RESUMEN
Sensory dendrites depend on cues from their environment to pattern their growth and direct them toward their correct target tissues. Yet, little is known about dendrite-substrate interactions during dendrite morphogenesis. Here, we describe MNR-1/menorin, which is part of the conserved Fam151 family of proteins and is expressed in the skin to control the elaboration of "menorah"-like dendrites of mechanosensory neurons in Caenorhabditis elegans. We provide biochemical and genetic evidence that MNR-1 acts as a contact-dependent or short-range cue in concert with the neural cell adhesion molecule SAX-7/L1CAM in the skin and through the neuronal leucine-rich repeat transmembrane receptor DMA-1 on sensory dendrites. Our data describe an unknown pathway that provides spatial information from the skin substrate to pattern sensory dendrite development nonautonomously.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/genética , Clonación Molecular , Técnicas de Silenciamiento del Gen , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
During neural development, cellular adhesion is crucial for interactions among and between neurons and surrounding tissues. This function is mediated by conserved cell adhesion molecules, which are tightly regulated to allow for coordinated neuronal outgrowth. Here, we show that the proprotein convertase KPC-1 (homolog of mammalian furin) regulates the Menorin adhesion complex during development of PVD dendritic arbors in Caenorhabditis elegans. We found a finely regulated antagonistic balance between PVD-expressed KPC-1 and the epidermally expressed putative cell adhesion molecule MNR-1 (Menorin). Genetically, partial loss of mnr-1 suppressed partial loss of kpc-1, and both loss of kpc-1 and transgenic overexpression of mnr-1 resulted in indistinguishable phenotypes in PVD dendrites. This balance regulated cell-surface localization of the DMA-1 leucine-rich transmembrane receptor in PVD neurons. Lastly, kpc-1 mutants showed increased amounts of MNR-1 and decreased amounts of muscle-derived LECT-2 (Chondromodulin II), which is also part of the Menorin adhesion complex. These observations suggest that KPC-1 in PVD neurons directly or indirectly controls the abundance of proteins of the Menorin adhesion complex from adjacent tissues, thereby providing negative feedback from the dendrite to the instructive cues of surrounding tissues.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Ligandos , Membrana Celular , Animales Modificados Genéticamente , Transporte Biológico , Caenorhabditis elegans/genética , Dendritas , Mamíferos , Proteínas de la Membrana , Proteínas de Caenorhabditis elegans/genéticaRESUMEN
N-glycans are molecularly diverse sugars borne by over 70% of proteins transiting the secretory pathway and have been implicated in protein folding, stability, and localization. Mutations in genes important for N-glycosylation result in congenital disorders of glycosylation that are often associated with intellectual disability. Here, we show that structurally distinct N-glycans regulate an extracellular protein complex involved in the patterning of somatosensory dendrites in Caenorhabditis elegans. Specifically, aman-2/Golgi alpha-mannosidase II, a conserved key enzyme in the biosynthesis of specific N-glycans, regulates the activity of the Menorin adhesion complex without obviously affecting the protein stability and localization of its components. AMAN-2 functions cell-autonomously to allow for decoration of the neuronal transmembrane receptor DMA-1/LRR-TM with the correct set of high-mannose/hybrid/paucimannose N-glycans. Moreover, distinct types of N-glycans on specific N-glycosylation sites regulate DMA-1/LRR-TM receptor function, which, together with three other extracellular proteins, forms the Menorin adhesion complex. In summary, specific N-glycan structures regulate dendrite patterning by coordinating the activity of an extracellular adhesion complex, suggesting that the molecular diversity of N-glycans can contribute to developmental specificity in the nervous system.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Amantadina/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Polisacáridos/química , Polisacáridos/metabolismoRESUMEN
The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Neuronas/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Animales Modificados Genéticamente , Axones/fisiología , Tipificación del Cuerpo , Proteínas de Caenorhabditis elegans/genética , Movimiento Celular/genética , Dendritas/fisiología , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Vía de Señalización WntRESUMEN
Animals sample their environment through sensory neurons with often elaborately branched endings named dendritic arbors. In a genetic screen for genes involved in the development of the highly arborized somatosensory PVD neuron in C. elegans, we have identified mutations in kpc-1, which encodes the homolog of the proprotein convertase furin. We show that kpc-1/furin is necessary to promote the formation of higher order dendritic branches in PVD and to ensure self-avoidance of sister branches, but is likely not required during maintenance of dendritic arbors. A reporter for kpc-1/furin is expressed in neurons (including PVD) and kpc-1/furin can function cell-autonomously in PVD neurons to control patterning of dendritic arbors. Moreover, we show that kpc-1/furin also regulates the development of other neurons in all major neuronal classes in C. elegans, including aspects of branching and extension of neurites as well as cell positioning. Our data suggest that these developmental functions require proteolytic activity of KPC-1/furin. Recently, the skin-derived MNR-1/menorin and the neural cell adhesion molecule SAX-7/L1CAM have been shown to act as a tripartite complex with the leucine rich transmembrane receptor DMA-1 on PVD mechanosensory to orchestrate the patterning of dendritic branches. Genetic analyses show that kpc-1/furin functions in a pathway with MNR-1/menorin, SAX-7/L1CAM and DMA-1 to control dendritic branch formation and extension of PVD neurons. We propose that KPC-1/furin acts in concert with the 'menorin' pathway to control branching and growth of somatosensory dendrites in PVD.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Dendritas/metabolismo , Furina/genética , Células Receptoras Sensoriales/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Furina/química , Furina/metabolismo , Datos de Secuencia Molecular , Mutación , Fenotipo , Alineación de SecuenciaRESUMEN
Recently, mutations in the RNA polymerase III subunit A (POLR3A) have been described as the cause of the neonatal progeria or Wiedemann-Rautenstrauch syndrome (WRS). POLR3A has important roles in transcription regulation of small RNAs, including tRNA, 5S rRNA, and 7SK rRNA. We aim to describe the cellular and molecular features of WRS fibroblasts. Cultures of primary fibroblasts from one WRS patient [monoallelic POLR3A variant c.3772_3773delCT (p.Leu1258Glyfs*12)] and one control patient were cultured in vitro. The mutation caused a decrease in the expression of wildtype POLR3A mRNA and POLR3A protein and a sharp increase in mutant protein expression. In addition, there was an increase in the nuclear localization of the mutant protein. These changes were associated with an increase in the number and area of nucleoli and to a high increase in the expression of pP53 and pH2AX. All these changes were associated with premature senescence. The present observations add to our understanding of the differences between Hutchinson-Gilford progeria syndrome and WRS and opens new alternatives to study cell senesce and human aging.
