RESUMEN
Mediolateral cell intercalation is a morphogenetic strategy used throughout animal development to reshape tissues. Dorsal intercalation in the Caenorhabditis elegans embryo involves the mediolateral intercalation of two rows of dorsal epidermal cells to create a single row that straddles the dorsal midline, and thus is a simple model to study cell intercalation. Polarized protrusive activity during dorsal intercalation requires the C. elegans Rac and RhoG orthologs CED-10 and MIG-2, but how these GTPases are regulated during intercalation has not been thoroughly investigated. In this study, we characterized the role of the Rac-specific guanine nucleotide exchange factor (GEF) TIAM-1 in regulating actin-based protrusive dynamics during dorsal intercalation. We found that TIAM-1 can promote formation of the main medial lamellipodial protrusion extended by intercalating cells through its canonical GEF function, whereas its N-terminal domains function to negatively regulate the generation of ectopic filiform protrusions around the periphery of intercalating cells. We also show that the guidance receptor UNC-5 inhibits these ectopic filiform protrusions in dorsal epidermal cells and that this effect is in part mediated via TIAM-1. These results expand the network of proteins that regulate basolateral protrusive activity during directed rearrangement of epithelial cells in animal embryos.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Animales , Actinas/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptores de Superficie Celular , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismoRESUMEN
Animal development requires a carefully orchestrated cascade of cell fate specification events and cellular movements. A surprisingly small number of choreographed cellular behaviours are used repeatedly to shape the animal body plan. Among these, cell intercalation lengthens or spreads a tissue at the expense of narrowing along an orthogonal axis. Key steps in the polarization of both mediolaterally and radially intercalating cells have now been clarified. In these different contexts, intercalation seems to require a distinct combination of mechanisms, including adhesive changes that allow cells to rearrange, cytoskeletal events through which cells exert the forces needed for cell neighbour exchange, and in some cases the regulation of these processes through planar cell polarity.
Asunto(s)
Tipificación del Cuerpo , Desarrollo Embrionario , Animales , Adhesión Celular , Movimiento Celular , Polaridad Celular , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Células Epiteliales/fisiología , Humanos , Transducción de SeñalRESUMEN
A hallmark of gastrulation is the establishment of germ layers by internalization of cells initially on the exterior. In C. elegans the end of gastrulation is marked by the closure of the ventral cleft, a structure formed as cells internalize during gastrulation, and the subsequent rearrangement of adjacent neuroblasts that remain on the surface. We found that a nonsense allele of srgp-1/srGAP leads to 10-15% cleft closure failure. Deletion of the SRGP-1/srGAP C-terminal domain led to a comparable rate of cleft closure failure, whereas deletion of the N-terminal F-BAR region resulted in milder defects. Loss of the SRGP-1/srGAP C-terminus or F-BAR domain results in defects in rosette formation and defective clustering of HMP-1/âº-catenin in surface cells during cleft closure. A mutant form of HMP-1/âº-catenin with an open M domain can suppress cleft closure defects in srgp-1 mutant backgrounds, suggesting that this mutation acts as a gain-of-function allele. Since SRGP-1 binding to HMP-1/âº-catenin is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1/âº-catenin is constitutively open. A good candidate is AFD-1/afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1/afadin is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/afadin increases cleft closure defects in srgp-1/srGAP and hmp-1R551/554A/âº-catenin backgrounds. We propose that SRGP-1/srGAP promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1/âº-catenin opens, allowing maturing junctions to transition from recruitment of SRGP-1/srGAP to AFD-1/afadin. Our work identifies new roles for âº-catenin interactors during a process crucial to metazoan development.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Cateninas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , alfa Catenina/genética , Gastrulación/genética , Formación de Roseta , Cadherinas/genética , Cadherinas/metabolismo , Adhesión CelularRESUMEN
