Christopher Richie et al. (2008) Development & Evolution Meeting "Characterization of Genetic Suppressors of Separase"

Christopher Richie, Andy Golden

[Development & Evolution Meeting, 2008]

Accurate chromosome segregation requires separation of the sister chromatids during the metaphase-to-anaphase transition. This is achieved when the anaphase-promoting complex (APC) brings about the destruction of securin, an inhibitor of the protease known as separase. Once activated, separase can then cleave the SCC-1 protein, a member of the cohesion complex that holds the sister chromatids together. In C. elegans carrying a temperature-sensitive (ts) allele of separase (e2406), growth at 25oC causes a pleiotropic phenotype including embryonic lethality, sterility, and vulval defects. We have obtained two new alleles of sep-1 (ax110 and ax521) (Furuta and Schumacher, per. comm.). We have characterized their respective phenotypes, which include defective meiosis and embryonic lethality, but confer less severe developmental defects compared to e2406. In an effort to identify novel regulators and substrates of separase, we have isolated two suppressor mutants, av101 and av102, that rescue the lethality of the sep-1(e2406) allele when grown at 20oC, and neither suppressor allele has a phenotype in an otherwise wild type background. We have cloned av101, and identified it as pph-5, a conserved protein phosphatase that has been previously reported as an APC-interacting protein (Ollendorff and Donoghue, 1997). Interestingly, this allele also suppresses the embryonic lethality of sep-1(ax110) but not that of sep-1(ax521) or sep-1 RNAi. Using epifluorescence confocal microscopy we have observed GFP::PPH-5 to localize to the kinetochore microtubules of mitotic cells, a pattern similar to that seen with anti-SEP-1 antibodies. We have purified the PPH-5 protein complex using the tandem-affinity-purification (TAP) technique and identified DAF-21, the C. elegans ortholog to Hsp90, to be present in this complex. This interaction is conserved across species, and Hsp90 is known to regulate several kinase targets including Erk kinase and Cdk1/cyclin B (Hinds et al, 2007, Helmbrecht et al, 2000). Injection of daf-21 dsRNA results in Po sterility due to precocious activation of CDK-1, which is a direct negative regulator of separase activity (Inoue et al, 2006). We are currently testing a model in which reduction of PPH-5 activity results in the suppression of sep-1 phenotypes by deregulating CDK-1 via DAF-21.

Christopher Richie et al. (2006) Development & Evolution Meeting "Identification and characterization of separase cofactors in C. elegans"

Christopher Richie, Andy Golden

[Development & Evolution Meeting, 2006]

Accurate chromosome segregation requires separation of the sister chromatids during the metaphase-to-anaphase transition. This is achieved when the anaphase-promoting complex (APC) brings about the destruction of securin, an inhibitor of the cysteine protease known as separase. Once activated, separase can then cleave the SCC-1 protein, a member of the cohesin complex that holds the sister chromatids together. In C. elegans, the sep-1(e2406) temperature-sensitive mutant allele of separase causes a pleiotropic phenotype when worms are grown at 20°C (Siomos et al, 2001). In addition to complete sterility and embryonic lethality, sep-1(e2406) also causes developmental abnormalities in the germ line, somatic gonad, and the vulva. We have recently isolated new temperature sensitive alleles of sep-1 (ax110 and ax521) and are currently characterizing their respective phenotypes. In an effort to identify novel regulators and substrates of separase, we have performed a suppressor screen to select for mutations that can rescue the sterility and embryonic lethality associated with the sep-1(e2406) allele when grown at the restrictive temperature. One such allele, designated supE, suppresses sep-1(e2406 and ax110), but acts as an enhancer of ax521. We have mapped supE to a relatively small physical interval with 53 predicted coding sequences, and are currently attempting to identify candidates by RNAi. To date, RNAi of one candidate in the region appears to suppress both sep-1(e2406 and ax110) quite well. This data will be presented at the meeting.

Christopher T Richie et al. (2007) International Worm Meeting "Identification and characterization of separase cofactors in C. elegans."

