RESUMO
Despite a key role for dense core vesicles (DCVs) in neuronal function, there are major gaps in our understanding of DCV biogenesis. A genetic screen for Caenorhabditis elegans mutants with behavioral defects consistent with impaired DCV function yielded five mutations in UNC-108 (Rab2). A genetic analysis showed that unc-108 mutations impair a DCV function unrelated to neuropeptide release that, together with neuropeptide release, fully accounts for the role of DCVs in locomotion. An electron microscopy analysis of DCVs in unc-108 mutants, coupled with quantitative imaging of DCV cargo proteins, revealed that Rab2 acts in cell somas during DCV maturation to prevent the loss of soluble and membrane cargo. In Rab2 null mutants, two thirds of these cargoes move to early endosomes via a PI(3)P-dependent trafficking pathway, whereas aggregated neuropeptides are unaffected. These results reveal how neurons solve a challenging trafficking problem using the most highly conserved animal Rab.
Assuntos
Caenorhabditis elegans/enzimologia , Mutação , Neurônios/enzimologia , Vesículas Secretórias/enzimologia , Proteínas rab de Ligação ao GTP/deficiência , Proteína rab2 de Ligação ao GTP/deficiência , Animais , Axônios/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/genética , Endossomos/enzimologia , Potenciais Evocados , Potenciais Pós-Sinápticos Excitadores , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Genótipo , Locomoção , Potenciais Pós-Sinápticos em Miniatura , Músculos/inervação , Neurônios/ultraestrutura , Neuropeptídeos/metabolismo , Fenótipo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico , Vesículas Secretórias/ultraestrutura , Sinapses/enzimologia , Vesículas Sinápticas/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteína rab2 de Ligação ao GTP/genéticaRESUMO
For many organisms the ability to transduce light into cellular signals is crucial for survival. Light stimulates DNA repair and metabolism changes in bacteria, avoidance responses in single-cell organisms, attraction responses in plants, and both visual and nonvisual perception in animals. Despite these widely differing responses, in all of nature there are only six known families of proteins that can transduce light. Although the roundworm Caenorhabditis elegans has none of the known light transduction systems, we show here that C. elegans strongly accelerates its locomotion in response to blue or shorter wavelengths of light, with maximal responsiveness to ultraviolet light. Our data suggest that C. elegans uses this light response to escape the lethal doses of sunlight that permeate its habitat. Short-wavelength light drives locomotion by bypassing two critical signals, cyclic adenosine monophosphate (cAMP) and diacylglycerol (DAG), that neurons use to shape and control behaviors. C. elegans mutants lacking these signals are paralyzed and unresponsive to harsh physical stimuli in ambient light, but short-wavelength light rapidly rescues their paralysis and restores normal levels of coordinated locomotion. This light response is mediated by LITE-1, a novel ultraviolet light receptor that acts in neurons and is a member of the invertebrate Gustatory receptor (Gr) family. Heterologous expression of the receptor in muscle cells is sufficient to confer light responsiveness on cells that are normally unresponsive to light. Our results reveal a novel molecular solution for ultraviolet light detection and an unusual sensory modality in C. elegans that is unlike any previously described light response in any organism.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Proteínas de Membrana/fisiologia , Raios Ultravioleta , Animais , Proteínas de Caenorhabditis elegans/genética , AMP Cíclico/metabolismo , Diglicerídeos/metabolismo , Locomoção/fisiologia , Neurônios Motores/fisiologia , Células Musculares/metabolismo , Mutação , Transdução de SinaisRESUMO
The Galpha(q) pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cbeta [PLCbeta]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Galpha(q) pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCbeta). Strains containing null mutations in both EGL-8 (PLCbeta) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Galpha(q)-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Galpha(q) synergizes with Trio RhoGEF to activate RhoA. Activated Galpha(q) and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio's Rho-specific GEF domain is a major Galpha(q) effector that, together with PLCbeta, mediates the Galpha(q) signaling that drives the locomotion, egg laying, and growth of the animal.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Acetilcolina/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Modelos Biológicos , Mutação/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Fosfolipase C beta/genética , Fosfolipase C beta/fisiologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/genética , Transmissão Sináptica/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Forward genetic screens for mutations that rescue the paralysis of ric-8 (Synembryn) reduction-of-function mutations frequently reveal mutations that cause hyperactivation of one or more components of the G alpha(s) pathway. Here, we report that one of these mutations strongly reduces the function of the Dunce cAMP phosphodiesterase PDE-4 by disrupting a conserved active site residue. Loss of function and neural overexpression of PDE-4 have profound and opposite effects on locomotion rate, but drug-response assays suggest that loss of PDE-4 function does not affect steady-state acetylcholine release or reception. Our genetic analysis suggests that PDE-4 regulates both G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the neurons controlling locomotion rate. By immunostaining, PDE-4 is strongly expressed throughout the nervous system, where it localizes to small regions at the outside boundaries of synaptic vesicle clusters as well as intersynaptic regions. The synaptic subregions containing PDE-4 are distinct from those containing active zones, as indicated by costaining with an antibody against the long form of UNC-13. This highly focal subsynaptic localization suggests that PDE-4 may exert its effects by spatially regulating intrasynaptic cAMP pools.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Caenorhabditis elegans/metabolismo , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Sinapses/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/deficiência , Aldicarb/farmacologia , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte , Domínio Catalítico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Regulação da Expressão Gênica , Genes de Helmintos/genética , Levamisol/farmacologia , Locomoção , Dados de Sequência Molecular , Neurônios/metabolismo , Fenótipo , Estrutura Terciária de Proteína , Transporte Proteico , Deleção de Sequência , Vesículas Sinápticas/metabolismoRESUMO
C. elegans mutants lacking the dense-core vesicle priming protein UNC-31 (CAPS) share highly similar phenotypes with mutants lacking a neuronal G alpha(s) pathway, including strong paralysis despite exhibiting near normal levels of steady-state acetylcholine release as indicated by drug sensitivity assays. Our genetic analysis shows that UNC-31 and neuronal G alpha(s) are different parts of the same pathway and that the UNC-31/G alpha(s) pathway is functionally distinct from the presynaptic G alpha(q) pathway with which it interacts. UNC-31 acts upstream of G alpha(s) because mutations that activate the G alpha(s) pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (+) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the G alpha(s) pathway function in cholinergic motor neurons to regulate locomotion rate. Using immunostaining we show that UNC-31 is often concentrated at or near active zones of cholinergic motor neuron synapses. Our data suggest that presynaptic UNC-31 activity, likely acting via dense-core vesicle exocytosis, is required to locally activate the neuronal G alpha(s) pathway near synaptic active zones.