McGehee, Annette et al. (2013) International Worm Meeting "The DAF-7/TGF-b signaling pathway regulates abundance of the glutamate receptor GLR-1."

Moss, Benjamin, Juo, Peter, McGehee, Annette

[International Worm Meeting, 2013]

We study the regulation of the C. elegans glutamate receptor (GluR) GLR-1 using quantitative microscopy to analyze the localization and abundance of GFP-tagged GLR-1 (GLR-1::GFP) in various mutant backgrounds to identify genes involved in the regulation of GLR-1. In C. elegans the DAF-7/TGF-b signaling pathway senses environmental conditions and regulates entry into dauer. Here we show that multiple mutants in the DAF-7/TGF-b signaling pathway have increased abundance of GLR-1 at synapses in the ventral nerve cord (VNC). We found that GLR-1::GFP puncta fluorescence increased significantly in the VNC of multiple DAF-7/TGF-b pathway mutants as compared to wild type animals. Immunoblot analysis indicates that total levels of GLR-1::GFP are increased in DAF-7/TGF-b pathway mutants. Additionally, the rate of spontaneous reversals (a GLR-1-dependent behavior) is increased in both daf-7 and daf-8 mutants, suggesting that endogenous GLR-1 signaling is increased in these mutants. Together, these results suggest that the DAF-7/TGF-b pathway is required to regulate the proper abundance of GLR-1 in the VNC. The increase in GLR-1 observed in DAF-7/TGF-b pathway mutants could result from either increased synthesis or decreased degradation of GLR-1. Preliminary pulse-chase imaging experiments using the photoconvertible fluorescent protein Dendra fused to GLR-1 (GLR-1::Dendra) indicate that the rate of GLR-1 degradation in the VNC is unaltered in DAF-7/TGF-b pathway mutants. Thus, we hypothesize that GLR-1 synthesis is increased in DAF-7/TGF-b pathway mutants. Consistent with this idea, we found that daf-7 mutants exhibit increased levels of GFP driven by a glr-1 transcriptional reporter (Pglr-1::NLS-GFP). Taken together, these results suggest that the DAF-7/TGF-b signalling pathway negatively regulates the abundance of GLR-1 by controlling transcription of the receptor. These results identify a novel function for the DAF-7/TGF-b pathway in regulating GluRs, and provide an interesting potential connection between the sensing of environmental conditions and the regulation of GluRs and nervous system function.

McGehee, Annette et al. (2011) International Worm Meeting "The DAF-7/TGF-b signaling pathway regulates the abundance of the glutamate receptor GLR-1 in the ventral nerve cord."

McGehee, Annette, Juo, Peter

[International Worm Meeting, 2011]

The regulation of glutamate receptor (GluR) abundance and localization can be controlled by activity, and has been implicated in learning and memory. Thus, understanding the cell biological mechanisms that regulate GluRs is of significant interest. We use quantitative fluorescence microscopy to study genes and mechanisms involved in regulating the localization and abundance of GFP-tagged GluR GLR-1 (GLR-1::GFP) at synapses. GLR-1 is an AMPA-type GluR that is expressed in ventral nerve cord (VNC) interneurons where it localizes to sensory-interneuron and interneuron-interneuron synapses (1-3). Here we describe a novel role for the DAF-7/TGF-b signaling pathway in regulating the abundance of GLR-1 in the VNC. TGF-b signaling regulates multiple cell biological and developmental processes in many organisms, including several aspects of neuronal development. In C. elegans, the DAF-7/TGF-b pathway has been well studied as a regulator of dauer formation (4). We used temperature-sensitive (ts) mutants to show that multiple components of the DAF-7/TGF-b signaling pathway are involved in regulating GLR-1 levels in the adult VNC. Specifically, we found that the abundance of GLR-1::GFP increases significantly in daf-7 (TGF-b/ligand), daf-1 (type I receptor), daf-8 (Smad), and daf-14 (Smad) ts mutants compared to wild type animals. DAF-3 is a Smad whose transcriptional activity is antagonized by DAF-8 (5). We found that mutation of daf-3 suppresses the increase in GLR-1::GFP observed in daf-8 mutants, suggesting that the effect of DAF-8 on GLR-1 is mediated through DAF-3. The effect on GLR-1::GFP is specific for the DAF-7/TGF-b signaling pathway because animals with mutations in sma-6, a type I receptor that acts in a parallel TGF-b signaling pathway to regulate body size, showed no increase in the abundance of GLR-1::GFP in the VNC. We also found no change in the distribution of a presynaptic marker, GFP-tagged synaptobrevin, in daf-8 or daf-14 mutant animals suggesting that the number of presynaptic inputs is likely not affected by the DAF-7/TGF-b pathway. These results identify a novel function for the DAF-7/TGF-b signaling pathway in regulating the abundance of GLR-1 in the VNC, and suggest that an extracellular factor (TGF-b) is required to maintain GluR levels in the mature nervous system. (1) Hart et al. (1995). Nature 378:82-85. (2) Maricq et al. (1995). Nature 378:78-81. (3) Rongo et al. (1998). Cell 94:751-759. (4) Patterson and Padgett (2000). TIGS 16:27-33. (5) Patterson et al. (1997) Genes & Dev 11:2679-2690.

Dorlean, Shad-Lee et al. (2021) International Worm Meeting "Investigating the connection between the DAF-7/TGF-beta signaling pathway and the dense core vesicle protein IDA-1"

McLaughlin, Shannon, Rojas Rodriguez, Paola A, McGehee, Annette, Dorlean, Shad-Lee

[International Worm Meeting, 2021]

The DAF-7/TGF-beta signaling pathway in C. elegans is affected by the environment. This includes factors such as inadequate food supply, high temperature, or overpopulation. We previously found that the DAF-7/TGF-beta signaling pathway regulates the abundance of the glutamate receptor GLR-1. Mutants in the DAF-7/TGF-beta signaling pathway have increased levels of GLR-1 and an increased rate of spontaneous reversals, a behavior which is regulated by GLR-1. However, the exact mechanism of how DAF-7/TGF-beta signaling regulates GLR-1 is unknown. We tested to see whether ida-1, which is expressed in cells that express glr-1, might be involved in this regulation. IDA-1 is normally found on the surface of dense core vesicles (DCV's) and is involved in normal neuropeptide and DCV signaling. To test the involvement of ida-1 in the DAF-7/TGF-beta-dependent regulation of GLR-1 we assessed spontaneous reversal frequency and GLR-1::GFP levels in single and double mutants. The increased rate of spontaneous reversals and the increased GLR-1::GFP levels that are detected in daf-7 mutants are blocked in daf-7 ida-1 double mutants. These results support the idea that IDA-1 does indeed play a role in the DAF-7/TGF-beta-dependent regulation of GLR-1. We then focused on testing whether IDA-1 expression and localization are affected in daf-7 mutants. We investigated this question using strains expressing IDA-1::GFP. We imaged neurons in the head of C. elegans worms of the appropriate genotypes at both the cell body and neurite in order to measure the intensity of the IDA-1::GFP. We found increased levels of IDA-1::GFP in both cell bodies and neuronal puncta in the daf-7 mutant as compared to wildtype. These results suggest that the DAF-7 pathway regulates levels of IDA-1. The exact mechanism however, is yet to be discovered.

Patterson GI et al. (1996) East Coast Worm Meeting "Identification of new genes in dauer/TGF-beta signalling"

Ruvkun GB, Patterson GI

[East Coast Worm Meeting, 1996]

We are interested in understanding the mechanism by which food and pheromone signals are transduced to signal dauer arrest or non-dauer development. Don Riddle's group has identified a TGF-beta homologous pathway that is involved in this signal transduction process. daf-7 encodes a peptide ligand, and daf-1 and daf-4 encode transmembrane serine/threonine kinases that may be receptors for the daf-7 protein. Recent work by Takao Inoue, Annette Estevez and our laboratory (see abstract by A. Koweek and G. Patterson) has identified a family of genes that are likely to be involved in the signal transduction from these receptors; daf-8, daf-14, and daf-3 are all homologues of the drosophila gene Mad, which transduces signals from serine/threonine kinase receptors. The mechanism by which these receptors and Mad homologues transduce signal is unknown; we are identifying new genes that act in this pathway. In previous screens for suppressors of daf-7, the genes daf-3, daf-5 and daf-12 were identified by D. Riddle. We have identified four new genes that suppress daf-7. Two of the genes were identified by mutations that strongly suppress daf-7; the other two show weak suppression. We are trying to clone a strong suppressor called scd-1 (Suppressor of Constitutive Dauer formation--pronounced "scud"). We isolated two alleles as suppressors of daf-7; Takao Inoue provided two alleles he isolated as suppressors or daf-14 and daf-8. We mapped the gene between lin-2 and unc-9 on X, and we are doing finer mapping as well as injecting candidate cosmids for rescue. We have also begun a screen for suppressors of daf-3. We are mutagenizing a daf-7 daf-3 strain (daf-7 is dauer constitutive, daf-3 is dauer defective, and the double mutant is dauer defective). We will screen for dauers at 25 degrees C. daf-2, daf-19 and daf-23 are the known dauer constitutive mutants epistatic to daf-3. We hope to get new mutations that, while not dauer constitutive on their own, suppress the daf-3 mutation and allow the daf-7 mutation to force dauer formation. We will screen with both a null daf-3 allele and a partial loss-of function daf-3 allele.