Tsang SY et al. (2007) Biochem Biophys Res Commun "Ectopic expression of systemic RNA interference defective protein in embryonic stem cells."

Li RA, Tsang SY, Moore JC, Huizen RV, Chan CW

[Biochem Biophys Res Commun, 2007]

RNA interference (RNAi), a post-transcriptional gene silencing mechanism originally described in Caenorhabditis elegans, involves sequence-specific mRNA degradation mediated by double-stranded RNAs (dsRNAs). Passive dsRNA uptake has been uniquely observed in C. elegans due to the expression of systemic RNA interference defective-1 (SID-1). Here we investigated the ability of ectopic SID-1 expression to enable passive cellular uptake of short interfering RNA (siRNA) or double stranded RNA (dsRNA) in pluripotent mouse embryonic stem cells (mESCs). When SID-1-GFP and the Firefly luciferase reporter gene (luc(Fir)) were co-expressed in mESCs, luc(Fir) activity could be suppressed by simple incubation with dsRNAs/siRNAs that were designed to specifically target luc(Fir). By contrast, suppression was not observed in mESCs expressing luc(Fir) and GFP alone or when either GFP- or SID-1-GFP-expressing cells were incubated with control dsRNAs/siRNAs (non-silencing or Renilla luciferase-specific). These results may lead to high-throughput experimental strategies for studying ESC differentiation and novel approaches to genetically inhibit or eliminate the tumorigenicity of ESCs.

Kara Chin et al. (2005) International Worm Meeting "Who needs mitochondrial mtDNA anyway? Fitness effects of the 3.1kb UaDf-5 mtDNA deletion."

Cleonique Deshommes, Aidyl Gonzalez-Serricchio, Craig LaMunyon, Kara Chin

[International Worm Meeting, 2005]

Mitochondrial chromosomes have very little non-coding DNA, and non-mutated chromosomes are generally considered to be associated with increased metabolic fitness. In C. elegans, the UaDf5 mtDNA deletion removes 3.1kb encompassing 11 genes, and yet this deletion can be maternally transmitted for at least 100 generations in a stable heteroplasmic state at all developmental stages (Tsang and Lemire , 2002). Why should a mitochondrial deletion persist? We investigated this question by looking at several measures of metabolism and fitness in relation to the proportion of UaDf-5 deleted mtDNA. Our data support Tsang and Lemires (2002) findings of stable heteroplasmy between wild-type and deleted mtDNA, even though the proportion of deleted mtDNA can vary greatly among siblings and over the course of several generations. We also looked at individual tissues of worms and found no mosaicism among the gut, testes and other tissues for this deletion. We found a positive correlation between the proportion of deleted mtDNA and measures of metabolism (defecation, feeding, and egg-laying rates), indicating that worms bearing this deletion of the mitochondrial chromosome are more fit than wild-type, at least under laboratory conditions.Tsang WY, Lemire BD, 2002. Biochem Cell Biol 80:645-54.

Peterson, Sagen et al. (2015) International Worm Meeting "Elucidating the mechanisms for purifying selection of mitochondrial DNA."

Goyal, Sidhartha, Torres Cleuren, Yamila, Peterson, Sagen, Rothman, Joel

[International Worm Meeting, 2015]

