Laura F. Ficociello et al. (2007) International Worm Meeting "Neuronal Migration: Investigating MIG-10 interaction with Ras GTPases in C. elegans."

Laura F. Ficociello, Elizabeth F. Ryder

[International Worm Meeting, 2007]

The gene mig-10 is necessary for the long range anteroposterior migration during embryogenesis of the neurons CAN, ALM, and HSN. Mutations in mig-10 result in incomplete migrations of all three neurons. Since ALM and CAN migrate posteriorly and HSN migrates anteriorly, mig-10 mutations are believed to affect the basic mechanism of migration rather than affecting positional information. Mutations in mig-10 also affect axon guidance of several neurons, including the CAN, HSN, AVM, PVM, and ADL neurons, and overexpression of MIG-10 promotes axon outgrowth in vivo. Like its vertebrate homologs lamellipodin and RIAM, MIG-10 binds to an Ena/VASP protein (UNC-34); Ena/VASP proteins are involved in regulating actin dynamics. Overexpression of MIG-10 or its vertebrate homologs in cultured cells causes lamellipodia formation. Several lines of evidence suggest that MIG-10 might interact with members of the Ras GTPase family. MIG-10 contains a Ras-association (RA) domain as well as a pleckstrin homology (PH) domain. A similar RAPH domain in RIAM is known to interact with Rap1, a member of the Ras GTPase family. In addition, double mutants of mig-10 with either of two C. elegans rac genes, mig-2 or ced-10, show an enhanced migration defect, suggesting an interaction between mig-10 and the rac genes. We are testing the hypothesis that MIG-10 interacts with one or more of the members of the Ras GTPase family to transduce environmental signals, ultimately resulting in oriented neuronal migration or axon outgrowth. We are currently using a yeast two-hybrid system to investigate potential interactions between Ras-family proteins and MIG-10. Our initial results suggest that the MIG-10 RAPH domain on its own does not interact with any of the Ras family members tested. We plan to test the full length MIG-10 protein for interactions with the Ras family, as well as screening the entire C. elegans cDNA library for other potential MIG-10 interactors.

Yang, Huan et al. (2009) International Worm Meeting "Genetic characterization of rde-10 in RNAi and transgene silencing pathways."

Mak, Ho Yi, Yang, Huan

[International Worm Meeting, 2009]

We identified rde-10, a new gene that is required for RNAi and transgene silencing. In search for transgenes that allow monitoring of intracellular lipid droplets, we unexpectedly found that a vha-6p::atgl-1::gfp transgene was reproducibly silenced in every generation from L2. We screened for mutants that abolish such silencing and isolated more than 10 alleles. Among these, one is a new allele of rde-1. Genetic complementation and molecular cloning indicated that we also recovered three alleles that defined a new gene rde-10. RDE-10 appears to have no obvious homology to proteins outside the Caenorhabditis genus. Nevertheless, RDE-10 is strictly required for RNAi in multiple tissues. And similar to rde-1, rde-10 acts antagonistically to eri-1 and eri-3 for transgene silencing. To further explore the molecular function of RDE-10 and its interaction with other small RNA pathway components, we are conducting proteomic analysis on RDE-10 associated proteins.

Johanna Arnadottir et al. (2005) International Worm Meeting "Role of MEC-10 in the MEC-4 channel complex"

Robert O'Hagan, Martin Chalfie, Miriam B Goodman, Johanna Arnadottir

[International Worm Meeting, 2005]

Sensitivity to gentle body touch is mediated by the MEC-4 channel complex, which contains two pore-forming components, the DEG/ENaC proteins MEC-4 and MEC-10. MEC-4 is essential for the function of the complex, but the role of MEC-10 has been difficult to interpret. The mec-10(u20) missense allele changes the ion selectivity of the mechanoreceptor current (MRC), suggesting that MEC-10 is part of the complex in vivo. Yet, unlike MEC-4, MEC-10 does not form a functional channel alone or with MEC-2 and MEC-6 in Xenopus oocytes. MEC-10, however, alters the amiloride sensitivity and reduces the total current of the MEC-4d, MEC-2, MEC-6 channel complex in oocytes. This last property suggests that wild-type MEC-10 may negatively regulate channel function. Although mec-4 and mec-10 encode similar proteins, initial screens for touch insensitive mutants yielded approximately ten times more mec-4 mutations than mec-10 mutations. Subsequent sequence analysis revealed that the mec-4 mutations included nonsense and missense mutations and deletions. The five characterized mec-10 alleles are all recessive and contain only missense mutations, four of which are clustered around the putative pore region of the channel. These results suggest that the existing mec-10 alleles may not be loss-of-function mutations. A mutagenesis for non-complementing alleles of mec-10(u20) was inconclusive, yielding only a few alleles that produced a Mec phenotype. A deletion mutation [mec-10(ok1104)] produced by the Knockout Consortium results in animals that are weakly touch insensitive (responding at least half the time when touched). MRCs in these animals are qualitatively similar to those observed in wild-type animals, suggesting that, despite contributing to the pore of the mechanotransduction channel, MEC-10 is not an essential component. We have investigated the effects of mutant MEC-10 proteins, corresponding to the characterized mec-10 mutants, on the MEC-4d and MEC-2 current in Xenopus oocytes. As before, we find that wild-type MEC-10 reduces the amiloride-sensitive current produced by MEC-4d and MEC-2. The current is further reduced by one of the MEC-10 mutant proteins and virtually abolished by the other four. This reduction is not explained by a decrease in the surface expression of MEC-4d, since surface expression of myc-tagged MEC-4d is unchanged in the presence of MEC-10. In addition, GFP-tagged wild-type and mutant alleles of MEC-10 all co-immunoprecipitate equivalent amounts of myc-tagged MEC-4d. These results support the hypothesis that the Mec alleles of mec-10 are recessive gain-of-function mutations and are consistent with a role for MEC-10 as a negative regulator of the MEC-4 channel complex.

