Sean M. O'Rourke

The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.

Dynein modifiers in C. elegans: light chains suppress conditional heavy chain mutants.

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis.

Caenorhabditis elegans EFA-6 limits microtubule growth at the cell cortex

Microtubules are polymers of tubulin heterodimers that exhibit dynamic instability: periods of growth followed by periods of shrinkage. However, the molecular regulation of dynamic instability remains elusive. Here, we show that EFA-6, a cortically-localized protein, limits the growth of microtubules near the cell cortex of early embryonic cells from Caenorhabidits elegans, possibly by inducing microtubule catastrophes. Compared with wild type, embryos lacking EFA-6 had abnormally long and dense microtubules at the cell cortex, and growing microtubule plus ends resided at the cortex for up to five-fold longer. Loss of EFA-6 also caused excess centrosome separation and displacement towards the cell cortex early in mitosis, and subsequently a loss of anaphase spindle-pole oscillations and increased rates of spindle elongation. The centrosome separation phenotype was dependent on the motor protein dynein, suggesting a possible link between the modulation of microtubule dynamics at the cortex and dynein-dependent force production. EFA-6 orthologues activate ARF6-type GTPases to regulate vesicle trafficking. However, we show that only the C. elegans EFA-6 amino-terminus is both necessary and sufficient to limit microtubule growth along the cortex, and that this function is independent of ARF-6.

A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes.

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.

Maternally expressed and partially redundant β-tubulins in Caenorhabditis elegans are autoregulated

The mitotic spindle, which partitions replicated chromosomes to daughter cells during cell division, is composed of microtubule assemblies of α/β-tubulin heterodimers. Positioning of the mitotic spindle influences the size and location of daughter cells, and can be important for the proper partitioning of developmental determinants. We describe two semi-dominant mis-sense mutations in tbb-2, one of two C. elegans β-tubulin genes that are maternally expressed and together are required for microtubule-dependent processes in the early embryo. These mutations result in a posteriorly displaced and misoriented mitotic spindle during the first cell division. In contrast, a probable tbb-2 null allele is recessive, and when homozygous results in less severe spindle positioning defects and only partially penetrant embryonic lethality. Two of the tbb-2 mutations result in reduced levels of TBB-2 protein, and increased levels of a second maternally expressed β-tubulin, TBB-1. However, levels of TBB-1 are not increased in a tbb-2 mutant with an allele that does not result in reduced levels of TBB-2 protein. We conclude that feedback regulation influences maternal β-tubulin expression in C. elegans, but cannot fully restore normal microtubule function in the absence of one β-tubulin isoform.

Using RNA Interference to Identify Specific Modifiers of a Temperature-Sensitive, Embryonic-Lethal Mutation in the Caenorhabditis elegans Ubiquitin-Like Nedd8 Protein Modification Pathway E1-Activating Gene rfl-1

The essential Caenorhabditis elegans gene rfl-1 encodes one subunit of a heterodimeric E1-activating enzyme in the Nedd8 ubiquitin-like protein conjugation pathway. This pathway modifies the Cullin scaffolds of E3 ubiquitin ligases with a single Nedd8 moiety to promote ligase function. To identify genes that influence neddylation, we used a synthetic screen to identify genes that, when depleted with RNAi, enhance or suppress the embryonic lethality caused by or198ts, a temperature-sensitive (ts) mutation in rfl-1. We identified reproducible suppressor and enhancer genes and employed a systematic specificity analysis for each modifier using four unrelated ts embryonic lethal mutants. Results of this analysis highlight the importance of specificity controls in identifying genetic interactions relevant to a particular biological process because 8/14 enhancers and 7/21 suppressors modified lethality in other mutants. Depletion of the strongest specific suppressors rescued the early embryonic cell division defects in rfl-1(or198ts) mutants. RNAi knockdown of some specific suppressors partially restored Cullin neddylation in rfl-1(or198ts) mutants, consistent with their gene products normally opposing neddylation, and GFP fusions to several suppressors were detected in the cytoplasm or the nucleus, similar in pattern to Nedd8 conjugation pathway components in early embryonic cells. In contrast, depletion of the two strongest specific enhancers did not affect the early embryonic cell division defects observed in rfl-1(or198ts) mutants, suggesting that they may act at later times in other essential processes. Many of the specific modifiers are conserved in other organisms, and most are nonessential. Thus, when controlled properly for specificity, modifier screens using conditionally lethal C. elegans mutants can identify roles for nonessential but conserved genes in essential processes.