The mitochondrial genome (mtDNA) is present in many copies within each cell and follows a maternal pattern of inheritance. mtDNA experiences a high mutation rate which leads to heteroplasmy, in which a mixed population of both wildtype and altered mtDNA exist within the same cell. While each mtDNA can act as a "selfish DNA" element, purifying selection is used to eliminate from the germline mtDNAs that are defective and debilitate mitochondrial function. We are investigating the mechanisms used in purifying selection in the germline. One level at which purifying selection might act is during the early embryonic divisions that establish the germline (P lineage). We have obtained preliminary evidence that the most active mitochondria (those with the highest membrane potential) are enriched in the germline lineage during the early embryonic cell divisions. Following this early phase, we found that germline mitochondria are then fully quiescent (no detectable membrane potential) until after the initiation of larval development. We have taken advantage of a 3 kb mtDNA deletion, uaDf5 [Tsang et al 2002], to investigate further the mechanisms that remove defective mtDNAs. This deletion exhibits stable heteroplasmy, raising the suggestion that it is maintained as a result of complementing mutations on the non-deleted mtDNAs in the strain [Tsang et al 2002]. However, we found that the uaDf5-bearing strain appears to contain fully intact, wild-type mtDNA, suggesting that the deletion may be maintained as a result of replicative advantage. Further, we found that the uaDf5 strain gives rise to arresting embryos with many cell corpses, reminiscent of cells that have undergone programmed cell death. Lines that generate arresting embryos at highest frequency also show extended lifespan in the survivors, consistent with a trade-off between mitochondrial function required for survival and lifespan extension. Finally, we have obtained preliminary results suggesting that physiological state of worms influences the rate of removal of mtDNAs encoding defective proteins. We are investigating the trans-generational dynamics of this apparent purifying selection.

Lee JH et al. (1991) International C. elegans Meeting "ROLE OF UNC-101 IN C.ELEGANS VULVAL DEVELOPMENT"

Jongeward GD, Lee JH, Sternberg PW

[International C. elegans Meeting, 1991]

We previously isolated two unc-101 alleles (e.g. syl O8) in a screen for suppressors of vulvaless phenotype of let-23. unc-101 mutations also confer an uncoordinated phenotype as well as subviability, defecation and FITC staining defects. We are interested m the genetics and molecular biology of this locus because of its broad range of phenotypes and lts role in vulval development. We isolated a putative null allele of unc-101. We used trimethyl- psoralen as a mutagen in a screen for mutations that fail to complement unc-l Ol (rh6). One new allele, sy 216, was recovered from 11,000 Fl cross-progeny hermaphrodites. This allele is a homozygous Ll larval lethal. Unlike the visible alleles, the lethality of thls allele cannot be maternally rescued. Animals of the genotype sy2161syl O8 are less viable than animals bearing syl O8 in trans to any visible allele. The sy216 Isyl O8 heterozygote is also a good suppressor of the let-23(syl ) vulval defect. Therefore we consider sy216 a putative null allele of unc-101. We believe that the null phenotype of unc-101 is lethality and suppression of let-23. sy216 does not delete unc-75, ced-l and unc-S9, nor does it overlap eDf3. We are currently trying to clone unc-101 using parallel RFLP mapping with the congenic strain used for cloning ced-l, which was kindly provided by Bob Horvitz's lab. We obtained 37 unc-59 nUnc-75 recombinants from unc-75(e950)ced-1(nlS06) +unc-S9 (e290)1 ++unc-lOl( sylO8) + heterozygotes,and southern hybridization analysis with Tcl as probe is underway. [See Figure]

Knapp-Wilson, A. et al. (2017) International Worm Meeting "Investigating Complex I activity and mitochondrial protein import in the multi-cellular model organism C. elegans."

Collinson, I., Knapp-Wilson, A., Kuwabara, P.

[International Worm Meeting, 2017]

