Mitchell, Diana et al. (2013) International Worm Meeting "Methods to Study Toxic Transgenes: Analysis of Protease-Dead Separase in Membrane Trafficking."

Uehlein, Lindsey, Mitchell, Diana, Bembenek, Joshua

[International Worm Meeting, 2013]

The C. elegans embryo provides an ideal system to study the processes of egg activation and cell division. We have demonstrated a novel role for separase, a conserved protease that facilitates chromosome segregation, in the regulation of membrane trafficking during cortical granule exocytosis and cytokinesis. We are interested in determining the role of separase's protease function in the regulation of membrane trafficking. To address this, we generated protease-dead separase mutant tagged with GFP (GFP::SEP-1-PD). We find that GFP::SEP-1-PD accumulates more strongly than wt GFP::SEP-1 at the furrow and midbody during anaphase and cytokinesis, suggesting it could be substrate trapping and have a role in membrane trafficking. The expression of GFP::SEP-1-PD causes embryonic lethality, suggesting that it is a dominant-negative transgene that interferes with endogenous separase function. To enable further study of GFP::SEP-1-PD, we developed methods that allow propagation of worm lines containing toxic transgenes. We are using these methods to enable the study of the protease function of separase in membrane trafficking in the C. elegans embryo. The first method involves feeding worms gfp(RNAi) bacteria to silence the transgene. Transgene expression takes on average 5 generations after removal from gfp(RNAi). Transgene re-expression is also temperature sensitive, allowing further control over its re-expression. We are using this method to obtain large numbers of embryos expressing GFP::SEP-1-PD for biochemical and proteomics analysis, which may allow us to identify novel separase substrates. The second method takes advantage of the pie-1 promotor, which drives transgene expression, and is not expressed in the male germline. Male worms containing GFP::SEP-1-PD can used to propagate the transgene and circumvent the generation delay in the gfp(RNAi) feeding method. We are employing this method to examine genetic interactions of PD-separase with genes in the membrane trafficking pathway known to be involved in cortical granule exocytosis and/or cytokinesis. The general application of these methods facilitates the study of dominant-negative/toxic transgenes using standard techniques in the C. elegans model system.

Vlachos IS et al. (2016) Nucleic Acids Res "DIANA-mirExTra v2.0: Uncovering microRNAs and transcription factors with crucial roles in NGS expression data."

Vlachos IS, Hatzigeorgiou AG, Chatzopoulos S, Lykokanellos F, Paraskevopoulou MD, Georgiou P, Karagkouni D, Vergoulis T, Georgakilas G, Christodoulou F, Dalamagas T

[Nucleic Acids Res, 2016]

Differential expression analysis (DEA) is one of the main instruments utilized for revealing molecular mechanisms in pathological and physiological conditions. DIANA-mirExTra v2.0 (http://www.microrna.gr/mirextrav2) performs a combined DEA of mRNAs and microRNAs (miRNAs) to uncover miRNAs and transcription factors (TFs) playing important regulatory roles between two investigated states. The web server uses as input miRNA/RNA-Seq read count data sets that can be uploaded for analysis. Users can combine their data with 350 small-RNA-Seq and 65 RNA-Seq in-house analyzed libraries which are provided by DIANA-mirExTra v2.0.The web server utilizes miRNA:mRNA, TF:mRNA and TF:miRNA interactions derived from extensive experimental data sets. More than 450 000 miRNA interactions and 2 000 000 TF binding sites from specific or high-throughput techniques have been incorporated, while accurate miRNA TSS annotation is obtained from microTSS experimental/in silico framework. These comprehensive data sets enable users to perform analyses based solely on experimentally supported information and to uncover central regulators within sequencing data: miRNAs controlling mRNAs and TFs regulating mRNA or miRNA expression. The server also supports predicted miRNA:gene interactions from DIANA-microT-CDS for 4 species (human, mouse, nematode and fruit fly). DIANA-mirExTra v2.0 has an intuitive user interface and is freely available to all users without any login requirement.

