Fazzio RT et al. (1998) West Coast Worm Meeting "INITIAL CHARACTERIZATION OF VAV-1: AN INTRACELLULAR SIGNAL TRANSDUCER IN C. ELEGANS"

Fazzio RT, Beckerle MC, Maricq AV

[West Coast Worm Meeting, 1998]

Central to the regulation of cellular proliferation and differentiation are the mechanisms by which external stimuli influence intracellular processes. Signal transduction at sites of membrane-substrate contact are mediated by transmembrane receptors, and several associated intracellular cytoskeletal proteins. The proto-oncogene encoded protein, Vav, is a multifunctional protein that has been shown to mediate signaling in several Rho/Rac pathways involved in cytoskeletal control. To gain a better understanding of the molecular mechanisms involved in such signaling, we have undertaken a genetic and molecular analysis of Vav in the nematode Caenorhabditis elegans. In mammals, Vav, and its family member, Vav-2, exhibit GEF activity for the Rac GTPase. Rac has been implicated in the control of cytoskeletal reorganization through the stimulation of actin polymerization at focal contacts and newly forming lamellipodia. In addition to GEF function, Vav contains a number of other structural motifs. Vav is potentially localized to the membrane through its pleckstrin-homology (PH) domain, where it could participate in a variety of protein-protein interactions. For example, Vav has been shown to bind to the focal adhesion scaffolding protein, zyxin, through its carboxy-terminal SH3 domain. Lastly, Vav contains an amino-terminal calponin-homology (CH) domain that could function in actin binding. Deletion of this domain results in oncogenesis, producing dense, non-refractile foci in NIH3T3 cells, and tumors in nude mice. Clearly, such a multifunctional protein will play key roles in the control of diverse intracellular signaling events. The study of Vav in C. elegans will contribute a novel approach to the understanding of such signaling mechanisms. Recently, the C. elegans genome sequencing project identified a vav homolog in the nematode that is located on linkage group III. We have screened a cDNA library provided by Robert Barstead to isolate a full-length cDNA encoding vav-1 in C. elegans. This vav-like protein (Vav-1) is highly homologous to mammalian Vav, containing an overall 55% identity with the murine protein. The predicted protein contains many of the structural motifs found in mammalian Vav, such as an amino terminal calponin homology (CH) domain, central GEF and PH domains, and a carboxy-terminal SH2 domain. To identify the cellular localization of this signaling protein, we constructed several fusions of vav-1 to a reporter gene encoding Green Fluorescent Protein (GFP). In transgenic strains that express these fusion proteins, we identified expression primarily in neurons and muscle. In order to determine the function of Vav-1, in vivo, we have taken two different approaches to disrupt Vav function: RNA-mediated interference, and Tc1-transposon based mutagenesis. Using a PCR-based strategy, we identified a strain containing a Tc1 transposon in exon 4 of the vav-1 gene. This strain is viable and exhibits no obvious phenotype on preliminary analysis. Rigorous phenotypic analysis of this strain, and strains derived from imprecise excision of the transposon, are currently in progress.

RT Fazzio et al. (1999) International C. elegans Meeting "vav Function in C. elegans"

MC Beckerle, RT Fazzio, AV Maricq

[International C. elegans Meeting, 1999]

