Cheng, Eric et al. (2021) International Worm Meeting "The role of the Insulin Signaling Pathway in C. elegans Germline Stem Cell Mitosis"

Gerhold, Abigail, Cheng, Eric

[International Worm Meeting, 2021]

The spindle assembly checkpoint (SAC) monitors kinetochore-microtubule attachments and halts mitotic progression in the presence of unattached or incorrectly attached chromosomes. The SAC ensures the fidelity of chromosome segregation during mitosis by guarding against chromosome mis-segregation and aneuploidy. While the events and mechanisms that control the SAC are relatively well understood, how they are influenced by developmental and environmental signaling networks remains unclear. The insulin signaling (IIS) pathway is a universally important and highly conserved pathway that integrates cell proliferation and development in many organisms. We are using C. elegans germline stem cells (GSCs) to ask how the IIS pathway affects mitotic duration and fidelity in vivo via the in situ live imaging of these mitotically dividing cells. This pathway has a known role in cell cycle progression, with daf-2 mutants having fewer germ cells and a lower mitotic index, compared to stage-matched N2 animals, due primarily to a delay in the G2 phase of the cell cycle. Removal of daf-18 or daf-16 restores normal germ cell numbers suggesting canonical signaling from DAF-2 to DAF-16 affecting the rate of cell cycle progression. While clear links have been established between IIS and GSC proliferation, IIS involvement in events during mitosis, including the SAC, remains unclear. Evidence from cell culture models suggests that IIS may play a role during mitosis; however, it is not clear whether this occurs under in vivo physiological conditions. We have found that daf-2 mutants have an increased duration of mitosis and decreased number of cells entering mitosis. Both effects can be rescued by subsequent deletion of daf-18 or daf-16. In addition, daf-2 mutants have an increased incidence of chromosome segregation errors when the SAC is compromised, suggesting impairment of spindle assembly. Ongoing work will assess whether these effects are cell autonomous and will determine the underlying cause of the observed increase in chromosome segregation errors.

Henderson ST et al. (1994) Worm Breeder's Gazette "The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell"

Gao DL, Henderson ST, Lambie EJ, Kimble JE

[Worm Breeder's Gazette, 1994]

The lag-2 gene encodes a protein with homology to Drosophila Delta and is expressed in the distal tip cell. Sam Henderson, Dali Gao, Eric Lambie#, and Judith Kimble, University of Wisconsin, Madison WI 53706, Dartmouth College, Hanover, NH 03755

Tonks NK (2009) Cell "Pseudophosphatases: grab and hold on."

Tonks NK

[Cell, 2009]

Catalytically inactive pseudophosphatases are able to signal in the absence of enzymatic activity. Analyzing the oocyte-to-zygote transition in the worm, Cheng et al. (2009) and Parry et al. (2009) now show how two pseudophosphatases, EGG-4 and EGG-5, can regulate signaling by the DYRK family kinase MBK-2.

Yasuda H et al. (1987) Worm Breeder's Gazette "screening C elegans expression libraries with nerve specific anit-tubulin antibodies."

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1987]

C. elegans, like many other multicellular organisms, has multiple tubulin genes. Linda Gremke has shown that there are at least 4 and 5 alpha tubulin genes in the nematode, based on homology to the chicken tubulin gene probes(1). Similarly, it has been shown that purified tubulin from the nematode can be resolved in to at least 10 isotypes on isoelectric focusing, including 2 major alpha, 2 major , and about 6 minor alpha and isotypes(2). We have screened C. elegans cDNA and genomic expression libraries (kindly provided by Bob Barstead and Bob Waterston), with a set of anti-tubulin monoclonal antibodies, which preferentially stain neurons cytochemically(2). In the present screen 44 clones have been identified, which test positive on rescreening. The insert size of these clones range from 0.6 to 1.7 kb. Among these 29 clones are specific to anti- tubulin, and 14 are specific to anti-alpha tubulin antibodies. Our attention has been drawn to the problem of false positives in screening expression libraries with antibodies(e.g. C. Link and W. Wood, WBG Nov. 1986, and Eric Aamodt, personal communication). These workers have found E. coli DNA in the 'positive' clones, as a major artefact. Affinity purification of antibodies with the fusion protein, may not preclude the inclusion of false positives (Eric Aamodt, personal communication) either. Therefore, we plan to subclone the inserts in an appropriate vector, and determine the nucleotide sequence of the inserts to distinguish the true from the false positives. This is possible for the tubulin clones, as the nucleotide sequence of tubulin variants is available, including that of C. elegans tubulin(1). We plan to use this strategy for other genes for which we have collected 'positives' using expression libraries, including N-CAM like antigen, and N- CADHERIN like antigen of C. elegans, as these genes have conserved sequences, and their nucleotide sequence data are available.