Rieko Imae et al. (2007) International Worm Meeting "Functional analysis of acl-10, a novel acyltransferase in C. elegans."

Takao Inoue, Naoko Hara, Hiroyuki Arai, Rieko Imae, Keiko Gengyo-Ando, Shohei MItani

[International Worm Meeting, 2007]

Alignment of amino acid sequences from phospholipids acyltransferases, such as glycerol-3-phosphate acyltransferase, acl-glycerol-3-phosphate acyltransferase and dihydroxyacetonephosphate acyltransferase, reveals four regions of strong homology that comprise a glycerolipid acyltransferase signature sequence. There are more than 10 genes containing the acyltransferase signature sequence in human genome, of which physiological functions and substrate specificities are largely unknown. We identified 14 genes containing the signature sequence from C. elegans genome sequences (acl-1~14), and generated mutants lacking these genes. Among the genes, acl-10 shows ~40% identity with the corresponding mammalian gene, but until recently, its functions have not been elucidated. In this study, we analyzed the physiological function of acl-10 using the mutants lacking this gene. The acl-10 deletion mutant, acl-10 (tm1045), exhibited dumpy morphology and severely uncoordinated movement at adult stage. In addition, acl-10 (tm1045) displayed hypersensitivity to the acetylcholine receptor agonist levamisole, suggesting that the signaling pathway leading to muscle contraction is activated in the mutants. These defects were fully rescued by acl-10 expression at L4 stage, indicating that acl-10 is required for muscle function rater than muscle formation. To further elucidate how acl-10 controls muscle contraction, we performed rescue experiments using tissue-specific promoters (unc-119p, myo-3p, dpy-7p). Interestingly, all these constructs could rescue the movement defects of acl-10 (tm1045). The hypersensitivity to levamisole was also rescued by neuronal or epithelial expression of acl-10, indicating that acl-10 functions in a cell nonautonomous manner. We are currently investigating the mechanism of acl-10 function in muscle contraction.

El Bejjani, Rachid et al. (2013) International Worm Meeting "Genes that function downstream of Notch define a novel mechanism for inhibiting axon regeneration."

Hammarlund, Marc, El Bejjani, Rachid

[International Worm Meeting, 2013]

We have previously shown that Notch/lin-12 signaling functions cell autonomously in motor neurons to inhibit axonal regeneration. We now show that the conserved neuronal scaffolding protein Mint/lin-10 is a likely target of anti-regeneration Notch signaling. Like Notch, Mint/lin-10 functions cell-autonomously to inhibit axonal regeneration in GABA motor neurons. Further, Mint/lin-10 is necessary for Notch to inhibit regeneration: lin-10 null mutations fully suppress the decreased regeneration caused by lin-12/Notch gain of function alleles or by overexpression of the Notch Intracellular Domain (NICD). Finally, lin-10 contains LAG-1 binding consensus elements in its 5' UTR. Together, these data suggest that LIN-10 is a transcriptional target of Notch signaling during axonal regeneration. LIN-10 may inhibit regeneration by regulating trafficking of other signaling molecules. We find that in axotomized GABA neurons, LIN-10 GFP localizes to the proximal stump of the axotomized neurons (as well as at synapses and the Golgi). LIN-10-dependent inhibition of axonal regeneration requires retrograde transport, as inhibition of retrograde transport in a rab-6.2 null significantly increases regeneration and suppresses the effect of LIN-10 overexpression. Further, the increase in regeneration in rab-6.2 mutants is fully suppressed by mutations that affect clathrin-mediated endocytosis. We propose that LIN-10 and retrograde trafficking function in damaged axons to maintain membrane localization of anti-regeneration factors. We are currently testing candidate binding partners of MINT/LIN-10 to identify these unknown factors.

