Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion

The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.PostprintPeer reviewe

Identificación del mecanismo funcional de Ariadne-1a de Drosophila melanogaster

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Biología Molecular. Fecha de lectura: 14-07-2005La subfamilia de proteínas Ariadne pertenece a la familia RBR (RiNG finger-In Between RíNG fingers-RING Finger) que funcionan como ligasas de ubiquitina (también llamadas enzimas de tipo E3) mediante su interacción con enzimas E2. En Drosophila melanogaster existen tres miembros de esta familia: Ariadne-la, lb y 2. En este trabajo, estudiamos el papel de Ariadne-la como enzima E3 en el contexto celular y tisular; finalizando con un primer acercamiento a las relaciones funcionales entre esta proteína y los otros miembros de la subfamilia Ariadne. Trabajos previos habían demostrado que la falta de función de Ariadne-la es letal durante la pupación; nosotros encontramos que este fenómeno se repite en el caso del exceso de función, indicando que la estequiometna de esta proteína es primordial para una fisiología normal. En el estadio de pupa tiene lugar la metamorfosis y este proceso está regulado por la señalización de la hormona esteroidea Ecdisona. La Ecdisona regula la expresión de genes durante el desarrollo mediante su unión a sus receptores nucleares, principalmente el heterodímero formado por el Receptor de Ecdisona y el receptor nuclear Ultraspiracles. Existen tres isoformas del Receptor de Ecdisona, las cuales mediante su expresión diferencial confieren especificidad temporal y espacial a la señalización hormonal. Durante e 1 desarrollo de esta tesis, hemos encontrado que Ariadne-la está involucrada en esta vía de señalización regulando a la isoforma A del Receptor de Ecdisona repercutiendo, finalmente, en posibles cambios en la estructura de la cromatina. Por otro lado, comprobamos que el fenotipo de reducción de retículo endoplásmico descrito para mutantes puntuales en los motivos RiNG finger de ariadnela también se produce en mutaciones nulas, corroborando la importancia funcional de dichos motivos estructurales. Además, analizamos este fenotipo en condiciones de exceso de función y observamos un incremento en el tamafio de este orgánulo al aumentar la cantidad de producto. En organismos completos y sistemas celulares describimos que la deficiencia de ariadne-la reduce la resistencia al estrés oxidativo producido por metales pesados y al estrés celular provocado por la inanición, y tiene un efecto neuro-protector en procesos neurodegenerativos durante el envejecimiento. En cuanto a otros miembros de este grupo de proteínas, la sobre-expresión de Ariadne-lb también produce fenotipos relacionados con la ruta de la Ecdisona, por lo que cabe proponer que estas dos proteínas posiblemente están relacionadas funcionalmente en la regulación de esta vía de señalización.Ariadne proteins are part of the larger family of gene products, RBR (RING finger-in Between RING fingers-RING fmger) that function as ubiquitin ligases E3 through their direct interaction with E2 enzymes. in Drosophila melanogaster there are three different Ariadnes: Ariadne-la, lb, and 2. In this work we study the role of Ariadne-1 a as an E3 at the cellular and whole organism levels, finishing with a first approach to the functional connections between this protein and other members of the Ariadne subfamily. Earlier studies showed that a lack of function of Ariadne-la is lethal during the pupal stages. We also found pupal lethality during overexpression of the gene, suggesting that the stoichiometry of this protein is crucial for the normal physiology. Metamorphosis takes place during pupation and it is regulated by the steroid hormone Ecdysone. Ecdysone controls gene expression throughout d evelopment b y binding t o nuclear r eceptors, m ainly the h eterodymer formed by the Ecdysone Receptor and Ultraspiracles. The Ecdysone Receptor gene produces three isofotms providing both temporal and spatial specificity to the hormonal signalling. We found that Ariadne-la is involved in the Ecdysone pathway by regulating the isoform A of the Ecdysone Receptor which ends up affecting chromatin structure. in addition, we proved that the previously described phenotype of point mutations in ariadne-la, a reduction of the endoplasmic reticulum, also takes place in null mutants, íürther emphasising the functional irnportance of the RING finger motifs. Furthermore, we found that with excessive function of Ariadne-la the amount of endoplasmic reticulum increases. We also demonstrated that a deficiency of Ariadne-la reduces the resistance to oxidative stress caused by heavy metals, and to cell stress due to starvation. interestingly, this deficiency also has a neuroprotecting effect in neurodegeneration processes. The effects of Ariadne-la deficiency may be influenced by and related to the protein's functions in both the Ecdysone pathway and the endoplasmic reticulum. Finally, the over-expression of ariadne-lb also produces phenotypes related to the Ecdysone signal transduction chain, so it is plausible that the hvo Ariadne-1 proteins are functionally linked in this cell process

