potential blood-based biomarker in prostate cancer. Furthermore this workflow could serve as a template to be used in other cancer entities.


Assuntos
Biomarcadores Tumorais/metabolismo , Claudina-3/metabolismo , Exossomos/metabolismo , Neoplasias da Próstata/metabolismo , Idoso , Biomarcadores Tumorais/sangue , Linhagem Celular Tumoral , Claudina-3/sangue , Bases de Dados Factuais , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Gradação de Tumores , Hiperplasia Prostática/sangue , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia
8.
Bioinformatics ; 31(6): 933-9, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25388151

RESUMO

MOTIVATION: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.


Assuntos
Biologia Computacional , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Factuais , Exossomos/metabolismo , Espaço Extracelular/metabolismo , Software , Pesquisa Biomédica , Humanos , Interface Usuário-Computador
9.
BMC Biol ; 12: 44, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24885675

RESUMO

BACKGROUND: Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed. RESULTS: Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner. CONCLUSIONS: We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.


Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Via de Sinalização Wnt , Proteína Wnt3A/química , Proteína Wnt3A/metabolismo , Sequência de Aminoácidos , Animais , Embrião não Mamífero/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação Puntual/genética , Ligação Proteica , Estrutura Terciária de Proteína , Solubilidade , Relação Estrutura-Atividade , Peixe-Zebra/embriologia
10.
J Extracell Vesicles ; 11(9): e12263, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36103151

RESUMO

Extracellular vesicle (EV) secretion enables cell-cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. Here, we present an in vivo model to study EV secretion using the fat body and the haemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue-specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualisation of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterisation of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. We furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV biogenesis and secretion.


Assuntos
Proteínas de Drosophila , Vesículas Extracelulares , Animais , Secreções Corporais , Drosophila melanogaster , Endossomos , Cinesinas , Transdução de Sinais , Proteínas rab de Ligação ao GTP
11.
Bio Protoc ; 11(11): e4040, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34250206

RESUMO

Secretory Wnt trafficking can be studied in the polarized epithelial monolayer of Drosophila wing imaginal discs (WID). In this tissue, Wg (Drosophila Wnt-I) is presented on the apical surface of its source cells before being internalized into the endosomal pathway. Long-range Wg secretion and spread depend on secondary secretion from endosomal compartments, but the exact post-endocytic fate of Wg is poorly understood. Here, we summarize and present three protocols for the immunofluorescence-based visualization and quantitation of different pools of intracellular and extracellular Wg in WID: (1) steady-state extracellular Wg; (2) dynamic Wg trafficking inside endosomal compartments; and (3) dynamic Wg release to the cell surface. Using a genetic driver system for gene manipulation specifically at the posterior part of the WID (EnGal4) provides a robust internal control that allows for direct comparison of signal intensities of control and manipulated compartments of the same WID. Therefore, it also circumvents the high degree of staining variability usually associated with whole-tissue samples. In combination with the genetic manipulation of Wg pathway components that is easily feasible in Drosophila, these methods provide a tool-set for the dissection of secretory Wg trafficking and can help us to understand how Wnt proteins travel along endosomal compartments for short- and long-range signal secretion. Graphic abstract: Figure 1. Visualization of extracellular and intracellular Wg trafficking in Drosophila wing imaginal discs. While staining of extracellular Wg without permeabilization exclusively visualizes Wg bound to the extracellular surface (left), Wg uptake and endosomal trafficking can be visualized using an antibody uptake assay (middle). Dynamic Wg release can be visualized by performing a non-permeabilizing staining at a permissive temperature that sustains secretory Wg transport (right).

12.
Biomolecules ; 10(11)2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33207719

RESUMO

Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fusão de Membrana/fisiologia , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Drosophila , Células HCT116 , Células HEK293 , Humanos , Fosforilação/fisiologia , Proteínas SNARE/genética , Proteínas SNARE/metabolismo
13.
Sci Rep ; 10(1): 5516, 2020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251349

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

14.
Cancers (Basel) ; 12(1)2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31877768

RESUMO

Extracellular vesicles (EVs) are shed by many different cell types. Their nucleic acids content offers new opportunities for biomarker research in different solid tumors. The role of EV RNA in prostate cancer (PCa) is still largely unknown. EVs were isolated from different benign and malignant prostate cell lines and blood plasma from patients with PCa (n = 18) and controls with benign prostatic hyperplasia (BPH) (n = 7). Nanoparticle tracking analysis (NTA), Western blot, electron microscopy, and flow cytometry analysis were used for the characterization of EVs. Non-coding RNA expression profiling of PC3 metastatic PCa cells and their EVs was performed by next generation sequencing (NGS). miRNAs differentially expressed in PC3 EVs were validated with qRT-PCR in EVs derived from additional cell lines and patient plasma and from matched tissue samples. 92 miRNAs were enriched and 48 miRNAs were depleted in PC3 EVs compared to PC3 cells, which could be confirmed by qRT-PCR. miR-99b-5p was significantly higher expressed in malignant compared to benign EVs. Furthermore, expression profiling showed miR-10a-5p (p = 0.018) and miR-29b-3p (p = 0.002), but not miR-99b-5p, to be overexpressed in plasma-derived EVs from patients with PCa compared with controls. In the corresponding tissue samples, no significant differences in the miRNA expression could be observed. We thus propose that EV-associated miR-10a-5p and miR-29b-3p could serve as potential new PCa detection markers.