Bai, Xiaofei et al. (2015) International Worm Meeting "The Protease Activity of Separase Is Required for Both of Chromosome Segregation and Regulation of Membrane Trafficking During Cytokinesis."

Klebanow, Lindsey, Mitchell, Diana, Bembenek, Joshua, Bai, Xiaofei

[International Worm Meeting, 2015]

Chromosomal segregation and cytokinesis are tightly regulated processes during cell division. Separase is a cysteine protease that is involved in the cleavage of cohesin, which allows chromosome segregation from the onset of mitotic anaphase. Besides the canonical proteolytic function of separase in chromosome segregation, we also found that separase is localized to vesicles and is required for exocytosis during cytokinesis. To better understand the functions of the separase protease activity in chromosome segregation and cytokinesis, we generated a protease-dead separase mutant with a point mutation fused to green fluorescent protein GFP (SEP-1PD::GFP). We recently demonstrated that SEP-1PD::GFP expression is dominant negative. In order to better understand this phenotype, we investigated the SEP-1PD::GFP cellular phenotypes using live-cell imaging. Consistent with the dominant negative phenotype, we found that SEP-1PD::GFP expression causes chromosome nondisjunction, possibly due to impaired cohesin cleavage by the inactive protease. To test this, we used RNAi to partially knock down the cohesin scc-1, the subunit cleaved by separase, which significantly rescues embryo lethality and chromosome segregation defects. Additionally, SEP-1PD::GFP also shows enhanced accumulation to the ingressing furrow and the midbody during cytokinesis compared to wild-type protein (SEP-1WT::GFP). Previously, we have shown that RNAi depletion of separase causes an accumulation of RAB-11 positive vesicle at the cleavage furrow and midbody. To test whether SEP-1PD::GFP impairs RAB-11 trafficking during cytokinesis, we generated a strain with RAB-11::mCherry and SEP-1PD::GFP. Interestingly, we found that the expression of SEP-1PD::GFP also causes the abnormal accumulation of RAB-11 vesicles at the cleavage furrow. This result suggests that the protease activity of separase affects exocytosis of RAB-11 vesicles with the plasma membrane. Collectively, we favor the possibility that protease-dead separase traps substrates that should be cleaved by endogenous separase which impairs cohesin cleavage and RAB-11-vesicle exocytosis. In chromosome segregation, the protease activity of separase will cleave the cohesion SCC-1, whereas during cytokinesis, the protease activity of separase may cleave a putative substrate allowing RAB-11-vesicles to undergo exocytosis during cytokinesis. In the future, we hope to identify the putative substrates of separase required for exocytosis using GFP trap beads to purify both SEP-1WT::GFP and SEP-1PD::GFP from C. elegans..

Bai, Xiaofei et al. (2017) International Worm Meeting "A potential role for midbodies in developing tissues of C. elegans."

Simmons, Ryan, Chen, Bi-Chang, Turpin, Chris, Bembenek, Joshua, Klebanow, Lindsey, Bai, Xiaofei, Betzig, Eric, Mitchell, Diana

[International Worm Meeting, 2017]

