The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor.
Ontology highlight
ABSTRACT:
SUBMITTER: Ahmed S
PROVIDER: S-EPMC1133105 | biostudies-other | 1992 Nov
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode Caenorhabditis elegans. Molecular cloning of the gene and sequencing of the cDNA revealed that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C. The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium; diacylglycerol inhibited the binding in a competitive manner. These findings confirm that the unc-13 gene product has binding sites similar to those of protein kinase C and may be a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system.
| S-EPMC51951 | biostudies-other
Project description:Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.
| S-EPMC5588468 | biostudies-literature
Project description:The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products.
| S-EPMC15005 | biostudies-literature
Project description:To investigate the role of environmental factor-temperature in the regulation of aging process by unc-13 and sbt-1 in Caenorhabditis elegans.The lifespan, the speed of pharynx pumping, and the intestinal autofluorescence of unc-13 and sbt-1 mutants were examined at different temperature conditions. In addition, to exclude the possible influences from other factors in unc-13 and sbt-1 mutants, the dauer formation, the thermotaxis, the brood size and the population percentage of the mutants expressing hsp16.2-gfp were further investigated.Mutations of unc-13 and sbt-1 significantly increased the mean and the maximum lifespans of nematodes cultured at 20 degrees C and 25 degrees C, while no noticeable increase was found at 15 degrees C in either the mean or the maximum lifespan. Investigations on the speed of pharynx pumping and the intestinal autofluorescence suggested that at 20 degrees C and 25 degrees C, mutations of unc-13 and sbt-1 could slow the aging process and delay the accumulation of aging-related cellular damage. Meanwhile, mutations of unc-13 or sbt-1 did not affect the dauer formation or the thermotaxis to different temperatures in nematodes. In contrast, at 20 degrees C and 25 degrees C conditions, mutations of unc-13 and sbt-1 significantly decreased the brood size and the percentage of nematodes expressing hsp16.2-gfp, while no such differences were detected at 15 degrees C. Moreover, the thermotolerance of unc-13 and sbt-1 mutants could be greatly strengthened after the 16-h heat shock at 35 degrees C.The regulation of aging by unc-13 and sbt-1 is temperature-dependent. And the alterations in reproduction capability and stress response may be associated with the formation of this temperature-dependent property.
| S-EPMC5552505 | biostudies-other
Project description:Acute ethanol exposure affects the nervous system as a stimulant at low concentrations and as a depressant at higher concentrations, eventually resulting in motor dysfunction and uncoordination. A recent genetic study of two mouse strains with varying ethanol preference indicated a correlation with a polymorphism (D216N) in the synaptic protein Munc18-1. Munc18-1 functions in exocytosis via a number of discrete interactions with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin-1. We report that the mutation affects binding to syntaxin but not through either a closed conformation mode of interaction or through binding to the syntaxin N terminus. The D216N mutant instead has a specific impairment in binding the assembled SNARE complex. Furthermore, the mutation broadens the duration of single exocytotic events. Expression of the orthologous mutation (D214N) in the Caenorhabditis elegans UNC-18 null background generated transgenic rescues with phenotypically similar locomotion to worms rescued with the wild-type protein. Strikingly, D214N worms were strongly resistant to both stimulatory and sedative effects of acute ethanol. Analysis of an alternative Munc18-1 mutation (I133V) supported the link between reduced SNARE complex binding and ethanol resistance. We conclude that ethanol acts, at least partially, at the level of vesicle fusion and that its acute effects are ameliorated by point mutations in UNC-18.
| S-EPMC2613081 | biostudies-literature
Project description:unc-94 is one of about 40 genes in Caenorhabditis elegans that, when mutant, displays an abnormal muscle phenotype. Two mutant alleles of unc-94, su177 and sf20, show reduced motility and brood size and disorganization of muscle structure. In unc-94 mutants, immunofluorescence microscopy shows that a number of known sarcomeric proteins are abnormal, but the most dramatic effect is in the localization of F-actin, with some abnormally accumulated near muscle cell-to-cell boundaries. Electron microscopy shows that unc-94(sf20) mutants have large accumulations of thin filaments near the boundaries of adjacent muscle cells. Multiple lines of evidence prove that unc-94 encodes a tropomodulin, a conserved protein known from other systems to bind to both actin and tropomyosin at the pointed ends of actin thin filaments. su177 is a splice site mutation in intron 1, which is specific to one of the two unc-94 isoforms, isoform a; sf20 has a stop codon in exon 5, which is shared by both isoform a and isoform b. The use of promoter-green fluorescent protein constructs in transgenic animals revealed that unc-94a is expressed in body wall, vulval and uterine muscles, whereas unc-94b is expressed in pharyngeal, anal depressor, vulval and uterine muscles and in spermatheca and intestinal epithelial cells. By Western blot, anti-UNC-94 antibodies detect polypeptides of expected size from wild type, wild-type-sized proteins of reduced abundance from unc-94(su177), and no detectable unc-94 products from unc-94(sf20). Using these same antibodies, UNC-94 localizes as two closely spaced parallel lines flanking the M-lines, consistent with localization to the pointed ends of thin filaments. In addition, UNC-94 is localized near muscle cell-to-cell boundaries.