The proto-oncoprotein Vav is a guanine nucleotide exchange factor (GEF) for members of the Rho/Rac family of monomeric GTPases. Vav homologs have been identified in humans, mice, rat and C. elegans . Extensive studies in mammalian tissue culture cells and chimeric mice have implicated Vav in signal transduction pathways that lead to the reorganization of the actin cytoskeleton. Vav comprises several functional domains that are highly conserved in known signaling molecules. At the amino acid level, the C. elegans homolog, VAV-1, shares 33% identity with the human VAV protein and displays each of the domains thought to be important for signaling. VAV-1 contains a calponin-homology (CH) domain and an acidic domain (AD). Selective disruption of these regions of the mammalian proteins results in oncogenic transformation. VAV-1 also contains a Dbl homology (DH) domain, two SH3 domains and one SH2 domain. In humans, the DH domain is responsible for the GEF activation of Rho, Rac-1 and Cdc-42, and the Src-homology regions are known to interact with identified signaling molecules. To determine the function of VAV-1, in vivo , we used a Tc1 transposon-based mutagenesis approach to generate a genomic disruption of the vav-1 locus. This resulted in the deletion of the carboxy-terminal 80% of VAV-1, including the DH, SH3 and SH2 domains. The putative null was outcrossed multiple times to remove the Tc1 background, and the region was sequenced to confirm the presence of a genomic deletion. Phenotypic characterization of this mutant indicated that the disruption of vav-1 results in larval lethality. Experiments designed to rescue the vav-1 phenotype and better define the developmental basis for lethality are currently in progress. Future experiments will focus on the identification of proteins that interact with VAV-1. To complement the functional analysis, VAV-1 expression was studied using several constructs that fused regions of the vav-1 gene to the reporter gene encoding Green Fluorescent Protein (GFP). Transgenic lines expressing these constructs indicated the presence of VAV-1 in several neurons, body wall muscle, gonad, and vulval cells. We are currently generating monoclonal antibodies to this protein to further characterize vav-1 expression.

Madsen DM et al. (1998) West Coast Worm Meeting "GENETIC AND MOLECULAR ANALYSES OF A ZYXIN-LIKE MOLECULE IN C. ELEGANS"

Madsen DM, Fazzio RT, Beckerle MC, Maricq AV

[West Coast Worm Meeting, 1998]

Neuronal cells depend on cues from their environment, the substrate along which they migrate, to guide them to their correct targets. Focal adhesions are sites of contact between a cell and its substrate. These sites contain many specialized proteins that control and regulate cellular motility and shape. Amongst these is zyxin, a multifunctional LIM domain containing protein that colocalizes with components of the actin cytoskeleton and other proteins with LIM domains. Zyxin may function to recruit components of the actin assembly machinery to specific sites in the cell and to stimulate spatially restricted actin polymerization. Zyxin can also shuttle between the adhesion plaques and the nucleus, suggesting a mechanism by which changes in cell adhesion can trigger changes in gene expression and cellular differentiation. To gain a mechanistic understanding of signaling at the adhesion plaque we have undertaken a study of a zyxin homologue in C. elegans. We have isolated a cDNA that encodes a zyxin-like molecule in C. elegans. This gene, zyx-1, has the characteristic features of zyxin, including 3 LIM domains, each containing a perfect LIM consensus sequence, as well as a proline-rich N-terminal region believed to be important for protein-protein interactions. The zyx-1 gene has remarkably long intronic sequences compared to most C. elegans genes. These introns help regulate the cellular expression of the zyxin protein. Using various fusions of zyx-1 to the reporter gene Green Fluorescent Protein (GFP), one can observe markedly different expression patterns. Neuronal expression is predominant when GFP is fused to exon 5. However, when GFP is fused to downstream exons, expression is primarily muscular. To perturb the function of zyx-1 , we first mapped the genomic structure and, using a PCR-based strategy, identified two strains that contained Tc1 transposable elements in the zyxin gene. Recently, imprecise excision of the Tc1 elements allowed us to identify two deletion mutations that disrupt either the N-terminal or C-terminal regions. These strains are viable but have not yet been outcrossed. We anticipate that zyx-1 may function as part of a protein complex that transduces substrate adhesion into changes in cytoskeleton function. For each deletion mutation we will assess neuromuscular function by analyzing locomotion, both spontaneous and that induced by touch, chemical or osmotic stimuli. We will also examine neuronal differentiation, migration, and outgrowth using GFP reporters and antibody staining. Finally, we will assess the function of other behaviors and sensory modalities, including chemotaxis, chemosensory adaptation, egg laying, dauer formation, and defecation.