Andy Golden, Christopher T Richie

[International Worm Meeting, 2007]

Accurate chromosome segregation requires separation of the sister chromatids during the metaphase-to-anaphase transition. This is achieved when the anaphase-promoting complex (APC) brings about the destruction of securin, an inhibitor of the protease known as separase. Once activated, separase can then cleave the SCC-1 protein, a member of the cohesion complex that holds the sister chromatids together. In C. elegans the sep-1(e2406) temperature-sensitive (ts) mutant allele of separase causes a pleiotropic phenotype when worms are grown at 20oC (Siomos et al, 2001). In addition to sterility and embryonic lethality, sep-1(e2406) also causes developmental abnormalities in the germ line, somatic gonad, and the vulva. We have obtained two new ts alleles of sep-1 (ax110 and ax521) (Furuta and Schumacher, per. comm.). We are characterizing their respective phenotypes, which include defective meiosis and embryonic lethality, but confer less severe developmental defects compared to e2406. In an effort to identify novel regulators and substrates of separase, we have isolated two suppressor mutants, av101 and av102, that rescue the lethality of the sep-1(e2406) allele when grown at 20oC, and we are investigating the effects of these suppressor alleles in combination with other sep-1 mutants and in a wild type background. We have cloned av101, and identified it as pph-5, a conserved protein phosphatase that has been previously reported as an APC-interacting protein (Ollendorff and Donoghue, 1997). Interestingly, this allele also suppresses the embryonic lethality of sep-1(ax110) but not that of sep-1(ax521) or sep-1 RNAi. Using fluorescence confocal microscopy we have observed PPH-5 to localize to the kinetochore microtubules of mitotic cells, a pattern similar to that seen with anti-SEP-1 antibodies. We are currently investigating whether there is a physical interaction between SEP-1 and PPH-5 by isolating the PPH-5 protein complex using the tandem-affinity-purification (TAP) technique. Although the av102 suppressor remains unidentified, we have used SNP analysis to map it to an interval containing 23 genes on Linkage Group III. We are currently using an RNAi feeding strategy to phenocopy the suppression of sep-1(e2406). Our future efforts will include the TAP-purification of the SEP-1 protein complex and the isolation of additional suppressor mutations in the background of sep-1(ax110 and ax521).

Christopher T Richie et al. (2005) International Worm Meeting "Identification and characterization of separase cofactors in C. elegans"

Andy Golden, Tokiko Furuta, Christopher T Richie, Jill M Schumacher, Bonnielin Scuerman, Barry Chestnut

[International Worm Meeting, 2005]

Accurate chromosome segregation requires separation of the sister chromatids during the metaphase-to-anaphase transition. This is achieved when the anaphase-promoting complex (APC/cyclosome) brings about the destruction of securin, an inhibitor of the cysteine protease known as separase. Once activated, separase can then cleave the SCC-1 protein, a member of the cohesion complex that holds the sister chromatids together.In C. elegans, the sep-1(e2406) temperature-sensitive mutant allele of separase causes a pleiotropic phenotype when worms are grown at 20C (Siomos et al, 2001). In addition to complete sterility and embryonic lethality, sep-1(e2406) also causes developmental abnormalities in the germ line, somatic gonad, and the vulva. We have recently isolated new temperature sensitive alleles of sep-1 (ax110 and ax521) and are currently characterizing their respective phenotypes.In an effort to identify novel regulators and substrates of separase, we have performed a suppressor screen to select for mutations that can rescue the sterility and embryonic lethality associated with the sep-1(e2406) allele when grown at the restrictive temperature. Mutant alleles representing three different loci were recovered and we are currently mapping their position using molecular and classical genetic techniques. We are investigating the ability of these suppressor alleles to modulate the new sep-1 mutant phenotypes. We are also determining the phenotype of these suppressor mutants alone, and whether they act in a similar process by constructing double mutants.