15.
Front Cardiovasc Med ; 5: 10, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29564334

RESUMO

Wnt signaling is an important pathway in health and disease and a key regulator of stem cell maintenance, differentiation, and proliferation. During heart development, Wnt signaling controls specification, proliferation and differentiation of cardiovascular cells. In this regard, the role of activated Wnt signaling in cardiogenesis is well defined. However, the knowledge about signaling transmission has been challenged. Recently, the packaging of hydrophobic Wnt proteins on extracellular vesicles (EVs) has emerged as a mechanism to facilitate their extracellular spreading and their functioning as morphogens. EVs spread systemically and therefore can have pleiotropic effects on very different cell types. They are heavily studied in tumor biology where they affect tumor growth and vascularization and can serve as biomarkers in liquid biopsies. In this review we will highlight recent discoveries of factors involved in the release of Wnts on EVs and its potential implications in the communication between physiological and pathological heart cells.

16.
J Extracell Vesicles ; 6(1): 1378056, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29184623

RESUMO

Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g ("microvesicles" (MV)) and 100,000 g ("exosomes") are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that - despite the different vesicle biogenesis - the routes of vesicular export are adaptable.

17.
Methods Mol Biol ; 1481: 17-28, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27590148

RESUMO

Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading.


Assuntos
Exossomos/química , Vesículas Extracelulares/química , Biologia Molecular/métodos , Proteínas Wnt/isolamento & purificação , Animais , Retículo Endoplasmático/química , Complexo de Golgi/química , Humanos , Transdução de Sinais , Proteínas Wnt/química
18.
PLoS One ; 11(3): e0150377, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26953690

RESUMO

Cryptochromes, blue-light absorbing proteins involved in the circadian clock, have been proposed to be the receptor molecules of the avian magnetic compass. In birds, several cryptochromes occur: Cryptochrome 2, Cryptochrome 4 and two splice products of Cryptochrome 1, Cry1a and Cry1b. With an antibody not distinguishing between the two splice products, Cryptochrome 1 had been detected in the retinal ganglion cells of garden warblers during migration. A recent study located Cry1a in the outer segments of UV/V-cones in the retina of domestic chickens and European robins, another migratory species. Here we report the presence of cryptochrome 1b (eCry1b) in retinal ganglion cells and displaced ganglion cells of European Robins, Erithacus rubecula. Immuno-histochemistry at the light microscopic and electron microscopic level showed eCry1b in the cell plasma, free in the cytosol as well as bound to membranes. This is supported by immuno-blotting. However, this applies only to robins in the migratory state. After the end of the migratory phase, the amount of eCry1b was markedly reduced and hardly detectable. In robins, the amount of eCry1b in the retinal ganglion cells varies with season: it appears to be strongly expressed only during the migratory period when the birds show nocturnal migratory restlessness. Since the avian magnetic compass does not seem to be restricted to the migratory phase, this seasonal variation makes a role of eCry1b in magnetoreception rather unlikely. Rather, it could be involved in physiological processes controlling migratory restlessness and thus enabling birds to perform their nocturnal flights.


Assuntos
Criptocromos/genética , Regulação da Expressão Gênica , Passeriformes/genética , Células Ganglionares da Retina/metabolismo , Estações do Ano , Migração Animal , Animais
19.
Sci Rep ; 6: 21848, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26898837

RESUMO

Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown, and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae, and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein.


Assuntos
Ritmo Circadiano/fisiologia , Criptocromos/genética , Mamíferos/fisiologia , Filogenia , Células Fotorreceptoras Retinianas Cones/fisiologia , Animais , Aves/fisiologia , Canidae/fisiologia , Ritmo Circadiano/efeitos da radiação , Opsinas dos Cones/genética , Criptocromos/química , Expressão Gênica , Hominidae/fisiologia , Soros Imunes/química , Imuno-Histoquímica , Luz , Campos Magnéticos , Mamíferos/classificação , Mustelidae/fisiologia , Conformação Proteica , Domínios Proteicos , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Ursidae/fisiologia
20.
Curr Opin Genet Dev ; 23(4): 385-90, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23540564

RESUMO

Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion.


Assuntos
Diferenciação Celular/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Animais , Retículo Endoplasmático , Exossomos/genética , Exossomos/metabolismo , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Wnt/genética
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