The midbody forms at the end of cytokinesis and facilitates abscission, the final separation of the daughter cells. Recently, the midbody has been implicated in several developmental processes, including cell fate specification, dorsoventral axis formation, neurite growth, cilium formation, and apical polarity during epithelial lumen formation. To investigate developmental roles of the midbody, we examined midbody fate in the invariant lineage of the C. elegans embryo. Cytokinesis occurs in stereotypical patterns that are unique and tissue specific with a defined pattern of midbody inheritance. In the first mitosis, a relatively symmetric furrow results in a centrally positioned midbody that is always internalized by the P1 daughter cell. In the next AB cell division, a highly asymmetric furrow positions the midbody next to EMS, which internalizes it instead of either AB daughter cell. A dramatic shift in midbody behavior is observed in several tissues at around the 300-cell stage when the embryo undergoes global morphogenetic changes. In two lumen-forming tissues, the intestine and the pharynx, midbodies form after symmetric furrowing and migrate across the cell to the future apical midline. This coordinated midbody migration event coincides with polarization events in intestinal epithelia. At the apical midline, the midbody ring is internalized and disappears. However, Aurora B kinase (AIR-2) remains on the apical surface for over an hour after cytokinesis and polarization. A similar apical localization pattern is observed for AIR-2 in the pharyngeal primordium. Finally, in cells that form the inner labial sensilla (ILS), we observe symmetrical cytokinesis followed by a midbody migration event that leads to a focal aggregation of AIR-2. AIR-2 persists along the leading edge of developing dendrites (which also may be apical) as they migrate towards the anterior end of the embryo, anchor at the tip of the embryo and elongate. Other midbody markers are either internalized or maintained with AIR-2 in a tissue-specific manner. Inactivating several fast-inactivating temperature sensitive cytokinesis mutants late in embryogenesis causes severe defects in positioning, continuity and shaping of the intestinal and pharyngeal lumen. Many animals fail to hatch, but among those that do, a high percentage show defects in neuronal DiI staining. These data suggest that the proper execution of cytokinesis, which shows surprising flexibility during development, and specific cytokinesis regulators such as AIR-2, may regulate different aspects of development including the final interphase architecture of a terminally dividing cell.

Maragkakis M et al. (2011) Nucleic Acids Res "DIANA-microT Web server upgrade supports Fly and Worm miRNA target prediction and bibliographic miRNA to disease association."

Alexiou P, Reczko M, Vergoulis T, Kourtis K, Maragkakis M, Plomaritou K, Koziris N, Hatzigeorgiou AG, Dalamagas T, Gousis M

[Nucleic Acids Res, 2011]

microRNAs (miRNAs) are small endogenous RNA molecules that are implicated in many biological processes through post-transcriptional regulation of gene expression. The DIANA-microT Web server provides a user-friendly interface for comprehensive computational analysis of miRNA targets in human and mouse. The server has now been extended to support predictions for two widely studied species: Drosophila melanogaster and Caenorhabditis elegans. In the updated version, the Web server enables the association of miRNAs to diseases through bibliographic analysis and provides insights for the potential involvement of miRNAs in biological processes. The nomenclature used to describe mature miRNAs along different miRBase versions has been extensively analyzed, and the naming history of each miRNA has been extracted. This enables the identification of miRNA publications regardless of possible nomenclature changes. User interaction has been further refined allowing users to save results that they wish to analyze further. A connection to the UCSC genome browser is now provided, enabling users to easily preview predicted binding sites in comparison to a wide array of genomic tracks, such as single nucleotide polymorphisms. The Web server is publicly accessible in www.microrna.gr/microT-v4.

Gilchrist EJ et al. (1995) International C. elegans Meeting "A C. ELEGANS MYOTONIC DYSTROPHY GENE HOMOLOGUE."

Gilchrist EJ, Baillie DL

[International C. elegans Meeting, 1995]

Myotonic Dystrophy is an inherited, autosomal dominant disease that results in a progressive wasting of the skele- tal muscles (and sometimes heart and smooth muscles) in humans. The gene has been cloned and sequenced (R. Korneluk, personal communication) and encodes a large protein that has been localised to neuromuscular and myotendinous junctions. The N-terminus domain bears similarity to cAMP-dependent serine/threonine protein kinases. Yuji Kohara, in his cDNA sequencing project, identified a C. elegans gene that is 75% similar (if conserved amino acids are considered) to the kinase domain of the human myotonic dystrophy gene. He mapped the cDNA to LG I (personal communication), and, through Southern analysis, we have placed the cDNA on cosmid K06G1. This cosmid was injected into wild-type N2 hermaphrodites by Diana Collins, and we are currently trying to rescue some of the genes in this region with the extrachromosomal array. One candidate gene was unc-57. Animals homozygous for mutations in unc-57 show no obvious structural muscle defects. This is consistent with unc-57 being a muscle, or neuro- muscular kinase. However, the Unc-57 phenotype is not rescued in animals carrying the K06G1 extrachromo- somal array. We are now focusing on some of the lethal mutations in this region that have been isolated in the laboratory of Ann Rose at UBC.