Christopher T Richie et al. (2004) West Coast Worm Meeting "Identification and Characterization of Separase Cofactors in C. elegans"

Andy Golden, Barry Chestnut, Bonnielin Sceurman, Christopher T Richie

[West Coast Worm Meeting, 2004]

Accurate chromosome segregation requires separation of the sister chromatids during the metaphase-to-anaphase transition. This is achieved when the anaphase-promoting complex (APC/cyclosome) brings about the destruction of securin, an inhibitor of the cysteine protease known as separase. Once activated, separase can then cleave the SCC-1 protein, a member of the cohesion complex that holds the sister chromatids together. In C. elegans, the sep-1(e2406) temperature-sensitive mutant allele of separase causes a pleiotropic phenotype when worms are grown at 20degC (Siomos et al, 2001). In addition to complete sterility and embryonic lethality, sep-1(e2406) also causes developmental abnormalities in the germ line, somatic gonad, and the vulva. In an effort to identify novel regulators and substrates of separase, we have performed a suppressor screen to select for mutations that can rescue the sterility and embryonic lethality associated with the sep-1(e2406) allele when grown at the restrictive temperature. Mutant alleles representing three different loci were recovered and we are currently mapping their position using molecular and classical genetic techniques. We are also using a proteomics approach to identify separase cofactors and substrates by both coimmunoprecipitation with anti-separase antibodies, and affinity-tag pull-down of complexes containing ectopically expressed separase derived from transgenic worms. Mass spectroscopy will be used to characterize any co-purifying proteins.

Page K E et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Genes Affecting Aluminium Toxicity in Caenorhabditis elegans"

McCrohan C R, Page K E, White K N, Lithgow G J

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Aluminium (Al) is a highly abundant crustal metal with known toxic effects in multiple biological systems. Using the nematode C. elegans we have found a set of genetic modulators of Al toxicity. C. elegans is widely used for toxicity and aging studies due to its small size, large progeny numbers, short lifespan, and simple methods of genetic manipulation. This makes C. elegans a good model to study the toxicity mechanism of Al in animals. The exposures were carried out on agar plates, with Al in the form of Al(NO3)3, mixed with a concentrated Escherichia coli food source. Here we show that a selection of genes involved in the mechanisms of Al toxicity and/or metabolism have potential relevance in the metallostasis (metal homeostasis) of other metals, or are key regulators of known stress resistance pathways. Al negatively affects C. elegans developmental progression from 3 microM Al, fertility at 30 microM Al, and body size from 1.9 mM Al. The developmental delay phenotype caused by Al exposure can be passed on to the un-exposed next generation. At 4.8mM exposures Al affects the levels of Al and other elements in C. elegans; shown using ICP-OES. We are also investigating the effect of Al exposure to the course of normal aging in C. elegans.

Shelli N Williams et al. (2002) Mid-west Worm Meeting "Cytoskeletal defects associated with NUD-1 knockdown in early embryogenesis of C. elegans"

Shelli N Williams, Kim A Caldwell, Guy A Caldwell

[Mid-west Worm Meeting, 2002]

Dawe et al. Dev Genes Evol. (2001) 211:434-441.

Page, Kathryn E. et al. (2009) International Worm Meeting "Exploring Aluminium Toxicity in C. elegans."

Page, Kathryn E., McCrohan, Catherine R., White, Keith N., Lithgow, Gordon J.

[International Worm Meeting, 2009]

Aluminium (Al) is a highly abundant crustal metal with known toxic effects in multiple biological systems. Caenorhabditis elegans (C. elegans) is widely used for toxicity and aging studies due to its small size, large progeny numbers, short lifespan, and simple methods of genetic manipulation. C. elegans is a good model to study the toxic mechanism of Al in animals, and has been widely used for the study of environmental metal toxicology. Here we show Al effects fertility, and development progression, and that these phenotypes are transferable to the progeny of exposed worms. The exposures were carried out on agar plates with the Al mixed with concentrated OP50 which was then fed to the worms. Al negatively affects C. elegans developmental progression at 0.1 to 300 mM Al food concentration, and fertility at 1 mM Al food concentration. The developmental delay phenotype caused by Al exposure can be passed on to the next generation. It is possible that the mechanism of Al toxicity is similar to that of other toxic metals.