Asunto(s)
Retardo del Crecimiento Fetal , Fibroblastos , Progeria , ARN Polimerasa III , Proteína p53 Supresora de Tumor/metabolismo , Nucléolo Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Daño del ADN , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/patología , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Expresión Génica , Humanos , Mutación , Progeria/genética , Progeria/patología , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN Ribosómico 5S/metabolismoRESUMEN
Dendritic arbors are crucial for nervous system assembly, but the intracellular mechanisms that govern their assembly remain incompletely understood. Here, we show that the dendrites of PVD neurons in Caenorhabditis elegans are patterned by distinct pathways downstream of the DMA-1 leucine-rich transmembrane (LRR-TM) receptor. DMA-1/LRR-TM interacts through a PDZ ligand motif with the guanine nucleotide exchange factor TIAM-1/GEF in a complex with act-4/Actin to pattern higher order 4° dendrite branches by localizing F-actin to the distal ends of developing dendrites. Surprisingly, TIAM-1/GEF appears to function independently of Rac1 guanine nucleotide exchange factor activity. A partially redundant pathway, dependent on HPO-30/Claudin, regulates formation of 2° and 3° branches, possibly by regulating membrane localization and trafficking of DMA-1/LRR-TM. Collectively, our experiments suggest that HPO-30/Claudin localizes the DMA-1/LRR-TM receptor on PVD dendrites, which in turn can control dendrite patterning by directly modulating F-actin dynamics through TIAM-1/GEF.
Asunto(s)
Actinas/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de la Membrana/genética , Plasticidad Neuronal/genética , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/genética , Actinas/metabolismo , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Claudinas/genética , Claudinas/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Sistema Nervioso/citología , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Neurogénesis/genética , Neuronas/citología , Neuronas/metabolismo , Dominios PDZ , Unión Proteica , Transporte de Proteínas , Transmisión Sináptica , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The mechanisms that pattern and maintain dendritic arbors are key to understanding the principles that govern nervous system assembly. The activity of presynaptic axons has long been known to shape dendrites, but activity-independent functions of axons in this process have remained elusive. Here, we show that in Caenorhabditis elegans, the axons of the ALA neuron control guidance and extension of the 1° dendrites of PVD somatosensory neurons independently of ALA activity. PVD 1° dendrites mimic ALA axon guidance defects in loss-of-function mutants for the extracellular matrix molecule MIG-6/Papilin or the UNC-6/Netrin pathway, suggesting that axon-dendrite adhesion is important for dendrite formation. We found that the SAX-7/L1CAM cell adhesion molecule engages in distinct molecular mechanisms to mediate extensions of PVD 1° dendrites and maintain the ALA-PVD axon-dendritic fascicle, respectively. Thus, axons can serve as critical scaffolds to pattern and maintain dendrites through contact-dependent but activity-independent mechanisms.
Asunto(s)
Axones/metabolismo , Moléculas de Adhesión Celular/metabolismo , Dendritas/metabolismo , Plasticidad Neuronal/fisiología , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismoRESUMEN
Channelopsins and photo-regulated ion channels make it possible to use light to control electrical activity of cells. This powerful approach has lead to a veritable explosion of applications, though it is limited to changing membrane voltage of the target cells. An enormous potential could be tapped if similar opto-genetic techniques could be extended to the control of chemical signaling pathways. Photopigments from invertebrate photoreceptors are an obvious choice-as they do not bleach upon illumination -however, their functional expression has been problematic. We exploited an unusual opsin, pScop2, recently identified in ciliary photoreceptors of scallop. Phylogenetically, it is closer to vertebrate opsins, and offers the advantage of being a bi-stable photopigment. We inserted its coding sequence and a fluorescent protein reporter into plasmid vectors and demonstrated heterologous expression in various mammalian cell lines. HEK 293 cells were selected as a heterologous system for functional analysis, because wild type cells displayed the largest currents in response to the G-protein activator, GTP-γ-S. A line of HEK cells stably transfected with pScop2 was generated; after reconstitution of the photopigment with retinal, light responses were obtained in some cells, albeit of modest amplitude. In native photoreceptors pScop2 couples to Go; HEK cells express poorly this G-protein, but have a prominent Gq/PLC pathway linked to internal Ca mobilization. To enhance pScop2 competence to tap into this pathway, we swapped its third intracellular loop-important to confer specificity of interaction between 7TMDRs and G-proteins-with that of a Gq-linked opsin which we cloned from microvillar photoreceptors present in the same retina. The chimeric construct was evaluated by a Ca fluorescence assay, and was shown to mediate a robust mobilization of internal calcium in response to illumination. The results project pScop2 as a potentially powerful optogenetic tool to control signaling pathways.