The cadherin-catenin complex (CCC) is central to embryonic development and tissue repair, yet how CCC binding partners function alongside core CCC components remains poorly understood. Here, we establish a previously unappreciated role for an evolutionarily conserved protein, the slit-robo GTPase-activating protein SRGP-1/srGAP, in cadherin-dependent morphogenetic processes in the Caenorhabditis elegans embryo. SRGP-1 binds to the M domain of the core CCC component, HMP-1/α-catenin, via its C terminus. The SRGP-1 C terminus is sufficient to target it to adherens junctions, but only during later embryonic morphogenesis, when junctional tension is known to increase. Surprisingly, mutations that disrupt stabilizing salt bridges in the M domain block this recruitment. Loss of SRGP-1 leads to an increase in mobility and decrease of junctional HMP-1. In sensitized genetic backgrounds with weakened adherens junctions, loss of SRGP-1 leads to late embryonic failure. Rescue of these phenotypes requires the C terminus of SRGP-1 but also other domains of the protein. Taken together, these data establish a role for an srGAP in stabilizing and organizing the CCC during epithelial morphogenesis by binding to a partially closed conformation of α-catenin at junctions.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Cadherinas/genética , Cadherinas/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Morfogénesis/genética , alfa Catenina/genética , alfa Catenina/metabolismoRESUMEN
The Slit-Robo GTPase-activating proteins (srGAPs) were first identified as potential Slit-Robo effectors that influence growth cone guidance. Given their N-terminal F-BAR, central GAP and C-terminal SH3 domains, srGAPs have the potential to affect membrane dynamics, Rho family GTPase activity and other binding partners. Recent research has clarified how srGAP family members act in distinct ways at the cell membrane, and has expanded our understanding of the roles of srGAPs in neuronal and non-neuronal cells. Gene duplication of the human-specific paralog of srGAP2 has resulted in srGAP2 family proteins that may have increased the density of dendritic spines and promoted neoteny of the human brain during crucial periods of human evolution, underscoring the importance of srGAPs in the unique sculpting of the human brain. Importantly, srGAPs also play roles outside of the nervous system, including during contact inhibition of cell movement and in establishing and maintaining cell adhesions in epithelia. Changes in srGAP expression may contribute to neurodevelopmental disorders, cancer metastasis and inflammation. As discussed in this Review, much remains to be discovered about how this interesting family of proteins functions in a diverse set of processes in metazoans and the functional roles srGAPs play in human disease.
Asunto(s)
Encéfalo/metabolismo , Conos de Crecimiento/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Humanos , Proteínas RoundaboutRESUMEN
Cell intercalation is a highly directed cell rearrangement that is essential for animal morphogenesis. As such, intercalation requires orchestration of cell polarity across the plane of the tissue. CDC-42 is a Rho family GTPase with key functions in cell polarity, yet its role during epithelial intercalation has not been established because its roles early in embryogenesis have historically made it difficult to study. To circumvent these early requirements, in this paper we use tissue-specific and conditional loss-of-function approaches to identify a role for CDC-42 during intercalation of the Caenorhabditis elegans dorsal embryonic epidermis. CDC-42 activity is enriched in the medial tips of intercalating cells, which extend as cells migrate past one another. Moreover, CDC-42 is involved in both the efficient formation and orientation of cell tips during cell rearrangement. Using conditional loss-of-function we also show that the PAR complex functions in tip formation and orientation. Additionally, we find that the sole C. elegans Eph receptor, VAB-1, functions during this process in an Ephrin-independent manner. Using epistasis analysis, we find that vab-1 lies in the same genetic pathway as cdc-42 and is responsible for polarizing CDC-42 activity to the medial tip. Together, these data establish a previously uncharacterized role for polarized CDC-42, in conjunction with PAR-6, PAR-3 and an Eph receptor, during epithelial intercalation.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Unión al GTP/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Movimiento Celular/genética , Polaridad Celular/genética , Desarrollo Embrionario/genética , Efrinas/genética , Epidermis/crecimiento & desarrollo , Epidermis/metabolismo , Epistasis Genética , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Morfogénesis/genética , Especificidad de Órganos , Transducción de SeñalRESUMEN
Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and ß-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/ß-catenin is higher than that of α-catenin for ß-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalian α-catenin·ß-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40-100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans Our data provide novel insights into how cadherin-dependent cell-cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Comunicación Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Complejos Multiproteicos/metabolismo , alfa Catenina/metabolismo , Sustitución de Aminoácidos , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Adhesión Celular/fisiología , Cristalografía por Rayos X , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Mutación Missense , Estructura Cuaternaria de Proteína , alfa Catenina/química , alfa Catenina/genéticaRESUMEN
Intercellular epithelial junctions formed by classical cadherins, ß-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the ß-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds ß-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.