Irene Zelepuhin et al. (2005) International Worm Meeting "Genetics of sex Reversal of C. briggsae::C. remanei XO hybrids."

Scott Baird, Shannon Romer, Irene Zelepuhin

[International Worm Meeting, 2005]

Sterile F1 adult hybrids can be obtained from crosses of C. briggsae males to C. remanei females. Depending on the strains used, all hybrids are females. The absence of male hybrids results from sexual transformation, not male-specific lethality, as both XX and XO females are observed. Variant strains of C. briggsae and C. remanei suppress this hybrid sexual transformation, demonstrating genetic variation within both species the affects sex determination in F1 hybrids. This variation has been used to map hybrid sex reversal loci. In C. remanei, an ill-defined region of the X chromosome is implicated. In C. briggsae, three loci linked to hybrid sex reversal have been identified. The strongest linkage is to allelic variants in Cb-tra-2. A null mutation in Cb-tra-2 (thanks Eric) completely suppresses the transformation of XO hybrids. Other C. briggsae loci linked to sex reversal variants include Cb-daf-5 and Cb-mab-9.

Davidson EH et al. (1999) J Exp Zool "Themes from a NASA workshop on gene regulatory processes in development and evolution."

Davidson EH, Ruvkun GB

[J Exp Zool, 1999]

A memorable workshop, focused on causal mechanisms in metazoan evolution and sponsored by NASA, was held in early June 1998, at MBL. The workshop was organized by Mike Levine and Eric H. Davidson, and it included the PI and associates from 12 different laboratories, a total of about 30 people. Each laboratory had about two and one half hours in which to represent its recent research and cast up its current ideas for an often intense discussion. In the following we have tried to enunciate some of the major themes that emerged, and to reflect on their implications. The opinions voiced are our own. We would like to tender apologies over those contributions we have not been able to include, but this is not, strictly speaking, a meeting review. Rather we have focused on those topics that bear more directly on evolutionary mechanisms, and have therefore slighted some presentations (including some of our own), that were oriented mainly toward developmental processes. J. Exp. Zool. (Mol. Dev. Evol. ) 285:104-115, 1999.

Almeida MV et al. (2018) EMBO J "GTSF-1 is required for formation of a functional RNA-dependent RNA Polymerase complex in Caenorhabditis elegans."

Redl S, Hildebrandt A, Ulrich HD, Dietz S, Renz C, Almeida MV, Konig J, Ketting RF, Butter F, Karaulanov E

[EMBO J, 2018]

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the <i>Caenorhabditis elegans</i> Gtsf1 homolog, named it <i>gtsf-1</i> and characterized it in the context of the sRNA pathways of <i>C.elegans</i> We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, <i>gtsf-1</i> mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying <i>rrf-3</i> mutants. We show, both <i>invivo</i> and <i>invitro</i>, that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities.

Sawh, Ahilya et al. (2015) International Worm Meeting "Regulation of DCR-1-dependent RNAi pathways through DCR-1 phosphorylation."