Hart AC et al. (2000) East Coast Worm Meeting "Genetic characterization of osmosensation: structure-function analysis of OSM-10"

Sugimoto H, Hart AC

[East Coast Worm Meeting, 2000]

Osmosensation has been molecularly characterized in unicellular organisms but is poorly understood in multicellular organisms. When the nematode C. elegans encounters high osmolarity, it reverses direction to avoid the stimulus. Laser ablation studies demonstrated that the ASH sensory neurons are primarily responsible for the detection of high osmolarity, as well as nose touch and volatile repellents (1-octanol). osm-10 is specifically required for osmosensation as osm-10(nr2076), a null allele disrupts osmosensation but not mechanosensation or chemosensation. osm-10 encodes a novel protein of 419 amino acids which is expressed by the ASH, ASI, PHA and PHB sensory neurons. The OSM-10 protein contains 38 putative serine and threonine phosphorylation sites. osm-10(n1602), a recessive allele which changes an E to a K codon in a putative tyrosine phosphorylation site is completely defective for osmosensation. This suggests that phosphorylation of OSM-10 may be critical for signal transduction. The OSM-10 protein localizes to the cytoplasm, supporting this hypothesis. osm-10(n1602) protein expression is normal by western blot and immunohistochemistry. We are currently investigating the molecular function of OSM-10. First, the potential role of tyrosine phosphorylation in OSM-10 function will be addressed. In order to determine the role of tyrosine in OSM-10 function, tyrosine 199 (Y199) will be converted to a phenylalanine (Y202F) in the osm-10 rescue construct. In addition, a construct will be generated which replicates the n1602 mutation (E199K), perturbing the consensus tyrosine phosphorylation site. Both the Y202F and E199K encoding constructs should disrupt osmosensation if phosphorylation is important. We will also create a series of constructs encoding truncated OSM-10 proteins to identify the domains important for the function of OSM-10. Some of the mutant constructs may have a dominant negative effect on osmosensation. By studying the function of OSM-10, we hope to elucidate the molecular mechanism of osmosensation in C. elegans, which may shed a light into the molecular mechanism underlying osmosensation in multicellular organisms in general.

Wexler, Emily R. et al. (2015) International Worm Meeting "TGFß mediates the feeding state-dependent expression of the food chemoreceptor ODR-10 in males."

Portman, Douglas S., Wexler, Emily R., Ryan, Deborah A.

[International Worm Meeting, 2015]

The biological mechanisms by which neural circuits are modulated by an animal's sex ("gender") is poorly understood. We have previously found that C. elegans males and hermaphrodites differ in their response to several odorants detected by shared circuitry. In particular, hermaphrodites are more attracted than males to the odorant diacetyl, a food cue sensed by the chemoreceptor odr-10, expressed in the AWA sensory neuron. Recently, we have shown that regulated odr-10 expression underlies sex differences in diacetyl attraction. In adult males, odr-10 expression is significantly reduced compared to hermaphrodites, allowing these animals to prioritize exploratory behavior over feeding. Moreover, transient starvation upregulates odr-10 in males to promote feeding. Through a candidate screen, we identified the TGFbeta ligand daf-7 as a regulator of odr-10 expression. Males with mutations in daf-7 or the downstream SMAD daf-8 have increased expression of odr-10. In contrast, these mutations have no apparent effect on odr-10 expression in hermaphrodites. Animals with mutations in daf-5, a transcription factor inhibited by DAF-7 signaling, show decreased expression of odr-10; moreover, daf-5 males show no increase in odr-10 expression in response to starvation. These findings suggest that DAF-7 regulates feeding-state-mediated changes in odr-10 expression in a sex-dependent manner. We are currently working to understand how the sex determination pathway intersects with DAF-7 signaling to bring about state-dependent changes in odr-10 expression and behavior.

Frand, Alison et al. (2009) International Worm Meeting "Dysfunction of the novel MLT-10 family of repetitive, proline-rich secretory proteins blocks the molting cycle."

Ruvkun, Gary, Frand, Alison

[International Worm Meeting, 2009]

The molting cycle involves complete remodeling of collagen-rich extracellular matrices, but the signaling and enzymatic cascades that trigger and execute this remodeling are not well understood. Here, we describe the mlt-10 gene isolated in a forward genetic screen for mutants unable to molt. MLT-10 is the first reported member of a large family of annotated glycoproteins characterized by a domain of unknown function (#644) and a repetitive C-terminal region rich in serine/threonine and proline. The substitution mutation mlt-10(mg364), which disrupts the repetitive region, or increased expression of mlt-10(+) renders larvae unable to shed old cuticles. The development of mlt-10(mg364) larvae also requires a continuous supply of cholesterol, suggesting a reduced capacity to utilize sterols. Loss of mlt-10 has no obvious impact on development, possibly due to functional redundancy among the thirteen C. elegans homologs. Nevertheless, the conserved nuclear hormone receptors NHR-23 and NHR-25 promote expression of mlt-10 in the hypodermis whenever the exoskeleton is remodeled, showing that mlt-10 naturally relates to the molting cycle. Further, exposure of glycans on the surface of molting larvae corresponds with expression of mlt-10. We propose that MLT-10 is one of a suite of cuticular glycoproteins whose assembly and disassembly are highly regulated during the molting cycle.