Hedgehog On The Move: A Precise Spatial Control Of Hedgehog Dispersion Shapes The Gradient

Hedgehog (Hh) as morphogen directs cell differentiation during development activating various target genes in a concentration dependent manner. The mechanisms that permit controlled Hh dispersion and gradient formation remain controversial. New research in the Drosophila wing disc epithelium has revealed a crucial role of Hh recycling for its release and transportation from source cells. Lipid modifications on Hh mediate key interactions with different elements of the pathway, which balance the retention and release of the molecule through the basolateral side of the epithelium, allowing its tight spatial control. Dispersion of Hh is also determined by its hydrophobic nature, and the mechanisms that include membrane-tethered transport of Hh are increasingly proposed. © 2013 Elsevier Ltd.BFU2011-25987 and from Consolider ProgramCDS 2007-00008 from the Spanish MICINN; Fundación Areces; Marie Curie ITN FP7 contract (RTN 035528-2).Peer Reviewe

Cytoneme-mediated cell-to-cell signaling during development

Cell-to-cell communication is vital for animal tissues and organs to develop and function as organized units. Throughout development, intercellular communication is crucial for the generation of structural diversity, mainly by the regulation of differentiation and growth. During these processes, several signaling molecules function as messengers between cells and are transported from producing to receptor cells. Thus, a tight spatial and temporal regulation of signaling transport is likely to be critical during morphogenesis. Despite much experimental and theoretical work, the question as to how these signals move between cells remains. Cell-to-cell contact is probably the most precise spatial and temporal mechanism for the transference of signaling molecules from the producing to the receiving cells. However, most of these molecules can also function at a distance between cells that are not juxtaposed. Recent research has shown the way in which cells may achieve direct physical contact and communication through actin-based filopodia. In addition, increasing evidence is revealing the role of such filopodia in regulating spatial patterning during development; in this context, the filopodia are referred to as cytonemes. In this review, we highlight recent work concerning the roles of these filopodia in cell signaling during development. The processes that initiate and regulate the formation, orientation and dynamics of cytonemes are poorly understood but are potentially extremely important areas for our knowledge of intercellular communication.BFU2011-25987 and Consolider Program CDS 2007–00008 from the Spanish MICINN; Fundación Ramón ArecesPeer Reviewe

Isoform-specific regulation of a steroid hormone nuclear receptor by an E3 ubiquitin ligase in Drosophila melanogaster