Mitchell PH et al. (2008) European Worm Meeting "Modelling alcohol dependence in Caenorhabditis elegans"

James C, Mitchell PH, Mould RN, Hopper NA, Glautier SP, Dillon J, Oconnor VM, Holden-Dye LM,, Adrianakis I

[European Worm Meeting, 2008]

Ethanol is one of the most widely used and socially acceptable drugs in the. world. However its chronic use can lead to serious problems due to the. development of dependence. Ethanol has been shown have wide-ranging but. selective effects on many different neurotransmitter circuits including;. glutamatergic, GABAergic, serotonergic, dopaminergic and opioid peptidergic. systems. The basis for alcohol tolerance and withdrawal involves. counteradaptive neurochemical changes within these systems to adapt to the. prolonged presence of ethanol in the brain. Here we are using C. elegans to. enable an analysis at all levels of organization from gene, molecule, and. neurone through to neuronal circuit and behaviour. We want to use C.. elegans to investigate the development of neuroadaptation to ethanol. In. order to achieve this we have first provided evidence that ethanol rapidly. equilibriates across the cuticle of C. elegans (Mitchell et al. 2007) thus. enabling experiments in which the concentration-dependent effects of. ethanol on behaviour can be defined. In line with previous studies (Davies. et al. 2003) we have established that external concentrations of ethanol. >100mM are required to overtly affect the visually observable measures of. behaviour. This is equivalent to concentrations that are supra-intoxicating. in mammals. In order to determine whether ethanol elicits any effects on. C. elegans at concentrations equivalent to those that are intoxicating in. mammals we have performed more discreet analyses of behaviours. Pharyngeal. recordings have revealed that concentrations as low as 10mM ethanol have. significant effects on the activity of neural networks, thus providing an. opportunity to investigate the molecular determinants of acute ethanol. effects. We are also in the process of refining the analysis of locomotory. behaviour with the aim of determining whether ethanol elicits effects at. low mM concentrations. As part of this effort we have developed a paradigm. in which we can demonstrate withdrawal from ethanol, and relief from. withdrawal due to low concentrations of acute ethanol. We have used this to. study the onset and recovery rates of this neuroadaptation and the. concentration dependence of this effect in order to understand the. mechanisms involved. This has been extended to investigate if genes already. implicated in acute effects of ethanol in C. elegans are involved in. controlling the withdrawal and tolerance behaviours. These studies are. aimed at better defining how well C. elegans can model aspects of. alcoholism.. Funded by the BBSRC, UK.. References. Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele,. T. R., Bonci, A., Bargmann, C. I., & McIntire, S. L. 2003, "A central role. of the BK potassium channel in behavioral responses to ethanol in C.. elegans", Cell, vol. 115, no. 6, pp. 655-666.. Mitchell, P. H., Bull, K., Glautier, S., Hopper, N. A., Holden-Dye, L., &. O''connor, V. 2007, "The concentration-dependent effects of ethanol on. Caenorhabditis elegans behaviour", Pharmacogenomics.J., vol. 7, no. 6, pp.. 411-417.

Stewart HI et al. (1996) West Coast Worm Meeting "Rescue of New Essential Genes on LGIII(left), in the Nematode Caenorhabditis elegans."

Stewart HI, Franz NW, Barbazuk BW, Baillie DL, Ha TT

[West Coast Worm Meeting, 1996]

We utilized trangenic strains containing cosmids, sequenced by the sequencing consortium, to rescue the lethal phenotype of several essential genes (See presentation by Diana Janke et al, this session). The rescued genes are positioned on LGIII(left) balanced by the duplication sDp3(see poster; Stewart et al, this session). A set of overlapping deficiencies was utilized to define areas within which to focus our rescue attempts). PCR analysis was utilized to further define the end points of these deficiencies, facilitating our analysis ( see poster ; Norm Franz et al, this session). We concentrated our analysis on areas to the right of dpy-17, in the interval between dpy-17 to dpy-19. Lethals mapping to sDf125 that were not rescued by sDp8 are presented in a poster by Nigel O'Niel et al, this session. We have rescued several essential genes. Our rescues will facilitate the functional analysis of these new genes. These rescues have also helped us to align and correlate the physical and genetic maps. Our estimates are that there will be more than two essential genes per cosmid. A total of 202 recessive lethal mutations have been generated, using EMS as a mutagen. We intend to make molecular assignments of all of these elements, through our cosmid rescue experiments. As we have estimated that there is only a 28% saturation of the area for essential genes, we will be generating more recessive lethal genes, to facilitate this analysis. This work has been funded by grants from the National Institute of Health (N.I.H.), U. S.A. to Ann Rose; D. Riddle and David Baillie and Canadian Genome and Advanced Technologies (C.G.A.T.), Canada to Ann Rose and David Baillie.