Asunto(s)
Actinas/química , Cadherinas/química , Proteínas de Caenorhabditis elegans/química , Proteínas del Citoesqueleto/química , alfa Catenina/química , beta Catenina/química , Sitio Alostérico , Animales , Caenorhabditis elegans , Adhesión Celular , Cristalografía por Rayos X , Glutatión Transferasa/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , Conejos , Relación Estructura-Actividad , Vinculina/químicaRESUMEN
Cell intercalation is a fundamental, coordinated cell rearrangement process that shapes tissues throughout animal development. Studies of intercalation within epithelia have focused almost exclusively on the localized constriction of specific apical junctions. Another widely deployed yet poorly understood alternative mechanism of epithelial intercalation relies on basolateral protrusive activity. Using the dorsal embryonic epidermis of Caenorhabditis elegans, we have investigated this alternative mechanism using high-resolution live cell microscopy and genetic analysis. We find that as dorsal epidermal cells migrate past one another they produce F-actin-rich protrusions polarized at their extending (medial) edges. These protrusions are controlled by the C. elegans Rac and RhoG orthologs CED-10 and MIG-2, which function redundantly to polarize actin polymerization upstream of the WAVE complex and WASP, respectively. We also identify UNC-73, the C. elegans ortholog of Trio, as a guanine nucleotide exchange factor (GEF) upstream of both CED-10 and MIG-2. Further, we identify a novel polarizing cue, CRML-1, which is the ortholog of human capping Arp2/3 myosin I linker (CARMIL), that localizes to the nonprotrusive lateral edges of dorsal cells. CRML-1 genetically suppresses UNC-73 function and, indirectly, actin polymerization. This network identifies a novel, molecularly conserved cassette that regulates epithelial intercalation via basolateral protrusive activity.
Asunto(s)
Caenorhabditis elegans/embriología , Epidermis/embriología , Epitelio/embriología , Regulación del Desarrollo de la Expresión Génica , Actinas/metabolismo , Animales , Tipificación del Cuerpo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Movimiento Celular , Proteínas Fluorescentes Verdes/metabolismo , Proteínas del Tejido Nervioso/fisiología , Interferencia de ARN , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Adherens junctions play key roles in mediating cell-cell contacts during tissue development. In Caenorhabditis elegans embryos, the cadherin-catenin complex (CCC), composed of the classical cadherin HMR-1 and members of three catenin families, HMP-1, HMP-2 and JAC-1, is necessary for normal blastomere adhesion, gastrulation, ventral enclosure of the epidermis and embryo elongation. Disruption of CCC assembly or function results in embryonic lethality. Previous work suggests that components of the CCC are subject to phosphorylation. However, the identity of phosphorylated residues in CCC components and their contributions to CCC stability and function in a living organism remain speculative. Using mass spectrometry, we systematically identify phosphorylated residues in the essential CCC subunits HMR-1, HMP-1 and HMP-2 in vivo. We demonstrate that HMR-1/cadherin phosphorylation occurs on three sites within its ß-catenin binding domain that each contributes to CCC assembly on lipid bilayers. In contrast, phosphorylation of HMP-2/ß-catenin inhibits its association with HMR-1/cadherin in vitro, suggesting a role in CCC disassembly. Although HMP-1/α-catenin is also phosphorylated in vivo, phosphomimetic mutations do not affect its ability to associate with other CCC components or interact with actin in vitro. Collectively, our findings support a model in which distinct phosphorylation events contribute to rapid CCC assembly and disassembly, both of which are essential for morphogenetic rearrangements during development.
Asunto(s)
Blastómeros/metabolismo , Cadherinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Cateninas/metabolismo , Proteínas del Citoesqueleto/metabolismo , alfa Catenina/metabolismo , Animales , Cadherinas/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cateninas/genética , Proteínas del Citoesqueleto/genética , Embrión no Mamífero/embriología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosforilación/fisiología , alfa Catenina/genéticaRESUMEN
Stable intercellular adhesions formed through the cadherin-catenin complex are important determinants of proper tissue architecture and help maintain tissue integrity during morphogenetic movements in developing embryos. A key regulator of this stability is α-catenin, which connects the cadherin-catenin complex to the actin cytoskeleton. Although the C-terminal F-actin-binding domain of α-catenin has been shown to be crucial for its function, a more detailed in vivo analysis of discrete regions and residues required for actin binding has not been performed. Using Caenorhabditis elegans as a model system, we have characterized mutations in hmp-1/α-catenin that identify HMP-1 residues 687-742 and 826-927, as well as amino acid 802, as critical to the localization of junctional proximal actin during epidermal morphogenesis. We also find that the S823F transition in a hypomorphic allele, hmp-1(fe4), decreases actin binding in vitro. Using hmp-1(fe4) animals in a mutagenesis screen, we were then able to identify 11 intragenic suppressors of hmp-1(fe4) that revert actin binding to wild-type levels. Using homology modeling, we show that these amino acids are positioned at key conserved sites within predicted α-helices in the C terminus. Through the use of transgenic animals, we also demonstrate that HMP-1 residues 315-494, which correspond to a putative mechanotransduction domain that binds vinculin in vertebrate αE-catenin, are not required during epidermal morphogenesis but may aid efficient recruitment of HMP-1 to the junction. Our studies are the first to identify key conserved amino acids in the C terminus of α-catenin that modulate F-actin binding in living embryos of a simple metazoan.
