Bell RT et al. (2015) Genetics "Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans."

Fire AZ, Fu BX, Bell RT

[Genetics, 2015]

The proliferation of CRISPR/Cas9-based methods in Caenorhabditis elegans has enabled efficient genome editing and precise genomic tethering of Cas9 fusion proteins. Experimental designs using CRISPR/Cas9 are currently limited by the need for a protospacer adjacent motif (PAM) in the target with the sequence NGG. Here we report the characterization of two modified Cas9 proteins in C. elegans which recognize NGA and NGCG PAMs. We found that each variant could stimulate homologous recombination with a donor template at multiple loci, and that PAM specificity was comparable to wild type Cas9. In order to directly compare effectiveness, we used CRISPR/Cas9 genome editing to generate a set of assay strains with a common single-guide RNA (sgRNA) target sequence, but which differ in the juxtaposed PAM (NGG, NGA, or NGCG). In this controlled setting, we determined that the NGA PAM Cas9 variant can be as effective as wild type. We similarly edited a genomic target to study the influence of the base following the NGA PAM. Using four strains with four NGAN PAMs, differing only at the fourth position and adjacent to the same sgRNA target, we observed that efficient homologous replacement was attainable with any base in the fourth position, with an NGAG PAM being the most effective. In addition to demonstrating the utility of two Cas9 mutants in C. elegans and providing reagents which permit CRISPR/Cas9 experiments with fewer restrictions on potential targets, we established a means to benchmark the efficiency of different Cas9::PAM combinations that avoids variations due to differences in the sgRNA sequence.

Bell AD et al. (2023) G3 (Bethesda) "Beyond the reference: gene expression variation and transcriptional response to RNAi in C. elegans."

Bell AD, Paaby AB, Chou HT, Valencia F

[G3 (Bethesda), 2023]

Though natural systems harbor genetic and phenotypic variation, research in model organisms is often restricted to a reference strain. Focus on a reference strain yields great depth of knowledge, but potentially at the cost of breadth of understanding. Furthermore, tools developed in the reference context may introduce bias when applied to other strains, posing challenges to defining the scope of variation within model systems. Here, we evaluate how genetic differences among five wild C. elegans strains affect gene expression and its quantification, in general and after induction of the RNA interference (RNAi) response. Across strains, 34% of genes were differentially expressed in the control condition, including 411 genes that were not expressed at all in at least one strain; 49 of these were unexpressed in reference strain N2. Reference genome mapping bias caused limited concern: despite hyper-diverse hotspots throughout the genome, 92% of variably expressed genes were robust to mapping issues. The transcriptional response to RNAi was highly strain- and target gene-specific and did not correlate with RNAi efficiency, as the two RNAi insensitive strains showed more differentially expressed genes following RNAi treatment than the RNAi-sensitive reference strain. We conclude that gene expression, generally and in response to RNAi, differs across C. elegans strains such that choice of strain may meaningfully influence scientific inferences. Finally, we introduce a resource for querying gene expression variation in this dataset at https://wildworm.biosci.gatech.edu/rnai/.

Bell LR et al. (2006) Genetics "The molecular identities of the Caenorhabditis elegans intraflagellar transport genes dyf-6, daf-10 and osm-1."

Herman RK, Shaw JE, Yochem J, Stone S, Bell LR

[Genetics, 2006]

The Caenorhabditis elegans genes dyf-6, daf-10, and osm-1 are among the set of genes that affect chemotaxis and the ability of certain sensory neurons to take up fluorescent dyes from the environment. Some genes in this category are known to be required for intraflagellar transport (IFT), which is the bidirectional movement of raft-like particles along the axonemes of cilia and flagella. The cloning of dyf-6, daf-10, and osm-1 are described here. The daf-10 and osm-1 gene products resemble each other and contain WD and WAA repeats. DYF-6, the product of a complex locus, lacks known motifs, but orthologs are present in flies and mammals. Phenotypic analysis of dyf-6 mutants expressing an OSM-6::GFP reporter indicates that the cilia of the amphid and phasmid dendritic endings are foreshortened. Consistent with genetic mosaic analysis, which indicates that dyf-6 functions in neurons of the amphid sensilla, DYF-6::GFP is expressed in amphid and phasmid neurons. Movement of DYF-6::GFP along the ciliated endings of the neurons indicates that DYF-6 is a component of IFT particles. IFT can also be observed in dauer larvae.