Caroline Bowen et al. (2006) European Worm Meeting "RNA Degradation in the Nematode Worm C.elegans"

Caroline Bowen, Alison Woollard

[European Worm Meeting, 2006]

Caroline Bowen and Alison Woollard RNA turnover is necessary to maintain basal levels of proteins, through mRNA regulation, and to remove aberrant mRNA transcripts. Here we examine processes of RNA degradation in C.elegans. This approach is based on a functional investigation of the C.elegans components of the RNA degradation pathways, identified from homology to other organisms.. The 5-3 RNA degradation pathway in C.elegans has been previously identified (Newbury and Woollard 2004). Here we investigate the 3-5 pathway in C.elegans, starting with the identification of its core and accessory components. 3-5 exoribonucleic acid decay, first identified in S.cerevisae, is brought about by a complex of exoribonucleases, helicases and associated factors, known as the exosome, (Mitchell et al 1997). The exosome was hailed as a proteosome for RNA, due to the degradation properties of the heteromeric complex.. We have used RNAi and an available mutant to show that disruption of the exosome complex, through the targeted destruction of individual components, results in worms with severe developmental problems including slowed growth rates. A full length GFP tagged exosome component, has shown the component to be largely confined to the nucleus of all cells, including the germline. Phenotypes associated with depletion of the exosome components include slow growth and developmental problems. These are attributed to effects on rRNA processing. When exosome components are silenced, there is a failure of the degradation of specific cleaved rRNA precursors, which in turn effects ribosome function and subsequently protein synthesis. We will present our findings on the nature of these rRNA processing defects, which will help to clarify the molecular basis of the phenotypes we observe.

Stewart HI et al. (1995) International C. elegans Meeting "CAENORHABDITIS ELEGANS: BALANCERS, RECESSIVE LETHAL MUTATIONS AND DEFICIENCIES ON LGIII(LEFT) AND LGV(RIGHT)."

Ha TT, Stewart HI, Baillie DL, Rose AM, Collins D

[International C. elegans Meeting, 1995]

The new balancers sC1 and sDp3 along with the well documented eT1 balancer (Rosenbluth et al, 1981) completely balance LGIII. We have validated sC1 and sDp3 by isolating recessive lethal mutations and have generated a set of overlapping deficiencies spanning these balancer systems. A set of 91 new essential genes were isolated, while validating sDp3, as a balancer. These recessive lethal mutations were also utilized in the initial mapping of the new set of deficiencies. We have 11 confirmed deficiencies and have several other putative deficiencies, balanced by sDp3. Work validating the new balancer sC1 is also well underway. We have isolated over 70 recessive mutations and have also generated a set of deficiencies, in this region, two of which have been confirmed. As the sequencing of LGIII is rapidly being completed by the sequencing consortium, work on the correlation of the genetic and physical maps is also progressing. About 100 transgenic strains, using sequenced cosmids, have been constructed to aid in this work (See poster Diana Collins et al, this session). LGV(Right) is currently balanced by nT1 and sC4. sC4 is a new balancer, unc-76 rol-9/ sC4[dpy-21], which is not homozygous viable. It incompletly balances the right end of LGV. Work is in progress to develop a new balancer for the far right of LGV., using a derivative of nT1. The genetic tools provided by this reasearch are available to the C. elegans genetic community on request. Information on how to access these mutations, through the www site, is available at http: This research is being funded by The National Institutes of Health (NIH) U.S.A.