A stomatin and a degenerin interact in lipid rafts of the nervous system of Caenorhabditis elegans

In Caenorhabditis elegans, the gene unc-1 controls anesthetic sensitivity and normal locomotion. The protein UNC-1 is a close homolog of the mammalian protein stomatin and is expressed primarily in the nervous system. Genetic studies in C. elegans have shown that the UNC-1 protein interacts with a sodium channel subunit, UNC-8. In humans, absence of stomatin is associated with abnormal sodium and potassium levels in red blood cells. Stomatin also has been postulated to participate in the formation of lipid rafts, which are membrane microdomains associated with protein complexes, cholesterol, and sphingolipids. In this study, we isolated a low-density, detergent-resistant fraction from cell membranes of C. elegans. This fraction contains cholesterol, sphingolipids, and protein consistent with their identification as lipid rafts. We then probed Western blots of protein from the rafts and found that the UNC-1 protein is almost totally restricted to this fraction. The UNC-8 protein is also found in rafts and coimmunoprecipitates UNC-1. A second stomatin-like protein, UNC-24, also affects anesthetic sensitivity, is found in lipid rafts, and regulates UNC-1 distribution. Mutations in the unc-24 gene alter the distribution of UNC-1 in lipid rafts. Each of these mutations alters anesthetic sensitivity in C. elegans. Because lipid rafts contain many of the putative targets of volatile anesthetics, they may represent a novel class of targets for volatile anesthetics.     

The Caenorhabditis elegans Behavioral Gene unc-24 Encodes a Novel Bipartite Protein Similar to Both Erythrocyte Band 7.2 (Stomatin) and Nonspecific Lipid Transfer Protein

Abstract: We report here the positional cloning and molecular characterization of the unc-24 gene of Caenorhabditis elegans . This gene is required for normal locomotion and interacts with genes that affect the worm's response to volatile anesthetics. The predicted gene product contains a domain similar to part of two ion channel regulators (the erythrocyte integral membrane protein stomatin and the C. elegans neuronal protein MEC-2) juxtaposed to a domain similar to nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2). Sequence analysis suggests that the nsLTP-like domain of UNC-24 provides lipid carrier function and is tethered to the plasma membrane by the stomatin-like domain, which may be regulatory. We postulate that UNC-24 may be involved in lipid transfer between closely apposed membranes.     

The Caenorhabditis elegans Avermectin Resistance and Anesthetic Response Gene unc-9 Encodes a Member of a Protein Family Implicated in Electrical Coupling of Excitable Cells

Abstract: Mutations in the unc-9 gene of the nematode Caenorhabditis elegans cause abnormal forward locomotion and an egg-retention phenotype. unc-9 mutations also reduce the worms' sensitivity to avermectin and block a form of hypersensitivity to volatile anesthetics. We report here the cloning and molecular characterization of unc-9 and show that it encodes a member of the OPUS family of proteins that is 56% identical to another OPUS protein, UNC-7. It is significant that unc-9 mutants share all phenotypes with unc-7 mutants. Mutants in another gene, unc-124 , also share all tested phenotypes with unc-9 mutants, including identical locomotory and egg-laying defects, suggesting that multiple genes are required for the same biochemical function. OPUS proteins are implicated in the function of invertebrate gap junctions, and, based on a new alignment including 24 members from C. elegans , we present a refined model for the structure of OPUS proteins suggesting that oligomers could form a hydrophilic pore. We also show that alteration of highly conserved proline residues in UNC-9 leads to a cold sensitivity that likely affects a step in protein expression rather than function. Finally, we speculate on the basis of the avermectin resistance and anesthetic response phenotypes.     

unc-119 homolog required for normal development of the zebrafish nervous system

The UNC-119 proteins, found in all metazoans examined, are highly conserved at both the sequence and functional levels. In the invertebrates Caenorhabditis elegans and Drosophila melanogaster, unc-119 genes are expressed pan-neurally. Loss of function of the unc-119 gene in C. elegans results in a disorganized neural architecture and paralysis. The function of UNC-119 proteins has been conserved throughout evolution, as transgenic expression of the human UNC119 gene in C. elegans unc-119 mutants restores a wild-type phenotype. However, the nature of the conserved molecular function of UNC-119 proteins is poorly understood. Although unc-119 genes are expressed throughout the nervous system of the worm and fly, the analysis of these genes in vertebrates has focused on their function in the photoreceptor cells of the retina. Here we report the characterization of an unc-119 homolog in the zebrafish. The Unc119 protein is expressed in various neural tissues in the developing zebrafish embryo and larva. Morpholino oligonucleotide (MO)-mediated knockdown of Unc119 protein results in a "curly tail down" phenotype. Examination of neural patterning demonstrates that these "curly tail down" zebrafish experience a constellation of neuronal defects similar to those seen in C. elegans unc-119 mutants: missing or misplaced cell bodies, process defasciculation, axon pathfinding errors, and aberrant axonal branching. These findings suggest that UNC-119 proteins may play an important role in the development and/or function of the vertebrate nervous system.     

