RESUMEN
RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65 Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Encéfalo/metabolismo , Neurogénesis , Neuronas/metabolismo , Proteínas Nogo/metabolismo , Receptores Nogo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adipoquinas/metabolismo , Secuencia de Aminoácidos , Animales , Axones/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/metabolismo , Complemento C1q/metabolismo , Dendritas/metabolismo , Glicosilación , Células HEK293 , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ligandos , Ratones Endogámicos C57BL , Red Nerviosa/metabolismo , Polisacáridos/metabolismo , Unión Proteica , Dominios Proteicos , Eliminación de Secuencia , Sinapsis/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Canales de Calcio/genética , Diferenciación Celular/genética , Uniones Comunicantes/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Proteínas Musculares/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/biosíntesis , Canales de Calcio/biosíntesis , Señalización del Calcio/genética , Comunicación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Proteínas Musculares/biosíntesis , Neuronas Receptoras Olfatorias/metabolismo , Olfato/genéticaRESUMEN
Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed.
Asunto(s)
Sinapsis , Tomografía , Animales , Sinapsis/metabolismo , Sinapsis/ultraestructura , Tomografía/métodos , Imagenología Tridimensional/métodos , Coloración y Etiquetado/métodos , Ratones , Microscopía Electrónica de Rastreo/métodos , Colorantes Fluorescentes/química , Microinyecciones/métodos , Neuronas/metabolismo , RatasRESUMEN
Activation of voltage-gated calcium channels at presynaptic terminals leads to local increases in calcium and the fusion of synaptic vesicles containing neurotransmitter. Presynaptic output is a function of the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at Caenorhabditis elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters ~250 nm in diameter with the active zone proteins Neurexin, α-Liprin, SYDE, ELKS/CAST, RIM-BP, α-Catulin, and MAGI1. CaV2 channels are colocalized with the priming protein UNC-13L and mediate the fusion of vesicles docked within 33 nm of the dense projection. CaV2 activity is amplified by ryanodine receptor release of calcium from internal stores, triggering fusion up to 165 nm from the dense projection. By contrast, CaV1 channels are dispersed in the synaptic varicosity, and are colocalized with UNC-13S. CaV1 and ryanodine receptors are separated by just 40 nm, and vesicle fusion mediated by CaV1 is completely dependent on the ryanodine receptor. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.
Asunto(s)
Caenorhabditis elegans , Canal Liberador de Calcio Receptor de Rianodina , Vesículas Sinápticas , Animales , Caenorhabditis elegans/fisiología , Calcio/metabolismo , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismoRESUMEN
After axon outgrowth and synapse formation, the nervous system transitions to a stable architecture. In C. elegans, this transition is marked by the appearance of casein kinase 1δ (CK1δ) in the nucleus. In CK1δ mutants, neurons continue to sprout growth cones into adulthood, leading to a highly ramified nervous system. Nervous system architecture in these mutants is completely restored by suppressor mutations in ten genes involved in transcription termination. CK1δ prevents termination by phosphorylating and inhibiting SSUP-72. SSUP-72 would normally remodel the C-terminal domain of RNA polymerase in anticipation of termination. The antitermination activity of CK1δ establishes the mature state of a neuron by promoting the expression of the long isoform of a single gene, the cytoskeleton protein Ankyrin.
Asunto(s)
Ancirinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Núcleo Celular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Transcripción Genética , Animales , Ancirinas/genética , Axones/fisiología , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Quinasa Idelta de la Caseína/genética , Núcleo Celular/genética , Fosfoproteínas Fosfatasas/genética , Sinapsis/fisiologíaRESUMEN
Synapse formation defines neuronal connectivity and is thus essential for neuronal circuit assembly. Trans-synaptic interactions of cell adhesion molecules are thought to induce synapse assembly. Here we demonstrate that a recently discovered and conserved short form of neurexin, γ-neurexin, which lacks canonical extracellular domains, is nonetheless sufficient to promote presynaptic assembly in the nematode C. elegans. γ- but not α-neurexin is required for assembling active zone components, recruiting synaptic vesicles, and clustering calcium channels at release sites to promote evoked synaptic transmission. Furthermore, we find that neurexin functions in parallel with the transmembrane receptor Frizzled, as the absence of both proteins leads to an enhanced phenotype-the loss of most synapses. Frizzled's pro-synaptogenic function is independent of its ligand, Wnt. Wnt binding instead eliminates synapses by inducing Frizzled's endocytosis and the downregulation of neurexin. These results reveal how pro- and anti-synaptogenic factors converge to precisely sculpt circuit formation in vivo.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Receptores Frizzled/metabolismo , Neurogénesis/fisiología , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Caenorhabditis elegans , Endocitosis/fisiología , Neuronas Motoras/metabolismo , Isoformas de ProteínasRESUMEN
The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C. elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools.