RESUMEN
The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.
Asunto(s)
Proteínas de Unión al Calcio/genética , Embrión no Mamífero/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas Hedgehog/genética , Transducción de Señal/genética , Proteínas de Pez Cebra/genética , Animales , Western Blotting , Células COS , Proteínas de Unión al Calcio/metabolismo , Chlorocebus aethiops , ADN Complementario/química , ADN Complementario/genética , Embrión no Mamífero/embriología , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Hedgehog/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Fenotipo , Análisis de Secuencia de ADN , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Cullin/metabolismo , Meiosis , Microtúbulos , Mitosis , Animales , Caenorhabditis elegans/citología , KataninaRESUMEN
Many biological processes, such as development and cell cycle progression are tightly controlled by selective ubiquitin-dependent degradation of key substrates. In this pathway, the E3-ligase recognizes the substrate and targets it for degradation by the 26S proteasome. The SCF (Skp1-Cul1-F-box) and ECS (Elongin C-Cul2-SOCS box) complexes are two well-defined cullin-based E3-ligases. The cullin subunits serve a scaffolding function and interact through their C terminus with the RING-finger-containing protein Hrt1/Roc1/Rbx1, and through their N terminus with Skp1 or Elongin C, respectively. In Caenorhabditis elegans, the ubiquitin-ligase activity of the CUL-3 complex is required for degradation of the microtubule-severing protein MEI-1/katanin at the meiosis-to-mitosis transition. However, the molecular composition of this cullin-based E3-ligase is not known. Here we identified the BTB-containing protein MEL-26 as a component required for degradation of MEI-1 in vivo. Importantly, MEL-26 specifically interacts with CUL-3 and MEI-1 in vivo and in vitro, and displays properties of a substrate-specific adaptor. Our results suggest that BTB-containing proteins may generally function as substrate-specific adaptors in Cul3-based E3-ubiquitin ligases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin , Ligasas/química , Ligasas/metabolismo , Adenosina Trifosfatasas/genética , Alelos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/química , Sustancias Macromoleculares , Meiosis , Microtúbulos/metabolismo , Mitosis , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Subunidades de Proteína/metabolismo , Interferencia de ARN , Especificidad por Sustrato , Ubiquitina-Proteína LigasasRESUMEN
Primary cilia are ubiquitous antenna-like organelles that mediate cellular signaling and represent hotspots for human diseases termed ciliopathies. Within cilia, subcompartments are established to support signal transduction pathways, including Hedgehog signaling. How these compartments are formed and maintained remains largely unknown. Cilia use two mechanisms, a trafficking system and a diffusion barrier, to regulate the trafficking of proteins into, within, and out of cilia. The main ciliary trafficking machinery, intraflagellar transport (IFT), facilitates bidirectional transport of cargo, including signaling proteins, from the base (basal body) to the tip of the axoneme [1]. Anterograde IFT to the tip relies on kinesins, and cytoplasmic dynein enables retrograde transport back [2, 3]. To help confine proteins to cilia, a subdomain immediately distal to the basal body, called the transition zone (TZ), acts as a diffusion barrier for both membrane and soluble proteins [4-6]. Here, we show that in Caenorhabditis elegans a salt-sensing receptor-type guanylate cyclase, GCY-22, accumulates at a high concentration within a subcompartment at the distal region of the cilium. Targeting of GCY-22 to the ciliary tip is dynamic, requiring the IFT system. Disruption of the TZ barrier or IFT trafficking causes GCY-22 protein mislocalization and defects in the formation and maintenance of the ciliary tip compartment. Structure-function studies uncovered GCY-22 protein domains needed for entry and tip localization. Together, our findings provide mechanistic insights into the formation and maintenance of a novel subdomain at the cilium tip that contributes to the behavioral response to NaCl.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Células Quimiorreceptoras/metabolismo , Quimiotaxis/fisiología , Cilios/metabolismo , Guanilato Ciclasa/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Células Quimiorreceptoras/citología , Guanilato Ciclasa/genética , Cloruro de Sodio/metabolismoRESUMEN
Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling. Yet, the roles of cilia in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC-1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC-1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy.
Asunto(s)
Caenorhabditis elegans/fisiología , Cilios/metabolismo , Neuronas Dopaminérgicas/fisiología , Sinapsis/metabolismo , Transmisión Sináptica , AnimalesRESUMEN
An exciting discovery of the new millennium is that primary cilia, organelles found on most eukaryotic cells, play crucial roles in vertebrate development by modulating Hedgehog, Wnt and PDGF signaling. Analysis of the literature and sequence databases reveals that the ancient signal transduction pathway, which uses cGMP in eukaryotes or related cyclic di-GMP in bacteria, exists in virtually all eukaryotes. However, many eukaryotes that secondarily lost cilia during evolution, including flowering plants, slime molds and most fungi, lack otherwise evolutionarily conserved cGMP signaling components. Based on this intriguing phylogenetic distribution, the presence of cGMP signaling proteins within cilia, and the indispensable roles that cGMP plays in transducing environmental signals in divergent ciliated cells (e.g. vertebrate photoreceptors and Caenorhabditis elegans sensory neurons), we propose that cGMP signaling has a strong ciliary basis. cAMP signaling, also inherent to bacteria and crucial for cilium-dependent olfaction, similarly appears to have widespread usage in diverse cilia. Thus, we argue here that both cyclic nucleotides play essential and potentially ubiquitous roles in modulating ciliary functions.
Asunto(s)
Cilios/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Eucariontes/citología , Eucariontes/metabolismo , Transducción de Señal , Animales , Bacterias/metabolismo , Evolución Biológica , Caenorhabditis elegans/metabolismo , HumanosRESUMEN
Rho-binding kinase and the myosin phosphatase targeting subunit regulate nonmuscle contractile events in higher eukaryotes. Genetic evidence indicates that the C. elegans homologs regulate embryonic morphogenesis by controlling the actin-mediated epidermal cell shape changes that transform the spherical embryo into a long, thin worm. LET-502/Rho-binding kinase triggers elongation while MEL-11/myosin phosphatase targeting subunit inhibits this contractile event. We describe mutations in the nonmuscle myosin heavy chain gene nmy-1 that were isolated as suppressors of the mel-11 hypercontraction phenotype. However, a nmy-1 null allele displays elongation defects less severe than mutations in let-502 or in the single nonmuscle myosin light chain gene mlc-4. This results because nmy-1 is partially redundant with another nonmuscle myosin heavy chain, nmy-2, which was previously known only for its role in anterior/posterior polarity and cytokinesis in the early embryo. At the onset of elongation, NMY-1 forms filamentous-like structures similar to actin, and LET-502 is interspersed with these structures, where it may trigger contraction. MEL-11, which inhibits elongation, is initially cytoplasmic. In response to LET-502 activity, MEL-11 becomes sequestered away from the contractile apparatus, to the plasma membrane, when elongation commences. Upon completion of morphogenesis, MEL-11 again appears in the cytoplasm where it may halt actin/myosin contraction.