The steroid hormone 20-hydroxyecdysone (20E) regulates gene transcription through the heterodimeric nuclear receptor composed of ecdysone receptor (EcR) and Ultraspiracle (USP). The EcR gene encodes three protein isoforms-A, B1, and B2-with variant N-terminal domains that mediate tissue and developmental stage-specific responses to 20E. Ariadne-1a is a conserved member of the RING finger family of ubiquitin ligases first identified in Drosophila melanogaster. Loss-of-function mutations at key cysteines in either of the two RING finger motifs, as well as general overexpression of this enzyme, cause lethality in pupae, which suggests a requirement in metamorphosis. Here, we show that Ariadne-1a binds specifically the isoform A of EcR and ubiquitylates it. Coimmunoprecipitation experiments indicate that the full sequence of EcRA is required for this binding. Protein levels of EcRA and USP change in opposite directions when those of ARI-1a are genetically altered. This is an isoform-specific, E3-dependent regulatory mechanism for a steroid nuclear receptor. Further, qRT-PCR experiments show that the ARI-1a levels lead to the transcriptional regulation of Eip78C, Eip74EF, Eip75B, and Br-C, as well as that of EcR and usp genes. Thus, the activity of this enzyme results in the regulation of dimerizing receptors at the protein and gene transcription levels. This fine-tuned orchestration by a conserved ubiquitin ligase is required during insect metamorphosis and, likely, in other steroid hormone-controlled processes across species. © 2011 by the Genetics Society of America.Peer Reviewe

Parkin and relatives: The RBR family of ubiquitin ligases

Mutations in the parkin gene cause autosomal-recessive juvenile parkinsonism. Parkin encodes a ubiquitinprotein ligase characterized by having the RBR domain, composed of two RING fingers plus an IBR/DRIL domain. The RBR family is defined as the group of genes whose products contain an RBR domain. RBR family members exist in all eukaryotic species for which significant sequence data is available, including animals, plants, fungi, and several protists. The integration of comparative genomics with structural and functional data allows us to conclude that RBR proteins have multiple roles, not only in protein quality control mechanisms, but also as indirect regulators of transcription. A recently formulated hypothesis, based on a case of gene fusion, suggested that RBR proteins may be often part of cullin-containing ubiquitin ligase complexes. Recent data on Parkin protein agrees with that hypothesis. We discuss the involvement of RBR proteins in several neurodegenerative diseases and cancer.Peer Reviewe

Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion

© 2014 Macmillan Publishers Limited. All rights reserved. The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.This work was supported by grants from the Consolidation Program (CSD2007-008-25120) to R.B. and I.G., by grants BFU2011-25987 to I.G., BFU2011-25986 to R.B. and AGL2013-48998-C2-2-R to G.A. from the Spanish MICINN, by Marie Curie FP7 (ITN 238186) and by an institutional grant to the CBMSO from the Fundacion Areces to I.G. and by the Departments of Education and Industry of the Basque Government (PI2009-16 and PI2012/42), and the Bizkaia County to R.B. A.-C.G. and J.R.O.-F.Peer Reviewe

Balancing Hedgehog, a retention and release equilibrium given by Dally, Ihog, Boi and shifted/DmWif

Hedgehog can signal both at a short and long-range, and acts as a morphogen during development in various systems. We studied the mechanisms of Hh release and spread using the Drosophila wing imaginal disc as a model system for polarized epithelium. We analyzed the cooperative role of the glypican Dally, the extracellular factor Shifted (Shf, also known as DmWif), and the Immunoglobulin-like (Ig-like) and Fibronectin III (FNNIII) domain-containing transmembrane proteins, Interference hedgehog (Ihog) and its related protein Brother of Ihog (Boi), in the stability, release and spread of Hh. We show that Dally and Boi are required to prevent apical dispersion of Hh; they also aid Hh recycling for its release along the basolateral part of the epithelium to form a long-range gradient. Shf/DmWif on the other hand facilitates Hh movement restrained by Ihog, Boi and Dally, establishing equilibrium between membrane attachment and release of Hh. Furthermore, this protein complex is part of thin filopodia-like structures or cytonemes, suggesting that the interaction between Dally, Ihog, Boi and Shf/DmWif is required for cytoneme-mediated Hh distribution during gradient formation. © 2013 Elsevier Inc.BFU2008-03320/BMC,BFU2011-25987 from Consolider ProgramCDS2007-00008 from theSpanishMICINN; MarieCurieRTNFP6 and FP7; Fundacion Ramón Areces; JAE-CSICprogram; Spanish MICINNPeer Reviewe