Lim, Yun-Ki et al. (2011) International Worm Meeting "Regulation of calreticulin gene expression by sumoylation in C. elegance."

Lee, Sunkyung, Kim, Do Han, Ahnn, Joohong, Lim, Yun-Ki

[International Worm Meeting, 2011]

Sumoylation is a reversible post-translational modification which is involved in diverse biological processes including protein stability, nuclear-cytosolic transport, DNA binding activity, and gene expression. In C.elegans, sumoylation has been shown to be critical in gonadal development by modulating chromatin integrity and transcription (Broday et al., Genes & Dev. 18, 2380 2004). Calreticulin is a calcium binding protein residing in endoplasmic reticulum that plays pivotal roles in calcium homeostasis, chaperone activity, and glycosylation and transcriptionally up-regulated by unfolded protein response (UPR) in C.elegans. In order to understand the role of sumoylation on the UPR response in nematode, we attempted to study the function of SUMO conjugating enzyme UBC-9 on calreticulin gene regulation. ubc-9 mutant worms showed defective vulval development similar to smo-1, lack of SUMO protein. We found that, upon abolishing sumoylation, GFP fluorescence driven by calreticulin promoter was increased in C. elegans. Furthermore, the increased GFP fluorescence was attenuated by knockdown of xbp-1, indicating that sumoylation is required for transcriptional repression of calreticulin in C. elegans. We are currently investigating underlying mechanism to elucidate how sumoylation controls gene expression of calreticulin in C.elegans.

Peter Swoboda et al. (2002) European Worm Meeting "A multifaceted approach to elucidate the regulation of ciliogenesis in C. elegans"

Evgeni Efimenko, Peter Swoboda

[European Worm Meeting, 2002]

Cilia are evolutionarily conserved subcellular organelles functioning in cell motility, movement of extracellular fluids, sensory perception (e.g. smell) and determination of left-right asymmetry. While a great deal is known about the structure, function and motility of cilia, very little is known about the molecular mechanisms that regulate ciliogenesis in a cell-type specific and developmental manner. How do cilia become functional? How can they do what they do?

Song, Hyun-Ok et al. (2009) International Worm Meeting "Pleiotropic roles of a calcium binding protein, calumenin in C. elegans."

Song, Hyun-Ok, Sung, Hyun, Ahnn, Joohong, Singaravelu, Gunasekaran, Kim, Do Han, Dwivedi, Meenakshi, Kwon, Soonjae Kwon

[International Worm Meeting, 2009]

Calumenin is a Ca2+ binding protein located in the lumen of endoplasmic reticulum. Even though it has been implicated in various diseases, the in vivo functions of calumenin have not yet been reported. Here, we report the characterization of C. elegans calumenin loss of function mutant, calu-1(tm1783). Biochemical analysis revealed that CALU-1 binds with Ca2+ and the calu-1(tm1783) mutant exhibited pleiotropic defects such as decreased brood size, reduced egg laying rate, sluggish locomotion, and small body size suggesting the multiple roles of calumenin in C. elegans. Consistent to its ability to bind Ca2+, calu-1(tm1783) mutant shows reduced pumping rate and prolonged defecation cycle, which are known to be mediated by Ca2+ signaling in C. elegans. The knock-down of calu-1 phenocopies calu-1(tm1783) mutant, suggesting that specific loss of CALU-1 is responsible for the observed defects. Interestingly, calu-1(tm1783) mutant has severe defects in cuticle showing aberrant annuli and deformed alae indicating the possible role(s) of calumenin in cuticle formation and/or maintenance. To our knowledge, this is the first report of genetic analysis of calumenin illustrating its in vivo functions.