Asunto(s)
Actinas/metabolismo , Caenorhabditis elegans/metabolismo , Vinculina/metabolismo , Citoesqueleto de Actina/metabolismo , Alelos , Animales , Cadherinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Homocigoto , Modelos Biológicos , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Vinculina/química , alfa Catenina/metabolismoRESUMEN
Cadherin-mediated cell-cell interactions are dynamic processes, and cadherin function is tightly regulated in response to cellular context and signaling. Ultimately, cadherin regulation is likely to reflect the interplay between a range of fundamental cellular processes, including surface organization of receptors, cytoskeletal organization and cell trafficking, that are coordinated by signaling events. In this review we focus on recent advances in understanding how interplay with membrane trafficking and other cell-cell junctions can control cadherin function. The endocytosis of cadherins, and their post-internalization fate, influences surface expression and metabolic stability of these adhesion receptors. Similarly, at the surface, components of tight junctions provide a mode of cross-talk that regulates assembly of adherens junctions.
Asunto(s)
Cadherinas/metabolismo , Adhesión Celular/fisiología , Comunicación Celular/fisiología , Transducción de Señal/fisiología , Animales , Endocitosis/fisiología , Uniones Estrechas/fisiologíaRESUMEN
The ternary complex of cadherin, beta-catenin, and alpha-catenin regulates actin-dependent cell-cell adhesion. alpha-Catenin can bind beta-catenin and F-actin, but in mammals alpha-catenin either binds beta-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of alpha-catenin is evolutionarily conserved. The Caenorhabditis elegans alpha-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1-HMP-2(beta-catenin)-HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of alpha-catenin and suggests that multiple mechanisms regulate alpha-catenin binding to F-actin.
Asunto(s)
Cadherinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , alfa Catenina/metabolismo , Actinas/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cadherinas/química , Cadherinas/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Electroforesis en Gel de Poliacrilamida , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Difracción de Rayos X , alfa Catenina/química , alfa Catenina/genéticaRESUMEN
Mediolateral cell intercalation is a morphogenetic strategy used throughout animal development to reshape tissues. Dorsal intercalation in the C. elegans embryo involves the mediolateral intercalation of two rows of dorsal epidermal cells to create a single row that straddles the dorsal midline, and so is a simple model to study cell intercalation. Polarized protrusive activity during dorsal intercalation requires the C. elegans Rac and RhoG orthologs CED-10 and MIG-2, but how these GTPases are regulated during intercalation has not been thoroughly investigated. In this study, we characterize the role of the Rac-specific guanine nucleotide exchange factor (GEF), TIAM-1, in regulating actin-based protrusive dynamics during dorsal intercalation. We find that TIAM-1 can promote protrusion formation through its canonical GEF function, while its N-terminal domains function to negatively regulate this activity, preventing the generation of ectopic protrusions in intercalating cells. We also show that the guidance receptor UNC-5 inhibits ectopic protrusive activity in dorsal epidermal cells, and that this effect is in part mediated via TIAM-1. These results expand the network of proteins that regulate basolateral protrusive activity during directed cell rearrangement. Summary statement: TIAM-1 activates the Rac pathway to promote protrusion formation via its GEF domain, while its N-terminal domains suppress ectopic protrusions during dorsal intercalation in the C. elegans embryo.
RESUMEN
Protein phosphatase 2A (PP2A) functions in a variety of cellular contexts. PP2A can assemble into four different complexes based on the inclusion of different regulatory or targeting subunits. The B''' regulatory subunit "striatin" forms the STRIPAK complex consisting of striatin, a catalytic subunit (PP2AC), striatin interacting protein 1 (STRIP1), and MOB family member 4 (MOB4). In yeast and C. elegans, STRIP1 is required for formation of the endoplasmic reticulum (ER). Since the sarcoplasmic reticulum (SR) is the highly organized muscle-specific version of ER, we sought to determine the function of the STRIPAK complex in muscle using C. elegans . CASH-1 (striatin) and FARL-11 (STRIP1/2) form a complex in vivo , and each protein is localized to SR. Missense mutations and single amino acid losses in farl-11 and cash-1 each result in similar sarcomere disorganization. A missense mutation in farl-11 shows no detectable FARL-11 protein by immunoblot, disruption of SR organization around M-lines, and altered levels of the SR Ca +2 release channel UNC-68. Summary: Protein phosphatase 2A forms a STRIPAK complex when it includes the targeting B''' subunit "striatin" and STRIP1. STRIP1 is required for formation of ER. We show that in muscle STRIP1 is required for organization of SR and sarcomeres.