Doreen Glodowski et al. (2008) C.elegans Neuronal Development Meeting "RAB-10 Regulates Glutamate Receptor Recycling in a Cholesterol-dependent Endocytosis Pathway"

Christopher Rongo, Carlos Chih-Hsiung Chen, Doreen Glodowski, Barth Grant, Henry Schaefer

[C.elegans Neuronal Development Meeting, 2008]

Regulated endocytosis of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, the specific combination of clathrin-dependent and independent mechanisms that mediate AMPAR trafficking in vivo have not been fully characterized. Here, we examine the trafficking of the AMPAR subunit GLR-1 in Caenorhabditis elegans. GLR-1 is localized on synaptic membranes, where it regulates reversals of locomotion in a simple behavioral circuit. Animals lacking RAB-10, a small GTPase required for endocytic recycling of intestinal cargo, are similar in phenotype to animals lacking LIN-10, a postsynaptic density 95/disc-large/zona occludensdomain containing protein: GLR-1 accumulates in large accretions and animals display a decreased frequency of reversals. Mutations in unc-11 (AP180) or itsn-1 (Intersectin 1), which reduce clathrin-dependent endocytosis, suppress the lin-10 but not rab-10 mutant phenotype, suggesting that LIN-10 functions after clathrin-mediated endocytosis. By contrast, cholesterol depletion, which impairs lipid raft formation and clathrin-independent endocytosis, suppresses the rab-10 but not the lin-10 phenotype, suggesting that RAB-10 functions after clathrin-independent endocytosis. Animals lacking both genes display additive GLR-1 trafficking defects. We propose that RAB-10 and LIN-10 recycle AMPARs from intracellular endosomal compartments to synapses along distinct pathways, each with distinct sensitivities to cholesterol and the clathrin-mediated endocytosis machinery.

Hala Zahreddine et al. (2006) Development & Evolution Meeting "VAB-10 Links Cell-ECM Junctions To The Actin Remodeling LET-502/MEL-11 Pathway And May Act In Additional Pathways During C. elegans Morphogenesis"

Marie Diogon, Michel Labouesse, Hala Zahreddine, Frederic Landmann, Yasuko NAGAMATSU, Jerome Reboul

[Development & Evolution Meeting, 2006]

During C. elegans embryonic elongation, remodeling of the epidermal cytoskeleton transforms the ellipsoidal embryo into a long thin larva. We previously showed that mutations in the spectraplakin locus vab-10 strongly affect elongation, due to a failure to maintain epidermis attachment to the ECM and to muscles. vab-10, like other spectraplakin loci (shot, MACF and BPAG1), encodes multiple isoforms, with actin- microtubule- and/or intermediate filament-binding domains, which are major components of fibrous organelles (cell-ECM junctions). Given its modular structure and subcellular localization, VAB-10 might link junctions to cytoskeleton remodeling proteins. To identify genes acting with vab-10, we performed genetic enhancer screens using the fully viable vab-10 allele e698, and yeast two-hybrid screens with different VAB-10 regions. First, using mutations in known players of the embryonic elongation pathway, in particular the Rho-kinase (LET-502) and myosin phosphatase (MEL-11), we found that vab-10(e698) enhances the mel-11 mutant phenotype. Strikingly, the yeast two-hybrid screen revealed that the VAB-10 plakin domain interacts with the MEL-11 C-terminus. We next found that VAB-10 is necessary to localize MEL-11 to fibrous organelles. We speculate that VAB-10 may recruit MEL-11 to regulate its activity at cell-ECM junctions. Second, using a genome-wide feeding RNAi screen, looking for candidates that induce lethality in vab-10(e698) mutants but not in N2 controls, we identified 15 candidates encoding different classes of proteins (DNA or RNA binding proteins, cytoskeletal proteins, ubiquitin-conjugating enzymes). RNAi against these genes in vab-10(e698) mutant resembles vab-10 severe phenotypes with elongation or body morphology defects, and muscle detachment. Two genes identified in this screen, unc-52 and vab-19, act in the same pathway as vab-10, thus validating the screening strategy. We could confirm the genetic interaction with bona fide mutations for four new genes, and are currently analyzing at which step and where in the cell the two most interesting candidates act in the elongation process.