Maricq AV et al. (2000) West Coast Worm Meeting "VAV is required for pharyngeal muscle contraction in C. elegans"

Mellem JE, Fazzio RT, Maricq AV, Beckerle MC

[West Coast Worm Meeting, 2000]

In vertebrates, VAV, VAV-2 and VAV-3 are guanine nucleotide exchange factors for Rho-family GTPases. Nucleotide exchange is a key mechanism by which Rho GTPases are regulated during cytoskeletal remodeling. Disruption of this regulation by specific mutations in VAV results in the constituative activation of Rho-family members and oncogenesis. To better understand the role of VAV in cytoskeletal control, we have undertaken a genetic and molecular analysis of VAV in C. elegans. We have cloned the C. elegans homolog of vav, vav-1, from first-strand cDNA. vav-1 encodes a 975 amino acid protein that shares 34% overall identity with human VAV and contains the predicted exchange factor domain and other important functional motifs. We have previously reported vav-1 expression in pharyngeal tissue, body wall muscle, vulval tissue and somatic gonad. In the pharynx, VAV-1 is found in muscle, in some neuronal cells and in the g1 gland cells. Subcellular localization of VAV-1 will be studied using a mouse monoclonal antibody generated to this protein. We have engineered a deletion mutation in vav-1 that removes 85% of the mature protein product. This disruption yields a pharyngeal contraction defect that ultimately results in early larval lethality. Mutant animals display asynchronous pharyngeal muscle contraction or, in some cases, complete pharyngeal paralysis. Electrophysiological analysis of mutant pharyngeal muscle confirms the importance of VAV-1 for synchronous contraction. We can rescue this defect by the transgenic expression of a genomic DNA fragment that contains the vav-1 open reading frame. We are currently pursuing experiments designed to provide a better understanding of the vav-1 null phenotype: Through tissue-specific expression of vav-1 in the null background, and possibly mosaic analysis, we aim to identify the pharyngeal cells (and other cells) that require VAV-1. In addition, we aim to complete a mutational analysis of the vav-1 gene to determine the domains/residues of VAV-1 that are important for function. The generation of multiple alleles in vav-1 will provide the reagents necessary for a genetic screen designed to identify proteins that function in the VAV signal transduction pathway.

Fungtammasan A et al. (2016) Mol Biol Evol "Reverse Transcription Errors and RNA-DNA Differences at Short Tandem Repeats."

Fungtammasan A, Eckert K, Campos-Sanchez R, Makova KD, DeGiorgio M, Tomaszkiewicz M

[Mol Biol Evol, 2016]

Transcript variation has important implications for organismal function in health and disease. Most transcriptome studies focus on assessing variation in gene expression levels and isoform representation. Variation at the level of transcript sequence is caused by RNA editing and transcription errors, and leads to non-genetically encoded transcript variants, or RNA-DNA differences (RDDs). Such variation has been understudied, in part because its detection is obscured by reverse transcription (RT) and sequencing errors. It has only been evaluated for inter-transcript base substitution differences. Here we investigated transcript sequence variation for short tandem repeats (STRs). We developed the first maximum likelihood estimator (MLE) to infer RT error and RDD rates, taking next generation sequencing error rates into account. Using the MLE, we empirically evaluated RT error and RDD rates for STRs in a large-scale DNA and RNA replicated sequencing experiment conducted in a primate species. The RT error rates increased exponentially with STR length and were biased towards expansions. The RDD rates were approximately one order of magnitude lower than the RT error rates. The RT error rates estimated with the MLE from a primate data set were concordant with those estimated with an independent method, barcoded RNA sequencing, from a C. elegans data set. Our results have important implications for medical genomics, as STR allelic variation is associated with >40 diseases. STR non-allelic transcript variation can also contribute to disease phenotype. The MLE and empirical rates presented here can be used to evaluate the probability of disease-associated transcripts arising due to RDD.

