Thimo Kurz et al. (2003) International Worm Meeting "Neddylation and deneddylation of the C. elegans cullin CUL-3 are required for cytoskeletal regulation during cell division in early embryos."

Bruce Bowerman, Sarah Glaser, Sean ORourke, Lionel Pintard, Thimo Kurz, Matthias Peter, John Willis

[International Worm Meeting, 2003]

Ubiquitin-mediated degradation of proteins is emerging as a key regulatory mechanism in C. elegans embryos. The Nedd8 ubiquitin-like protein modification pathway is known to regulate cell cycle progression by conjugating Nedd8 to cullin proteins, which act in E3 ubiquitin ligases. We have found that the C. elegans NED-8 pathway also targets cytoskeletal regulatory proteins for degradation. The NED-8 pathway promotes microtubule stability during mitosis, and negatively regulates contractile ring activity outside the cleavage furrow during cytokinesis. We have identified the cullin CUL-3 as one target of NED-8 conjugation, and cul-3(RNAi) embryos exhibit defects identical to those observed in mutants lacking the NED-8 E1-activating enzyme RFL-1. Our data suggest that neddylated CUL-3 forms an E3 ubiquitin ligase required to degrade cytoskeletal regulators. One target for this putative E3 ligase is the microtubule severing activity katanin (in C. elegans, MEI-1 and MEI-2). C. elegans katanin is required for meiosis, which is mediated by short MTs, but katanin is degraded during the transition to mitosis, which is mediated by much longer MTs. In rfl-1 and cul-3(RNAi) mutant embryos, katanin accumulates on mitotic spindles, and is present at high levels in extracts. Genetic studies show that failure to down-regulate katanin accounts for the spindle defects observed in rfl-1 and cul-3 mutants. To extend our understanding of the NED-8 pathway, we have used RNAi to identify other genes required for katanin degradation. We have found that deneddylation of CUL-3 by the COP9 signalosome is also required. In mutant embryos lacking the COP9 signalosome, neddylated CUL-3 accumulates and katanin is stabilized. Simultaneous reduction of neddylation and de-neddylation restores viability, indicating that a balance is required for ligase function. We therefore suggest that poly-ubiquitination of target proteins by some E3 complexes may require repeated cycles of cullin neddylation and deneddylation. Using a MEI-1::GFP strain, we also have identified a new and conserved component of the NED-8 protein modification pathway. Inactivation of this gene by RNAi stabilizes katanin after meiosis, and a deletion of the S. cerevisiae homologue abolishes conjugation of the yeast NED-8 homologue to the cullin Cdc53p. We are investigating the possibility that this gene encodes an E3 ligase for Nedd8.

Prasad BC et al. (1997) International C. elegans Meeting "THE C. ELEGANS unc-3 GENE ENCODES A HOMOLOGUE OF THE O/E FAMILY OF MAMMALIAN TRANSCRIPTION FACTORS"

Prasad BC, Reed RR, Seydoux GC

[International C. elegans Meeting, 1997]

The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development, and are also expressed transiently in neurons in the developing nervous system during embryogenesis. We have identified a C. elegans homologue of the mammalian O/E proteins, which displays greater than 80% similarity over 350 amino acids. The CeO/E cDNA maps to cosmid F42D1, previously shown to encode unc-3(1). We demonstrate that CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons as well as defects in dauer formation, a process requiring chemosensory input. Like its mammalian homologues, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development, and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. Currently, we are defining the DNA sequence binding specificity of the CeO/E protein. These observations suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and neuronal differentiation. (1) Sean Eddy, WBG 12(5):86 (February 1, 1993)

Delia O'Rourke et al. (2007) International Worm Meeting "C-type lectins: elucidating their function in worm immunity."

Delia O'Rourke, Jonathan Hodgkin

[International Worm Meeting, 2007]

The nematode pathogen Microbacterium nematophilum infects C. elegans by attaching to the rectal and post-anal cuticle, which results in slowed growth and a characteristic tail swelling called the Dar response. The swelling is a protective host response and is induced by the ERK MAP kinase pathway (Nicholas et al 2004). Previously we investigated the genomic response of C.elegans to this infection, using microarrays to examine gene expression changes following infection. We found induction of genes encoding a number of putative defence proteins, including ten C-type lectins (ORourke et al 2006). C-type lectin genes (clec) are also induced by other infections in C. elegans including Serratia marcescens (Mallo et al 2002) and Pseudomonas aeruginosa PA14 (Shapira et al 2006, Troemel et al 2006), with each infection inducing a specific set of lectin genes. Their function in the immune response could be to act as antibacterial effectors and /or as pathogen recognition molecules. At least some of the clec genes are expressed in intestinal cells, consistent with a function in defence. We are now using a number of approaches to explore the role of clec genes in this host:pathogen interaction. We have shown that RNAi of clec-60, clec-17 and clec-86 affect C. elegans defence against M. nematophilum and we will present data on the effects of knockdown or knock out of the remaining seven M. nem-induced clec genes. We plan to express some of the induced clecs in the yeast Pichia pastoris, in order to study the CLEC proteins in isolation and conduct assays for bacterial binding and antibacterial activity. Within the worm, we are using TAP TAG technology to examine CLEC location (are these proteins secreted into the gut lumen?), to examine post-translational modifications and to identify interacting proteins. We will present progress and data from these experiments.