Bell AD et al. (2024) MicroPubl Biol "A web application for gene-based queries of CaeNDR RNA-seq data."

Bell AD, Paaby AB

[MicroPubl Biol, 2024]

Variation in gene expression is a feature of all living systems and has recently been characterized extensively among wild strains of the model organism <i>Caenorhabditis elegans.</i> To enable researchers to query gene expression and gene expression variation at any gene of interest, we have created a user-friendly web application that shares RNA-seq transcription data for 208 wild <i>C. elegans</i> strains generated by the <i>Caenorhabditis</i> Natural Diversity Resource (CaeNDR). Here, we describe the features of the web application and the details of the data and data processing underlying it. We hope that this website, wildworm.biosci.gatech.edu/cendrexp/ , will help <i>C. elegans</i> researchers better understand their favorite genes and strains.

Bell KR et al. (2020) Mol Biol Cell "Novel cytokinetic ring components drive negative feedback in cortical contractility."

Maddox AS, Cortes DB, Sattler A, Vuong-Brender T, Labouesse M, Werner ME, Bell KR, Doshi A

[Mol Biol Cell, 2020]

Actomyosin cortical contractility drives many cell shape changes including cytokinetic furrowing. While positive regulation of contractility is well characterized, counterbalancing negative regulation and mechanical brakes are less well understood. The small GTPase RhoA is a central regulator, activating cortical actomyosin contractility during cytokinesis and other events. Here we report how two novel cytokinetic ring components, GCK-1 (germinal center kinase-1) and CCM-3 (cerebral cavernous malformations-3), participate in a negative feedback loop among RhoA and its cytoskeletal effectors to inhibit contractility. GCK-1 and CCM-3 are recruited by active RhoA and anillin to the cytokinetic ring, where they in turn limit RhoA activity and contractility. This is evidenced by increased RhoA activity, anillin and nonmuscle myosin II in the cytokinetic ring, and faster cytokinetic furrowing, following depletion of GCK-1 or CCM-3. GCK-1 or CCM-3 depletion also reduced RGA-3 levels in pulses and increased baseline RhoA activity and pulsed contractility during zygote polarization. Together, our results suggest that GCK-1 and CCM-3 regulate cortical actomyosin contractility via negative feedback. These findings have implications for the molecular and cellular mechanisms of cerebral cavernous malformation pathologies.

Bell R et al. (2009) PLoS Genet "A human protein interaction network shows conservation of aging processes between human and invertebrate species."

Bell R, Kapahi P, Miller JP, Hubbard A, Melov S, Chettier R, Sahasrabuhde S, Chen D, Hughes RE, Tarnopolsky M

[PLoS Genet, 2009]

We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteome-scale human protein interaction Core Network generated through unbiased high-throughput yeast two-hybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33% extended life span when knocked-down in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins.

Rehain-Bell K et al. (2017) Curr Biol "A Sterile 20 Family Kinase and Its Co-factor CCM-3 Regulate Contractile Ring Proteins on Germline Intercellular Bridges."

Rehain-Bell K, Love A, Maddox AS, Macleod I, Werner ME, Yates JR

[Curr Biol, 2017]