Campbell G et al. (1995) International C. elegans Meeting "SCREENING FOR CELL DEATH GENES AS SUPPRESSORS OF lin-24 and lin-33 MUTATIONS"

Horvitz HR, Campbell G

[International C. elegans Meeting, 1995]

The mutations n432 and n1057 in lin-24 and n1043 and n1044 in lin-33 cause variable abnormal morphologies and deaths of the Pn.p cells, as well as some vulval lineage defects (Ferguson and Horvitz, Genetics 110:17-72, 1985). These abnormalities lead to a vulvaless (Vul) phenotype, which can be suppressed by mutations in the cell death engulfment genes ced-2, ced-5, and ced-10; mutations in other programmed cell death genes (ced-3, ced-4, ced-9(gf), ced-1, ced-6, ced-7, ced-8) do not suppress the Vul phenotype (Saechin Kim, personal communication). These data suggest that a suppressor screen of the Vul phenotype of lin-24 and lin-33 mutants could allow us to identify additional alleles of ced-2, ced-5 and ced-10 and to define new genes involved in programmed cell death.

M Kostrouchova et al. (1999) International C. elegans Meeting "Genetic dissection of molting pathway"

M Kostrouchova, JE Rall, Z Kostrouch, M Krause

[International C. elegans Meeting, 1999]

CHR3, a C. elegans nuclear hormone receptor similar to Drosophila DHR3 and the human ROR/RZR group, is important for regulation of molting. The CHR3 loss of function (RNAi) leads to a characteristic phenotype including an inability to completely shed the old cuticle during molting(1). There are only a few genes known to be involved in proper molting. These include let-454, let-462 and let-473(2) and in lrp-1 genes(3). To find additional genes involved in the molting pathway we performed a mutant screen to look for molting defects that were similar to the CHR3(RNAi) phenotype. We have examined the progeny from 1000 F1 animals (2000 haploid genomes) and found 4 mutations with molting defects; we continue to investigate one of them. The majority of these mutant worms do not complete the L4/adult molt and some animals are also affected in earlier stages. The worms do not shed and degrade the cuticle from the head area and they die several days later because of starvation. The mutant animals are also shorter and DPY. The adult hermaphrodites have only a few developing embryos inside that often do not hatch. We are now in the process of mapping the mutation. 1. Kostrouchova, M., Krause, M., Kostrouch, Z. & Rall, J. E. (1998) Development 125, 1617-26. 2. Johnsen, R. C. & Baillie, D. L. (1991) Genetics 129, 735-52. 3. Yochem, J., Tuck, S., Greenwald, I. & Han, M. (1999) Development 126, 597606. C. elegans, Drosophila

Yeh Chan-Hsien et al. (2007) International Worm Meeting "Screen for TLK-1-interacting proteins."

Yi-Chun Wu, Yeh Chan-Hsien, I-Ju Lee

[International Worm Meeting, 2007]

Tousled-like kinases (TLKs) form a conserved serine/threonine kinase family in multi-cellular organisms and have been implicated in transcription, DNA replication and repair and chromosome assembly and segregation. The tlk-1(RNAi) mutants exhibited defects in chromosome condensation and segregation. Previous studies of C. elegans TLK-1 indicates that tlk-1 may act in chromosome assembly by phosphorylating Asf-1 (anti-silencing function protein) and contribute to chromosome segregation as a substrate and activator of the Aurora B kinase AIR-2 (Han et al, 2005). To identify more TLK-1-interacting proteins, we performed a yeast-two hybrid screen. In the screen we isolated DLC-1 (Dynein light chain). DLC-1 is localized to spindle fiber during mitosis and this localization pattern is disrupted in tlk-1(RNAi) embryos. We are currently performing experiments to verify the interaction between DLC-1 and TLK-1 in vivo and investigating if TLK-1 may phosphorylate DLC-1. Reference Han, Z., Riefler, G.M., Saam, J.R., Mango, S.E., and Schumacher, J.M. (2005) Curr. Biol., 15, 894-904.