Iyengar, Anusha et al. (2021) MicroPubl Biol

Iyengar, Anusha, Diamantakis, Stavros, Norris, Adam

[MicroPubl Biol, 2021]

C. elegans was the first animal to have its genome completely sequenced. In the decades since, the genome continues to be actively curated, annotated, and improved. Here we report the discovery of a new gene in a region of the genome that is currently not associated with any annotated gene or feature. We present RNA-seq and RT-PCR evidence that this gene is expressed at detectable levels, and that it is alternatively spliced. The new gene (Y97E10C.2) shares an operon with two upstream genes. We provide RNA-seq and RT-PCR evidence for a missing exon in the upstream gene T05B11.7, as well as an alternatively-spliced exon.

Norman KR et al. (2005) Cell "The Rho/Rac-family guanine nucleotide exchange factor VAV-1 regulates rhythmic behaviors in C. elegans."

Mellem JE, Maricq AV, Fazzio RT, Norman KR, Strange K, Espelt MV, Beckerle MC

[Cell, 2005]

Rhythmic behaviors are a fundamental feature of all organisms. Pharyngeal pumping, the defecation cycle, and gonadal-sheath-cell contractions are three well-characterized rhythmic behaviors in the nematode C. elegans. The periodicities of the rhythms range from subsecond (pharynx) to seconds (gonadal sheath) to minutes (defecation). However, the molecular mechanisms underlying these rhythmic behaviors are not well understood. Here, we show that the C. elegans Rho/Rac-family guanine nucleotide exchange factor, VAV-1, which is homologous to the mammalian Vav proto-oncogene, has a crucial role in all three behaviors. vav-1 mutants die as larvae because VAV-1 function is required in the pharynx for synchronous contraction of the musculature. In addition, ovulation and the defecation cycle are abnormal and arrhythmic. We show that Rho/Rac-family GTPases and the signaling molecule inositol triphosphate (IP(3)) act downstream of VAV-1 signaling and that the VAV-1 pathway modulates rhythmic behaviors by dynamically regulating the concentration of intracellular Ca(2+).

Kushibiki Y et al. (1996) Pathophysiology "Molecular cloning of oxygen-inducible genes in Caenorhabditis elegans by RT-PCR differential display."

Kushibiki Y, Yanase S, Nakazawa H, Ishii N

[Pathophysiology, 1996]

To investigate the genetic response to oxidative stress in eukaryotic cells, we have applied the method RT-PCR differential display to the small, free-living nematode Caenorhabditis elegans (C. elegans) which was cultured under atmospheric or high oxygen concentrations. As a result, a new gene was cloned whose expression increased at high oxygen concentration.

Zhang Y et al. (2012) PLoS One "Selection of reliable reference genes in Caenorhabditis elegans for analysis of nanotoxicity."

Pan X, Zhang Y, Chen D, Zhang B, Smith MA

[PLoS One, 2012]

Despite rapid development and application of a wide range of manufactured metal oxide nanoparticles (NPs), the understanding of potential risks of using NPs is less completed, especially at the molecular level. The nematode Caenorhabditis elegans (C.elegans) has been emerging as an environmental model to study the molecular mechanism of environmental contaminations, using standard genetic tools such as the real-time quantitative PCR (RT-qPCR). The most important factor that may affect the accuracy of RT-qPCR is to choose appropriate genes for normalization. In this study, we selected 13 reference gene candidates (act-1, cdc-42, pmp-3, eif-3.C, actin, act-2, csq-1, Y45F10D.4, tba-1, mdh-1, ama-1, F35G12.2, and rbd-1) to test their expression stability under different doses of nano-copper oxide (CuO 0, 1, 10, and 50 g/mL) using RT-qPCR. Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative Ct method, were employed to evaluate these 13 candidates expressions. As a result, tba-1, Y45F10D.4 and pmp-3 were the most reliable, which may be used as reference genes in future study of nanoparticle-induced genetic response using C.elegans.

Chauve L et al. (2020) J Vis Exp "High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology."

Andrews S, Casanueva O, Miska EA, Chauve L, Le Pen J, Biggins L, Hodge F, Todtenhaupt P

[J Vis Exp, 2020]

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named 'Worm-to-CT', allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.