Germ cells in most animals are connected by intercellular bridges, actin-based rings that form stable cytoplasmic connections between cells promoting communication and coordination [1]. Moreover, these connections are required for fertility [1, 2]. Intercellular bridges are proposed to arise from stabilization of the cytokinetic ring during incomplete cytokinesis [1]. Paradoxically, proteins that promote closure of cytokinetic rings are enriched on stably open intercellular bridges [1, 3, 4]. Given this inconsistency, the mechanism of intercellular bridge stabilization is unclear. Here, we used the C.elegans germline as a model for identifying molecular mechanisms regulating intercellular bridges. We report that bridges are actually highly dynamic, changing size at precise times during germ cell development. We focused on the regulation of bridge stability by anillins, key regulators of cytokinetic rings and cytoplasmic bridges [1, 4-7]. We identified GCK-1, a conserved serine/threonine kinase [8],as a putative novel anillin interactor. GCK-1 works together with CCM-3, a known binding partner [9], to promote intercellular bridge stability and limit localization of both canonical anillin and non-muscle myosin II (NMM-II) to intercellular bridges. Additionally, we found that a shorter anillin, known to stabilize bridges [4, 7], also regulates NMM-II levels at bridges. Consistent with these results, negative regulators of NMM-II stabilize intercellular bridges in the Drosophila egg chamber [10, 11]. Together with our findings, this suggests that tuning of myosin levels is a conserved mechanism for the stabilization of intercellular bridges that can occur by diverse molecular mechanisms.

Salinas LS et al. (2007) Genesis "The DEAD box RNA helicase VBH-1 is required for germ cell function in C. elegans."

Salinas LS, Macias-Silva M, Blackwell TK, Navarro RE, Maldonado E

[Genesis, 2007]

Vasa and Belle are conserved DEAD box RNA helicases required for germ cell function. Homologs of this group of proteins in several species, including mammals, are able to complement a mutation in yeast (DED1) suggesting that their function is highly conserved. It has been proposed that these proteins are required for mRNA translation regulation, but their specific mechanism of action is still unknown. Here we describe functions of VBH-1, a C. elegans protein closely related to Belle and Vasa. VBH-1 is expressed specifically in the C. elegans germline, where it is associated with P granules, the C. elegans germ plasm counterpart. vbh-1(RNAi) animals produce fewer offspring than wild type because of defects in oocyte and sperm production, and embryonic lethality. We also find that VBH-1 participates in the sperm/oocyte switch in the hermaphrodite gonad. We conclude that VBH-1 and its orthologs may perform conserved roles in fertility and development. genesis 45:533-546, 2007. (c) 2007 Wiley-Liss, Inc.

Titanji VP et al. (1988) Parasitol Res "Studies on glucose-6-phosphate dehydrogenase from the human parasite, Onchocerca volvulus."

Tchoupe JR, Muluh JP, Titanji VP

[Parasitol Res, 1988]

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified from the extracts of adult Onchocerca volvulus by affinity chromatography on 2'5'ADP-Sepharose-4B. Kinetic studies revealed a typical bell-shaped pH profile with an optimum lying between pH 7.3 and 7.8. The apparent Km for glucose-6-phosphate was 5.66 x 10(-5) M, whereas that for NADP was 2.17 x 10(-6) M. Suramin, a filaricidal drug, inhibited the enzyme competitively with respect to NADP as a substrate: the apparent Ki values were 2.23 x 10(-6) M and 4.21 x 10(-7) M, respectively, for the crude and purified enzyme preparations. Glucose-6-phosphate dehydrogenase therefore, could be one of the targets of suramin in vivo.

Furuta T et al. (2023) MicroPubl Biol "sart-3 functions to regulate germline sex determination in C. elegans ."

Arur S, Furuta T

[MicroPubl Biol, 2023]

<i>Caenorhabditis elegans</i> gene <i>sart-3</i> was first identified as the homolog of human SART3 ( S quamous cell carcinoma A ntigen R ecognized by T -cells 3). In humans, expression of SART3 is associated with squamous cell carcinoma, thus most of the studies focus on its potential role as a target of cancer immunotherapy (Shichijo et al. 1998; Yang et al. 1999). Furthermore, SART3 is also known as Tip110 (Liu et al. 2002; Whitmill et al. 2016) in the context of HIV virus host activation pathway. Despite these disease related studies, the molecular function of this protein was not revealed until the yeast homolog was identified as spliceosome U4/U6 snRNP recycling factor (Bell et al. 2002). The function of SART3 in development, however, remains unknown. Here we report that the <i>C. elegans</i> <i>sart-3</i> mutant hermaphrodites exhibit a Mog ( M asculinization O f the G ermline) phenotype in adulthood suggesting that <i>sart-3</i> normally functions to regulate the switch from spermatogenic to oogenic gametic sex.