MV Sundaram et al. (1999) International C. elegans Meeting "A genetic screen for synthetic Vulvaless/Lethal mutations"

MV Sundaram, R Howard, A Goldman

[International C. elegans Meeting, 1999]

Vulval fate induction is controlled by several different signaling pathways, including an RTK/Ras/ERK pathway. Some genes that influence vulval development have wild-type or nearly wild-type null phenotypes, and their roles are only revealed in appropriate sensitized genetic backgrounds. For example, mutations in ksr-1 and sur-8 do not cause strong phenotypes singly, but do cause highly penetrant Vulvaless and Lethal phenotypes in combination with each other, or in combination with weak alleles of other Ras pathway genes such as lin-45 raf. We reasoned that we could identify additional genes acting with ksr-1 or sur-8 by screening for additional synthetic Vul/Let mutations. As a starting point for the screen, we used the weak lin-45 raf mutants ku51 or ku112, which have nearly wild-type phenotypes. In our initial screens of under 5000 EMS mutagenized genomes, we've identified five mutations that cause moderately penetrant Egl and Lethal phenotypes in these lin-45 mutant backgrounds, but little or no phenotype in a lin-45(+) background: cs1 is an allele of ksr-1, cs24 is an allele of sur-6, cs26 is a novel non-Vul allele of sur-2, and cs28 IV and cs30 X appear to define new genes (which we are cloning). In addition to our screen, we've also directly tested candidate genes for synthetic effects using existing mutations or RNAi, and we observed synthetic effects for mig-2 rac and a CNK homolog, among others. By testing for synthetic effects of different mutation/RNAi pairs, we are attempting to place these new genes into functional groups that may define different redundantly-acting pathways. We find that ksr-1 and sur-6 are members of the same functional group, since ksr-1(n2526) and sur-6(cs24) do not synergize with each other, although each synergizes strongly with either lin-45(ku112) or sur-8(ku167). These genetic interactions are consistent with a model in which ksr-1 and sur-6 function together to promote vulval fates. Derek Sieburth and Min Han previously demonstrated similar interactions between the ksr-1(ku68) and sur-6(ku123) alleles, and showed that sur-6 encodes a regulatory B subunit of protein phosphatase 2A (PP2A). Thus, KSR-1 might function to regulate PP2A, or might be regulated by PP2A. We are collaborating with Sieburth and Han to test such models.

Jason Pellettieri et al. (2003) International Worm Meeting "minibrain-kinase-2 Activates Maternal Protein Degradation and Germ Plasm Segregation During the Egg-to-Embryo Transition"

Geraldine Seydoux, Jason Pellettieri

[International Worm Meeting, 2003]

The transition from egg to embryo occurs in the absence of transcription yet requires significant changes in gene activity. In C. elegans, this transition is marked by two important changes: 1) a switch from meiosis to mitosis, and 2) the development of molecular asymmetries along the anterior/posterior axis.We have identified a new gene called minibrain-kinase-2 (mbk-2; formerly pom-1), that is required for both of these changes. Our results suggest that the primary defect in mbk-2 mutants is a failure to degrade several maternal proteins. mbk-2 mutants fail to degrade the meiosis-specific katanin subunits MEI-1 and MEI-2 at the meiosis-to-mitosis transition, and the first mitotic division fails. mbk-2 is also required for timely degradation of OMA-1, a regulator of oocyte maturation, and for the degradation of germ plasm components in anterior cells. Finally, mbk-2 is required for asymmetric segregation of germline determinants in the zygote, implicating regulated protein degradation in this process.MBK-2 distribution changes dramatically after fertilization during the transition from meiosis I to meiosis II. We have found that this change coincides with activation of mbk-2-dependent protein degradation. Mutants arrested in meiosis I do not relocalize MBK-2, and like mbk-2 mutants, do not degrade MEI-1, do not degrade OMA-1, and do not segregate P granules. mbk-2 mutants, however, complete the meiotic divisions normally. We conclude that mbk-2, while not required for meiosis, is activated by the meiotic divisions and serves to coordinate maternal protein degradation during the egg-to-embryo transition.MBK-2 belongs to the Dual-Specificity Yak1-Related Kinase (DYRK) family, which is conserved from yeast to humans. Intriguingly, DYRKs have been implicated in the regulation of cell polarity and protein degradation in other systems, suggesting that the function of MBK-2-related kinases may have been conserved throughout evolution.We thank Valerie Reinke, Stuart Kim, Bill Raich, Oliver Hobert, Thimo Kurz, Bruce Bowerman, Edward Kipreos, and Rueyling Lin for reagents and strains used in this work.