Duchaine, Thomas, Wohlschlegel, James, Lewis, Alexandra, Sawh, Ahilya

[International Worm Meeting, 2015]

RNA interference (RNAi) pathways are directed by small (18-22nt) non-coding RNAs and orchestrate a variety of evolutionarily-conserved gene-silencing programs. The type III RNase protein Dicer is at the center of most of these pathways, including responses triggered by exogenous sources of long double-stranded RNA (exoRNAi). Other pathways, such as microRNA-mediated silencing and endoRNAi are triggered by endogenous loci, and control developmental and homeotic events. Dicer's role is crucial and three-fold: it cleaves long dsRNA precursors into smaller RNAs that direct silencing events, it loads the small RNAs into Argonaute proteins to form an active RNA-induced silencing complex (RISC), and it acts as a scaffold for several protein co-factors required for substrate recognition and processing. In C. elegans, Dicer (DCR-1) is essential in three small-RNA mediated silencing mechanisms including exoRNAi and ERI endoRNAi, two RNAi pathways that compete for downstream effectors. DCR-1 is part of distinct multi-protein complexes in each of these RNAi pathways: RDE complex in exoRNAi and ERIC in ERI endoRNAi.We discovered and validated several phosphorylated determinants of DCR-1 in the C. elegans. Particular attention was placed on a cluster of phosphorylated sites between the PAZ and RNase III domains, one of which could implicate AMPK signaling. Recent crystal structures of human Dicer have shown that this region could be involved in recognition of dsRNA substrates. We thus hypothesized that phosphorylation in this cluster could impair substrate recognition, or alter its specificity.To study the effect of DCR-1 phosphorylation on the three DCR-1-dependent RNAi pathways in C. elegans, transgenic strains were engineered expressing phosphorylation-deficient, and phosphorylation-mimetic mutants of DCR-1 in this cluster of sites. Functional analysis on these strains revealed that phosphorylation-deficient mutants display an impaired exoRNAi response, and severe developmental defects. Phosphorylation-deficient DCR-1 mutants also enhance interaction with ERI-endoRNAi DCR-1 cofactors. Taken together, these results support a model wherein DCR-1 phosphorylation may promote its shift from the ERIC to the RDE complex. Post-translational regulation of Dicer and RNAi pathways are largely unexplored and could potentially be incredibly impactful in light of their crucial roles in development and disease. The present study will shed light on how RNAi pathways integrate signals through phosphorylation signaling cascades.

G Kao et al. (1999) International C. elegans Meeting "Characterization of Laminin beta and gamma Genes."

G Kao, C Huang, WG Wadsworth

[International C. elegans Meeting, 1999]

We have characterized laminin b (lam-1) and laminin g (lam-2) genes. The sequencing project has uncovered genes for one isoform each of the two subunits and two isoforms of laminin a , epi-1 and lam-3. (See abstract by Cheng et al.). This indicates that two types of trimers likely are form in basement membranes : a A bg and a B bg . A hypomorphic mutation (rh219) exists in lam-1, whose phenotypes we have described in previous meetings (IWM 1997). Both lam-1(RNAi) and lam-2(RNAi) animals arrest development during morphogenesis. Significantly, this phenotype is identical to the arrest seen in double knockout of epi-1 and lam-3, and is more severe than either knockout alone. We examined the expression patterns of lam-1 and lam-2 using promoter:GFP fusions. Both genes are expressed in body wall muscles, pharynx, vulval muscles, the rectal muscle group and the distal tip cells. The expression patterns of b and g subunit genes overlap with and are more widespread than either epi-1 or lam-3 alone. Taken together these results suggest that lam-1 and lam-2 are found in all basement membranes and indicate that trimers a A bg and a B bg are formed.

Goehring A et al. (2014) Nat Protoc "Screening and large-scale expression of membrane proteins in mammalian cells for structural studies."

Lee CH, Fischer S, Althoff T, Michel JC, Gouaux E, Garcia KC, Baconguis I, Claxton DP, Wang KH, Goehring A

[Nat Protoc, 2014]

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.