RESUMEN
Protein phosphatase 2A (PP2A) functions in a variety of cellular contexts. PP2A can assemble into four different complexes based on the inclusion of different regulatory or targeting subunits. The B''' regulatory subunit "striatin" forms the STRIPAK complex consisting of striatin, a catalytic subunit (PP2AC), striatin-interacting protein 1 (STRIP1), and MOB family member 4 (MOB4). In yeast and Caenorhabditis elegans, STRIP1 is required for formation of the endoplasmic reticulum (ER). Because the sarcoplasmic reticulum (SR) is the highly organized muscle-specific version of ER, we sought to determine the function of the STRIPAK complex in muscle using C. elegans. CASH-1 (striatin) and FARL-11 (STRIP1/2) form a complex in vivo, and each protein is localized to SR. Missense mutations and single amino acid losses in farl-11 and cash-1 each result in similar sarcomere disorganization. A missense mutation in farl-11 shows no detectable FARL-11 protein by immunoblot, disruption of SR organization around M-lines, and altered levels of the SR Ca+2 release channel UNC-68.
Asunto(s)
Caenorhabditis elegans , Retículo Sarcoplasmático , Animales , Caenorhabditis elegans/metabolismo , Retículo Sarcoplasmático/metabolismo , Sarcómeros/metabolismo , Proteína Fosfatasa 2/metabolismo , Retículo Endoplásmico/metabolismoRESUMEN
The simplicity of C. elegans makes it an outstanding system to study the role of Wnt signaling in development. Many asymmetric cell divisions in C. elegans require the Wnt/beta-catenin asymmetry pathway. Recent studies confirm that SYS-1 is a structurally and functionally divergent beta-catenin, and implicate lipids and retrograde trafficking in maintenance of WRM-1/beta-catenin asymmetry. Wnts also regulate short-range events such as spindle rotation and gastrulation, and a PCP-like pathway regulates asymmetric divisions. Long-range, cell non-autonomous Wnt signals regulate vulval induction. Both short-range and long-range Wnt signal s are regulated by recycling of MIG-14/Wntless via the retromer complex. These studies indicate that C. elegans continues to be useful for identifying new, conserved mechanisms underlying Wnt signaling in metazoans.
Asunto(s)
Tipificación del Cuerpo/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Transducción de Señal/fisiología , Proteínas Wnt/fisiología , Animales , Proteínas de Caenorhabditis elegans/fisiología , Proteínas Portadoras/fisiología , Polaridad Celular/genética , Embrión no Mamífero , Péptidos y Proteínas de Señalización Intracelular , Modelos Biológicos , Transducción de Señal/genética , Huso Acromático/fisiología , Sinapsis/fisiología , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/fisiología , beta Catenina/fisiologíaRESUMEN
Dorsal intercalation of the embryonic epidermis in the Caenorhabditis elegans embryo is a promising system for genetic analysis of convergent extension, a conserved process in animal embryos. We sought to identify functionally important actin regulators in dorsal epidermal cells. A promising candidate is MIG-10, the single MIG-10/RIAM/Lamellipodin (MRL) family member in C. elegans. We endogenously tagged all mig-10 isoforms with mNeonGreen and analyzed mig-10 mutants using 4-dimensional microscopy. MIG-10::mNG is expressed prominently in muscle progenitors but is not detectable in the dorsal epidermis. mig-10(ct41) homozygotes complete dorsal intercalation in a manner indistinguishable from wildtype, indicating MIG-10 is not essential during dorsal intercalation.
RESUMEN
The Caenorhabditis elegans embryo is well suited for analysis of directed cell rearrangement via modern microscopy, due to its simple organization, short generation time, transparency, invariant lineage, and the ability to generate engineered embryos expressing various fluorescent proteins. This chapter provides an overview of routine microscopy techniques for imaging dorsal intercalation, a convergent extension-like morphogenetic movement in the embryonic epidermis of C. elegans, including making agar mounts, low-cost four-dimensional (4D) Nomarski microscopy, laser microsurgery, and 4D fluorescence microscopy using actin and junctional fusion proteins, as well as tissue-specific promoters useful for studying dorsal intercalation.