Rapid Mapping and Identification of Mutations in Caenorhabditis elegans by Restriction Site-Associated DNA Mapping and Genomic Interval Pull-Down Sequencing

Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems.

Genomics: frontiers of gene function.

The technique of RNA interference continues to pay dividends. The latest application of the method to the nematode worm adds detail to the list of genes known to function in the early stages of development.Multi-screen testTo turn the wealth of information generated by genome sequencing into knowledge of the biological function of genes requires screening studies of the scale reported this week for Caenorhabditis elegans. This project to identify genes required for cell division used genome-wide RNA interference to block expression of 98% of the genes in the genome. Then over 4,000 time-lapse movies were made by differential interference contrast microscopy — and watched — to work out which gene hits affected the first two rounds of mitotic cell division after fertilization. Upwards of 650 genes are required for these early stages of embryogenesis.

Abstract LB-085: Protein phosphatase 1 and the RNA-binding protein CSTL2 function to constrain daughter centriole number

Centrioles are cylindrical microtubule-based structures that are required for the formation of cilia, flagella and the mitotic spindle. A centrosome is comprised of two centrioles (one mother and one daughter) that are organized in an orthogonal orientation. Centrioles are duplicated only once during each cell cycle and this involves the formation of a single daughter centriole next to each mother. Dysregulation of this process yields an abnormal centriole number and this can result in aneuploidy, a hallmark of cancer cells. Therefore, it is critical that centriole duplication (CD) is tightly regulated. The nematode C. elegans is an excellent model system to study the process of CD because the core components of the CD pathway in C. elegans are conserved in humans. The main purpose of this study is to identify novel regulators of CD using C. elegans as a model system. The kinase ZYG-1 is a functional ortholog of human PLK4 and is absolutely essential for CD. Using a zyg-1 suppression assay, we have identified Protein Phosphatase 1 (PP1) as a critical inhibitor of CD. Using super-resolution microscopy, we found that deactivating PP1 results in the formation of multiple daughter centrioles adjacent to a single mother. Western blot analysis indicated that the mechanism by which PP1 inhibits CD is by decreasing ZYG-1 levels. Our study has also identified an RNA-binding protein with homology to human cleavage stimulation factor subunit 2 tau variant (CSTL-2) as a potential PP1 substrate and a novel component of the C. elegans CD pathway. Utilizing a fluorescently tagged CSTL-2 transgenic worm line, we determined that CSTL-2 localizes to the centrosomes during mitosis. An immunoprecipitation assay followed by mass spectrometry has identified CSTL-2 as an interacting partner of PP1- thereby implicating it as a PP1 substrate. To evaluate the effect of CSTL-2 on ZYG-1-mediated CD, cstl-2-null; zyg-1-hypomorphic double mutants were constructed. At the restrictive temperature, the zyg-1-hypomorphic mutant worms show 100% embryonic lethality due to a failure of CD. However, in the cstl-2-null; zyg-1-hypomorphic double mutants, approximately 20% of the embryos survive indicating a role for CSTL-2 as an inhibitor of CD. We performed cytological analyses on cstl-2-null; zyg-1-hypomorphic double mutant embryos and confirmed that CSTL-2, like PP1, also functions as an inhibitor of CD. Therefore, based upon our data, we conclude that PP1 is an important negative regulator of CD which functions to ensure that only one daughter centriole forms adjacent to a mother centriole and we propose that CSTL-2 is a substrate through which PP1 regulates CD. In summary, this study highlights a novel pathway that controls centriole number in C. elegans, which may be dysregulated in cancer. Citation Format: Jyoti Iyer, Neena Peel, Sean O9Rourke, Bruce Bowerman, Kevin O9Connell. Protein phosphatase 1 and the RNA-binding protein CSTL2 function to constrain daughter centriole number. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-085. doi:10.1158/1538-7445.AM2015-LB-085

Pushing Your Back into Place

Establishment of the dorsal-ventral body axis during fly oogenesis depends on pushing forces provided by polymerizing microtubules. In multicellular animals, the localization of specific molecules to some but not other cells during development defines three body axes: anterior-posterior (AP; head to tail), dorsal-ventral (DV; back to belly), and left-right. On page 999 of this issue, Zhao et al. (1) describe an elegant application of live-cell imaging to investigate molecular polarity and body axis establishment in the fruit fly Drosophila melanogaster. Their insights debunk a long-standing linkage between the mechanisms that establish the AP and DV body axes, and illuminate a new role for microtubule polymerization pushing forces.