Richard S Allan et al. (2005) International Worm Meeting "High-resolution, digitally-encoded phenotypic analysis of gonadogenesis in C. elegans"

E Jane Albert Hubbard, Richard S Allan, Philip MacMenamin, Kristin C Gunsalus, Fabio Piano, Krishna Vijayendran

[International Worm Meeting, 2005]

A fundamental premise of genetic analysis is that genes with similar loss-of-function phenotypes likely act in similar developmental processes. Extending this concept to RNAi-based genome-scale functional characterization, Piano et al. (2002) defined 47 embryonic phenotypic characters in C. elegans and generated a digital phenotypic signature for each RNAi experiment. Genes conferring similar signatures were clustered (phenoclusters), placing previously uncharacterized genes in clusters with genes of known function. The primary data (high-magnification time-lapse movies), scoring criteria and results are publicly available and can be analyzed and queried using additional tools (e.g. Phenoblast) (Gunsalus et al., 2004; http://www.RNAi.org).We have applied this approach to gonadogenesis and germline development in C. elegans. In previous large-scale RNAi screens, over 800 genes are reported to confer a sterile (Ste) or sterile progeny (Stp) phenotype, based on low-magnification screening (Gonczy et al., 2000; Fraser et al. 2001; Piano et al., 2001; Maeda et al., 2001; Hanazawa et al. 2001; Piano et al., 2002; Kamath et al., 2003; Simmer et al., 2003). A subset of these have been examined under high magnification (Maeda et al., 2001; Hanazawa et al., 2001; Colaiacovo et al., 2002), but these data are not available in a systematic digital format amenable to analyses such as phenoclustering or Phenoblast, nor to phenotypic search criteria.We developed and are implementing a web-based digital scoring system (currently 262 characters) for high-magnification phenotypes in adult hermaphrodites. Characters include both quantitative and qualitative features of the gonad, germ line and gametes as well as several gonad-related characters. We generate a series of digital Z-plane images at high magnification from each RNAi experiment. All data will be made publicly available on the RNAi.org website. Our test set is 147 Stp genes and 260 Ste genes conferring a wide range of RNAi-induced defects.

Page, Kathryn E. et al. (2011) International Worm Meeting "Aluminium Toxicity and Elemental Composition Changes in Caenorhabditis elegans."

Killilea, David W., Lithgow, Gordon J., McCrohan, Catherine R., White, Keith N., Page, Kathryn E.

[International Worm Meeting, 2011]

Environmental metal contamination events such as the alumina sludge spill in Hungary last year highlight the importance of understanding metal toxicity and predicting detrimental effects. Aluminium (Al) is highly abundant in our environment, but can elicit a variety of toxic responses. Here we present the first characterization of the effects of Al exposure in the nematode worm Caenorhabditis elegans by identifying phenotypic abnormalities and disruption in whole body metal homeostasis (metallostasis) following Al exposure. Lifespan was decreased in worms exposed to low levels of Al, but was unexpectedly increased when the Al concentration reached higher levels. Interestingly, this bi-phasic phenotype was only observed when Al exposure occurred during development, as adult worm lifespan was unaffected by Al at similar exposures. Al negatively affected C. elegans developmental progression and self-fertility when exposed to as little as 30mM Al, and reduced body size when exposed to 1.9 mM Al. Significant developmental delay was observed even when Al exposure was restricted to embryogenesis. Widespread changes to the elemental content of adult nematodes was observed when chronically exposed to Al from L1. Specifically we saw increased Ba, Cr, Cu, and Fe content, and a reduction in Ca levels. Similar changes have been noted in human Al toxicity, suggesting that C. elegans may be exploited to understand the mechanisms of Al toxicity in human tissue. Elemental homeostasis is obviously highly regulated, and here we see that changing the levels of only one element can alter the whole elemental profile. This raises the question of how genes affect these elemental profiles. Here we determine how gene knockdown can alter metallostasis and elemental profiles in C. elegans. It is notable that these genetic modulators of elemental profiles are not only important for metallostasis, but are key regulators of known stress response mechanisms. Knock-down of these gene products alters the elemental profile of the worm, which suggests novel mechanisms of action for these genes in metal toxicity and general stress response.