Kim, Do-Young et al. (2019) International Worm Meeting "FMRFamide-related neuropeptide FLP-12 regulates head locomotion of C. elegans."

Li, Chris, Kim, Do-Young, Kim, Jinmahn, Park, Cheon-Gyu, Suh, Byung-Chang, Kim, Kyuhyung

[International Worm Meeting, 2019]

Neuropeptides regulate a multitude of behaviors in many organisms (Li et al., 2014). However, mechanisms by which functions of neuropeptides are coordinated to generate proper behaviors are not fully understood. In C. elegans, the SMB motor neurons consist of two pair of neurons that innervate head/neck muscles (White et al, 1986) and shape the amplitude of sinusoidal movement (Gray et al, 2005, Kim et al., 2014), indicating a role of SMB in head locomotion. The SMB neurons have shown to express FMRFamide-related neuropeptide FLP-12 (Kim and Li, 2004, Kim et al., 2014). To identify roles of FLP-12 neuropeptide in head locomotive behavior, we first observed phenotypic consequences by loss of FLP-12. We measured five locomotive parameters including omega turn, reversal, head lift, foraging and frequency of head movement. We found that all five locomotive parameters are increased in flp-12 mutants. We fully rescued defects in head locomotion of flp-12 mutants by expressing flp-12cDNA specifically in SMB. These results indicate that FLP-12 may act in the SMB to regulate head movement. To identify receptors for FLP-12, we performed RNAi screening against a total of 16 putative neuropeptide receptor genes. We found that knock-down of several genes including frpr-8 exhibits increases foraging behavior similarly to flp-12 mutants and frpr-8 mutants. Expressing frpr-8 cDNA with its own promoter successfully rescues foraging phenotype of frpr-8. Heterogously expressed FRPR-8 in HEK cells are sufficient to confer Ca2+ response upon FLP-12 exposure. Altogether, our data expand our understanding of how a single neuropeptide and its receptor modulate behaviors.

Lim, Yun-Ki et al. (2015) International Worm Meeting "Mitochondrial Ca2+ uniporter (mcu-1) regulates autophagy and mitochondrial UPR in Caenorhabditis elegans.."

Ahnn, Joohong, Kim, Do Han, Min, Choon Kee, Kim, Yeon-Soo, Lee, Sun-Kyung, Eom, Soo Hyun, Kang, Moon Kyung, Kim, Dongwook, Lim, Yun-Ki

[International Worm Meeting, 2015]

Mitochondrial Ca2+ uptake mediated by mitochondrial calcium uniporter (MCU) is fundamentally important in energy metabolism, cell signaling and survival. However, the role of MCU at the organismal level has not been fully explored. We have functionally characterized the newly identified CeMCU-1 encoding Caenorhabditis elegans ortholog of MCU by genetic manipulation and mitochondrial Ca2+ imaging. Overexpression of CeMCU-1 fully rescued mitochondrial Ca2+ flux in MCU-silenced HeLa cells, indicating that CeMCU-1 is a functional Ca2+ channel. Worms lacking mcu-1 showed reduced oxygen consumption, ATP and ROS levels. In addition, biochemical evidence indicated that the phosphorylation of AMP-activated protein kinase (AMPK) was significantly enhanced in the mcu-1 deficient condition. Furthermore, the mcu-1 deleted animals increased autophagy and mitochondrial UPR (UPRmt). Collectively, our findings suggest that mitochondrial Ca2+ homeostasis mediated by mcu-1 is closely associated with aging process involving pleiotropic effects of autophagy and UPRmt. This work was supported by the Bio & Medical Technology Development Program (NRF-2013M3A9A7046297) in the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Korean Ministry of Science, ICT & Future Planning and 2014 GIST-Infrastructure Establishment grant.