| S-EPMC2175264 | biostudies-literature
Project description:After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking.
| S-EPMC2441656 | biostudies-literature
Project description:BackgroundThe conserved Caenorhabditis elegans proteins NID-1/nidogen and PTP-3A/LAR-RPTP function to efficiently localize the presynaptic scaffold protein SYD-2/?-liprin at active zones. Loss of function in these molecules results in defects in the size, morphology and spacing of neuromuscular junctions.ResultsHere we show that the Cav2-like voltage-gated calcium channel (VGCC) proteins, UNC-2 and UNC-36, and the calmodulin kinase II (CaMKII), UNC-43, function to regulate the size and morphology of presynaptic domains in C. elegans. Loss of function in unc-2, unc-36 or unc-43 resulted in slightly larger GABAergic neuromuscular junctions (NMJs), but could suppress the synaptic morphology defects found in nid-1/nidogen or ptp-3/LAR mutants. A gain-of-function mutation in unc-43 caused defects similar to those found in nid-1 mutants. Mutations in egl-19, Cav1-like, or cca-1, Cav3-like, ?1 subunits, or the second ?2/? subunit, tag-180, did not suppress nid-1, suggesting a specific interaction between unc-2 and the synaptic extracellular matrix (ECM) component nidogen. Using a synaptic vesicle marker in time-lapse microscopy studies, we observed GABAergic motor neurons adding NMJ-like structures during late larval development. The synaptic bouton addition appeared to form in at least two ways: (1) de novo formation, where a cluster of vesicles appeared to coalesce, or (2) when a single punctum became enlarged and then divided to form two discrete fluorescent puncta. In comparison to wild type animals, we found unc-2 mutants exhibited reduced NMJ dynamics, with fewer observed divisions during a similar stage of development.ConclusionsWe identified UNC-2/UNC-36 VGCCs and UNC-43/CaMKII as regulators of C. elegans synaptogenesis. UNC-2 has a modest role in synapse formation, but a broader role in regulating dynamic changes in the size and morphology of synapses that occur during organismal development. During the late 4th larval stage (L4), wild type animals exhibit synaptic morphologies that are similar to those found in animals lacking NID-1/PTP-3 adhesion, as well as those with constitutive activation of UNC-43. Genetic evidence indicates that the VGCCs and the NID-1/PTP-3 adhesion complex provide opposing functions in synaptic development, suggesting that modulation of synaptic adhesion may underlie synapse development in C. elegans.
| S-EPMC3661369 | biostudies-literature
Project description:Axons navigating through the developing nervous system are instructed by external attractive and repulsive cues. Emerging evidence suggests the same cues control dendrite development, but it is not understood how they differentially instruct axons and dendrites. We studied a C. elegans motor neuron whose axon and dendrite adopt different trajectories and lengths. We found that the guidance cue UNC-6 (Netrin) is required for both axon and dendrite development. Its repulsive receptor UNC-5 repelled the axon from the ventral cell body, whereas the attractive receptor UNC-40 (DCC) was dendritically enriched and promotes antero-posterior dendritic growth. Although the endogenous ventrally secreted UNC-6 instructs axon guidance, dorsal or even membrane-tethered UNC-6 could support dendrite development. Unexpectedly, the serine-threonine kinase PAR-4 (LKB1) was selectively required for the activity of the UNC-40 pathway in dendrite outgrowth. These data suggest that axon and dendrite of one neuron interpret common environmental cues with different receptors and downstream signaling pathways.
| S-EPMC3908881 | biostudies-literature
Project description:The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand β6. It was also relatively less flexible with only five residues showing motions on a nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.
| S-EPMC4325984 | biostudies-literature