A stomatin and a degenerin interact to control anesthetic sensitivity in Caenorhabditis elegans

The mechanism of action of volatile anesthetics is unknown. In Caenorhabditis elegans, mutations in the gene unc-1 alter anesthetic sensitivity. The protein UNC-1 is a close homologue of the mammalian protein stomatin. Mammalian stomatin is thought to interact with an as-yet-unknown ion channel to control sodium flux. Using both reporter constructs and translational fusion constructs for UNC-1 and green fluorescent protein (GFP), we have shown that UNC-1 is expressed primarily within the nervous system. The expression pattern of UNC-1 is similar to that of UNC-8, a sodium channel homologue. We examined the interaction of multiple alleles of unc-1 and unc-8 with each other and with other genes affecting anesthetic sensitivity. The data indicate that the protein products of these genes interact, and that an UNC-1/UNC-8 complex is a possible anesthetic target. We propose that membrane-associated protein complexes may represent a general target for volatile anesthetics.     

The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans

UNC-51 and UNC-14 are required for the axon guidance of many neurons in Caenorhabditis elegans. UNC-51 is a serine/threonine kinase homologous to yeast Atg1, which is required for autophagy. The binding partner of UNC-51, UNC-14, contains a RUN domain that is predicted to play an important role in multiple Ras-like GTPase signaling pathways. How these molecules function in axon guidance is largely unknown. Here we observed that, in unc-51 and unc-14 mutants, UNC-5, the receptor for axon-guidance protein Netrin/UNC-6, abnormally localized in neuronal cell bodies. By contrast, the localization of many other proteins required for axon guidance was undisturbed. Moreover, UNC-5 localization was normal in animals with mutations in the genes for axon guidance proteins, several motor proteins, vesicle components and autophagy-related proteins. We also found that unc-5 and unc-6 interacted genetically with unc-51 and unc-14 to affect axon guidance, and that UNC-5 co-localized with UNC-51 and UNC-14 in neurons. These results suggest that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5, and that UNC-5 uses a unique mechanism for its localization; the functionality of UNC-5 is probably regulated by this localization.     

MEC-2 is recruited to the putative mechanosensory complex in C. elegans touch receptor neurons through its stomatin-like domain

BACKGROUND: The response to gentle body touch in C. elegans requires a degenerin channel complex containing four proteins (MEC-2, MEC-4, MEC-6, and MEC-10). The central portion of the integral membrane protein MEC-2 contains a stomatin-like region that is highly conserved from bacteria to mammals. The molecular function of this domain in MEC-2, however, is unknown. RESULTS: Here, we show that MEC-2 colocalizes with the degenerin MEC-4 in regular puncta along touch receptor neuron processes. This punctate localization requires the other channel complex proteins. The stomatin-like region of MEC-2 interacts with the intracellular cytoplasmic portion of MEC-4. Missense mutations in this region that destroy the interaction also disrupt the punctate localization and degenerin-regulating function of MEC-2. Missense mutations outside this region apparently have no effect on the punctate localization but significantly reduce the regulatory effect of MEC-2 on the MEC-4 degenerin channel. A second stomatin-like protein, UNC-24, colocalizes with MEC-2 in vivo and coimmunoprecipitates with MEC-2 and MEC-4 in Xenopus oocytes; unc-24 enhances the touch insensitivity of temperature-sensitive alleles of mec-4 and mec-6. CONCLUSION: Two stomatin homologs, MEC-2 and UNC-24, interact with the MEC-4 degenerin through their stomatin-like regions, which act as protein binding domains. At least in the case of MEC-2, this binding allows its nonstomatin domains to regulate channel activity. Stomatin-like regions in other proteins may serve a similar protein binding function.     

Two neuronal,nuclear-localized RNA binding proteins involved in synaptic transmission

While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using Caenorhabditis elegans as a model for studying neuron/target communication, we report that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. We show that unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. We found that human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, we report that the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, we identified a new neuronal, putative pre-mRNA splicing factor, UNC-75, and show that UNC-75, as well as the C. elegans homolog of ELAV, is required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function.     

MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion

The netrin UNC-6 repels motor axons by activating the UNC-5 receptor alone or in combination with the UNC-40/DCC receptor. In a genetic screen for C. elegans mutants exhibiting partial defects in motor axon projections, we isolated the max-1 gene (required for motor neuron axon guidance). max-1 loss-of-function mutations cause fully penetrant but variable axon guidance defects. Mutations in unc-5 and unc-6, but not in unc-40, dominantly enhance the mutant phenotypes of max-1, whereas overexpression of unc-5 or unc-6, but not of unc-40, bypasses the requirement for max-1. MAX-1 proteins contain PH, MyTH4, and FERM domains and appear to be localized to neuronal processes. Human MAX-1 and UNC5H2 colocalize in discrete subcellular regions of transfected cells. Our results suggest a possible role for MAX-1 in netrin-induced axon repulsion by modulating the UNC-5 receptor signaling pathway.     

Microtubule-based localization of a synaptic calcium-signaling complex is required for left-right neuronal asymmetry in C. elegans

The axons of C. elegans left and right AWC olfactory neurons communicate at synapses through a calcium-signaling complex to regulate stochastic asymmetric cell identities called AWC(ON) and AWC(OFF). However, it is not known how the calcium-signaling complex, which consists of UNC-43/CaMKII, TIR-1/SARM adaptor protein and NSY-1/ASK1 MAPKKK, is localized to postsynaptic sites in the AWC axons for this lateral interaction. Here, we show that microtubule-based localization of the TIR-1 signaling complex to the synapses regulates AWC asymmetry. Similar to unc-43, tir-1 and nsy-1 loss-of-function mutants, specific disruption of microtubules in AWC by nocodazole generates two AWC(ON) neurons. Reduced localization of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons strongly correlates with the 2AWC(ON) phenotype in nocodazole-treated animals. We identified kinesin motor unc-104/kif1a mutants for enhancement of the 2AWC(ON) phenotype of a hypomorphic tir-1 mutant. Mutations in unc-104, like microtubule depolymerization, lead to a reduced level of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons. In addition, dynamic transport of TIR-1 in the AWC axons is dependent on unc-104, the primary motor required for the transport of presynaptic vesicles. Furthermore, unc-104 acts non-cell autonomously in the AWC(ON) neuron to regulate the AWC(OFF) identity. Together, these results suggest a model in which UNC-104 may transport some unknown presynaptic factor(s) in the future AWC(ON) cell that non-cell autonomously control the trafficking of the TIR-1 signaling complex to postsynaptic regions of the AWC axons to regulate the AWC(OFF) identity.     

Regulatory machinery of UNC-33 Ce-CRMP localization in neurites during neuronal development in Caenorhabditis elegans

In Caenorhabditis elegans, unc-33 encodes an orthologue of the vertebrate collapsin response mediator protein (CRMP) family. We previously reported that CRMP-2 accumulated in the distal part of the growing axon of vertebrate neurons and played critical roles in axon elongation. unc-33 mutants show axonal outgrowth defects in several neurons. It has been reported that UNC-33 accumulates in neurites, whereas a missense mutation causes the mislocalization of UNC-33 from neurites to cell body, which suggests that the localization of UNC-33 in neurites is important for axonal outgrowth. However, it is unclear how UNC-33 accumulates in neurites and regulates neuronal development. In this study, to understand the regulatory mechanisms of localization of UNC-33 in neurites, we screened for the mutants that were involved in the localization of UNC-33, and identified three mutants: unc-14 (RUN domain protein), unc-51 (ULK kinase) and unc-116 (kinesin heavy chain). UNC-14 is known to associate with UNC-51. UNC-116 forms a complex with KLC-2 as Kinesin-1, a microtubule-dependent motor complex. We found that UNC-33 interacted with UNC-14 and KLC-2 in vivo. These results suggest that the UNC-14/UNC-51 complex and Kinesin-1 are involved in the localization of UNC-33 in neurites.     

Gap junctions synchronize action potentials and Ca2+ transients in Caenorhabditis elegans body wall muscle

The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients.     

Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescein dyes

Eight pairs of chemosensory neurons in Caenorhabditis elegans take up fluorescein dyes entering through the chemosensory organs. These are amphid neurons ADF, ASH, ASI, ASJ, ASK, and ADL and phasmid neurons PHA and PHB. When filled with dye, the processes and cell bodies of these neurons can be examined in live animals by fluorescence microscopy. Using this technique, we have identified five genes, unc-33, unc-44, unc-51, unc-76, and unc-106, that affect the growth of the amphid and phasmid axons. These genes were found to affect the axons of the mechanosensory PDE neurons as well. The unc-33 mutation specifically affects neuronal microtubules. Sensory dendrites in this mutant have a superabundance of microtubules. Moreover, many of these microtubules are abnormal in diameter, and some form hooks or multiple tubules.     

UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Caenorhabditis elegans

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ～50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the μ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as μ2 adaptin.     

FEZ2 has acquired additional protein interaction partners relative to FEZ1: functional and evolutionary implications

Background

The FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function.

Methodology/Principal Findings

Using the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom.

Conclusions/Significance

We found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins.