Roy, Peter J. (2022) MicroPubl Biol

Roy, Peter J.

[MicroPubl Biol, 2022]

Unsupervised Uniform Manifold Approximation and Projection (UMAP) plots of single cell sequencing data from synchronized Caenorhabditis elegans larvae yield tissue-specific data clusters, some of which are plotted as elongated archipelagos. These archipelagos likely represent a single cell type. I show that the pharyngeal archipelagos express a myriad of asynchronous temporally regulated genes, which likely accounts for their elongated topology. With one archipelago, I show that there is a high correlation between a) the base pair distance between the binding sites of an archipelago-specific transcription factor (HLH-6) and the transcriptional start site of the targeted genes and b) the timing of peak gene expression of those genes that are expressed in an archipelago-specific manner. Despite the correlation being made with only four genes, it prompts the hypothesis that the physical distance between a transcription factor and the relevant transcription start site may be an important factor in determining the temporal onset of transcription and transcript abundance.

Peter J Roy et al. (2005) International Worm Meeting "A Small Molecule Screen in C. elegans Yields a Potent Vertebrate L-Type Calcium Channel Inhibitor, Nemadipine A"

Elise F Stanley, Peter McCourt, Allen Chen, Regina Fraser, Trevor Kwok, Peter J Roy, Nicole Ricker, Sean Cutler

[International Worm Meeting, 2005]

We screened 10,000 small membrane permeable molecules from a diverse chemical library for the induction of defects in C. elegans. One compound that we call nemadipine A induced morphological, egg-laying, and slow growth defects in wild type worms (IC50<5.0uM). A subsequent screen of 15 related structures revealed a second molecule, nemadipine B, that induces the same phenotypes as nemadipine A. The structure of the nemadipines is similar to a class of molecules called 1,4-dihydropyridines (DHPs) that inhibit the 45;1 subunit of L-type calcium channels and are used to treat hypertension and ischemic heart disease in humans. Through candidate analysis we discovered that egl-19 loss-of-function mutants are hypersensitive to the nemadipines, while egl-19 gain-of-function mutants suppress nemadipine-induced defects. EGL-19 is the only C. elegans homologue of the 45;1 subunit of mammalian L-type calcium channels. These and other observations strongly suggest that EGL-19 is the primary target of the nemadipines. We tested four other DHP drugs, including felodipine, nicardipine, nifedipine, and nimodipine, together with representatives of three other classes of L-type calcium channel inhibitors, namely verapamil, diltiazem and bepredil. Unlike the nemadipines, none of these drugs elicited phenotype in wild type worms in our whole worm assays. Since many of these drugs can inhibit calcium channels in dissected worms, this result suggested that the nemadipines have unique properties that allow efficient entry into whole worms or are more effective than existing DHPs or both. We therefore compared the abilities of nemadipine A and nifedipine to inhibit vertebrate L-type channels. Our preliminary analysis shows that nemadipine A can specifically inhibit vertebrate L-type channels in the femtimolar range, and is at least 1000 fold more potent than nifedipine. Finally, we used nemadipine A to show that UNC-2, an 45;1 subunit homologue of N/P/Q calcium channels, functions redundantly with EGL-19 to control egg-laying. We are currently using the nemadipines for the genetic analysis of DHP action and the analysis of genetic variance in response to this class of widely-used drugs (see abstract by Kwok et al; this meeting). This study shows that C. elegans can be used to identify new small molecule tools to better understand biology and can reveal potential drug-leads.

Peter J Roy et al. (2001) International C. elegans Meeting "Identifying muscle genes that change transcription levels in response to varying cholinergic signals using full-genome microarrays."

Peter J Roy, Stuart K Kim

[International C. elegans Meeting, 2001]

The excitatory neurotransmitter acetylcholine (ACh) not only controls muscle cell contraction, but can also lead to a change in muscle cell physiology. For example, in mammals, chronic exposure to the ACh agonist nicotine can desensitize cells, leading to addiction. To better understand how a post-synaptic cell responds to ACh, we are examining the genome-wide transcriptional changes in C. elegans striated muscle cells in response to varying cholinergic signal. To do this, we first needed to develop a method to measure relative gene expression levels in single tissues in C. elegans. Previous DNA microarray experiments have compared gene expression levels from entire worms rather than from specific tissues. A major limitation to this approach is that gene expression changes in one tissue, such as muscle, could be obscured by expression in other tissues. To isolate muscle mRNAs, we use the myo-3 promoter (a gift from A. Fire) to drive expression of an epitope-tagged poly(A)-mRNA-binding protein in striated muscle. The mRNA/tagged-protein complex is co-immunoprecipitated from whole-animal lysates using antibodies against the epitope tag. mRNAs significantly enriched in muscle are identified by comparing the immunoprecipitated mRNA to the mRNA present in the initial worm extract using DNA microarrays. We call this technique "mRNA-tagging". L1-muscle mRNA-tagging (6 repeats) revealed 1453 genes that were significantly enriched, including most characterized muscle-specific genes (positive controls) and excluding most non-muscle genes (negative controls). We repeated the entire experiment using L2 larvae (6 repeats) and found that there were few significant differences between L1 and L2 muscle. Together, these results indicate that mRNA-tagging successfully identifies genes expressed in striated muscle. To profile transcriptional changes in striated muscle in response to ACh, we either excited muscle cells using the nicotinic ACh agonist levamisole or prevented normal muscle contraction using an ACh receptor mutant. We then used mRNA tagging to identify genes expressed in excited and mutant muscle, and compared those genes to each other and to those expressed in "normal" muscle. 304 genes show increased expression and 851 genes show decreased expression in levamisole-excited muscle, relative to normal or mutant muscle. The gene expression data indicates that ACh excitation decreases the relative expression of ACh receptors and other ligand-gated ion channels, which may represent new ACh receptor candidates. Specifically, the expression levels of seven ligand-gated receptors, two 2nd messenger-gated receptors, and two characterized ACh receptor genes (lev-1 and unc-29), were significantly lower in the levamisole-treated or normal worms than they were in the mutant. The overall effect may be to desensitize cells to acetylcholine following strong activation, and to sensitize cells in ACh receptor mutants.

Chan, Kevin K et al. (2011) International Worm Meeting "Integrin Cooperates with UNC-40 to Guide Muscle Arm Extension."

Roy, Peter J, Chan, Kevin K

[International Worm Meeting, 2011]

Body muscles of nematodes extend specialized plasma membranes, called muscle arms, to the dorsal and ventral nerve cords to form neuromuscular junctions. The extension of C. elegans muscle arms is stereotypical and likely guided by a chemoattractant that is secreted from motor axon targets. We recently found that an UNC-40/DCC pathway, which includes the UNC-40 receptor, the MADD-2 adaptor protein, and the UNC-73 Rho-GEF, functions cell-autonomously in C. elegans to direct muscle arm extension towards the motor axons (Alexander et al., 2009, 2010). Two lines of evidence suggest that this signalling pathway may be a non-canonical one. First, UNC-6/Netrin, which is the canonical ligand of UNC-40, is dispensable for muscle arm extension to motor axons. Second, the extra-cellular domains of UNC-40 are not required for UNC-40's role in directing muscle arm extension. These data suggest that a second receptor complex may co-operate with UNC-40 to guide muscle arms. To identify other plasma membrane-associated proteins that might cooperate with UNC-40, we screened candidate receptors for their ability to facilitate UNC-40-mediated muscle arm extension. We previously reported that PAT-2/a-integrin, but not INA-1/a-integrin, is required for muscle arm extension to the motor axons (Dixon et al., 2006). Here, we present evidence that the PAT-2/PAT-3 integrin heterodimer is required for UNC-40-dependent muscle arm extension. We are currently investigating the mechanism by which integrin facilitates UNC-40's role in directing muscle arm extension.

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

Labbe JC et al. (2006) Clin Genet "New developmental insights from high-throughput biological analysis in Caenorhabditis elegans."

Labbe JC, Roy R

[Clin Genet, 2006]

Labbe J-C, Roy R. New developmental insights from high-throughput biological analysis in Caenorhabditis elegans.The use of Caenorhabditis elegans as a model system for understanding animal development and human disease has long been recognized as an efficient tool of discovery. Recent developments, particularly in our understanding of RNA-mediated interference and its ability to modify gene activity, have facilitated the use of C. elegans in determining gene function via high-throughput analysis. These new strategies have provided a framework that allows investigators to analyse gene function globally at the genomic level and will likely become a prototypic model for biological analysis in the post-genome era.

Cunningham KA et al. (2009) Cell Metab "Fat rationing in dauer times."

Ashrafi K, Cunningham KA

[Cell Metab, 2009]

The fundamental task of maintaining energy balance is complex when nutrient levels are plentiful, but it becomes even more challenging when nutrients are dynamic or scarce. A recent Nature report delineates a role of the AMP kinase pathway in rationing energy stores for the long-term survival of Caenorhabditis elegans dauers (Narbonne and Roy, 2009).

Murakami S et al. (1999) Curr Biol "Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene."

Johnson TE, Murakami S

[Curr Biol, 1999]

In this Brief Communication, which appeared in the 14 September 1998 issue of Current Biology, the UV dose was reported erroneously. The dose reported was 20 J/m2 but the actual dose used was 0.4 J/cm2. Also, the gene formally referred to as tkr-1 has since been renamed old-1 (overexpression longevity determination).

Hotez PJ et al. (1993) Parasitol Today "Hookworm larval infectivity, arrest and amphiparatenesis: the Caenorhabditis elegans Daf-c paradigm."

Hotez PJ, Schad GA, Hawdon JM

[Parasitol Today, 1993]

Arrested development dramatically alters the life history of some species of soil-transmitted nematodes and elicits profound variations in the epidemiology of the infections they cause. Here, Peter Hotez, John Hawdon and Gerhard Schad show how an understanding of the cellular and molecular bases of arrested development may lead to new approaches for the control of ancylostomiasis and related infections.

Ramos CG et al. (2014) J Bacteriol "Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia."

Feliciano JR, da Costa PJ, Ramos CG, Grilo AM, Leitao JH

[J Bacteriol, 2014]

Volume 195, no. 16, p. 35143523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitao, J. Bacteriol. 193:15151526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitao, Microbiology 156:896908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitao, Microb. Pathog. 48:168177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.