Mitochondria are small cytoplasmic organelles essential for meeting the majority of cellular energy demands via the oxidative phosphorylation (OXPHOS) pathway. [1, 2]. They feature a double membrane that separates the organelle into four distinct compartments [3]. The vast majority of the work on mitochondria has been carried out in Saccharomyces cerevisiae, a single-celled genetically tractable eukaryote [4]. Although biochemically competent; the model has limitations in its applicability to humans. The mitochondrial structure, metabolism and bioenergetics of the nematode are very similar to the mammalian counterpart [5], this conservation of mitochondrial function and the technological and anatomical advantages offered by the nematode make it an excellent model organism to explore mitochondrial function and activity. [1, 6] My project is multidisciplinary, using a broad range of techniques including whole animal physiology, biochemical and structural analysis, and molecular dynamic simulations to gain a better understanding of the mitochondrial respiratory machinery, and the impact dysfunction of this machinery can have. Mitochondrial dysfunction leads to many serious human diseases and a better understanding of the mechanisms involved is essential in future medical applications. [1] Maglioni, S. & Ventura, N., 2016. C. elegans as a model organism for human mitochondrial associated disorders. Mitochondrion, 30, pp.117-125. [2] Lemire, B. Mitochondrial genetics (September 14, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.25.1, [3] McBride et al, 2006. Mitochondria: more than just a powerhouse., 16(14), pp.R551-60 [4] Tokatlidis, K., et al. (2000). "Membrane protein import in yeast mitochondria." Biochem Soc Trans 28(4): [5] Murfitt, R.R., Vogel, K. & Sanadi, D.R., 1976. Characterization of the mitochondria of the free-living nematode, Caenorhabditis elegans. Comparative biochemistry and physiology. B, Comparative biochemistry, 53(4), [6] Tsang, W.Y. & Lemire, B.D., 2003. The role of mitochondria in the life of the nematode, Caenorhabditis elegans. Biochimica et biophysica acta, 1638(2)

Aidyl S Gonzalez-Serricchio et al. (2005) International Worm Meeting "Paternal mitochondrial inheritance: No longer restricted to Moms"

Craig LaMunyon, Aidyl S Gonzalez-Serricchio

[International Worm Meeting, 2005]

A vast majority of eukaryotic organisms solely inherit maternal mitochondrial DNA (mtDNA) with the exception of mussels (males inherit paternal mtDNA and females inherit maternal mtDNA) (Zouros, 2000; Zouros et al., 1994). Paternal mtDNA disappear early in embryogenesis either due to selective destruction, inactivation or simple dilution by the numerous of oocyte mitochondria. Is there a deleterious effect if offspring inherits paternal mtDNA? Our aim is to further understand the mechanism of mitochondrial inheritance. We were able to isolate a strain with the capability to pass onto its offspring its paternal mtDNA. UadDf-5 is a stable heteroplasmic strain with a 3kb deletion on its mt genome (removes 11 genes; Tsang and Lemire, 2002). UaDf-5; him-8 (e1489) worms were mutagenized with ethylmethanesulfonate (EMS). F1 worms were isolated and allowed to clone in order to isolate F2 males. Next, the F2 males were crossed into fer-1 (hc13ts) hermaphrodites. Finally, F3 worms were isolated and underwent a nestedPCR assay to detect the mutant UaDf-5 band inherited from the Po: UaDf-5; him-8 (e1489) males. As we further confirm, homozygosis (if stable), and characterize the DNA lesion of the mutagenized strain(s), we will have a strong tool to use in explaining the mechanism that eliminates the sperm mitochondria from the embryo. Also, we will observe offspring that inherit some paternal mitochondria to see if it results in reduced fitness. Either initially, or after numerous generations, understand the evolution of maternal inheritance.Zouros E, 2000. The exceptional mitochondrial DNA system of the mussel family Mytilidae. Genes Genet Syst 75:313-8.Zouros E, Oberhauser Ball A, Saavedra C, Freeman KR, 1994. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proc Natl Acad Sci U S A 91:7463-7Tsang WY, Lemire BD, 2002. Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes. Biochem Cell Biol 80:645-54.

Hajjar, Connie et al. (2011) International Worm Meeting "Sperm Mitochondria are Associated with Ubiquitinated Vesicles After Fertilization."

Hajjar, Connie, Boyd, Lynn, Golden, Andy

[International Worm Meeting, 2011]

Studies in C. elegans and other organisms have indicated that mitochondria are maternally inherited.1 This has presented an interesting cell biological conundrum as to what becomes of sperm mitochondria that enter the egg upon fertilization. Studies in mammalian systems have suggested that sperm mitochondria are eliminated from the fertilized egg via a mechanism involving ubiquitination.2 We have investigated this possibility in the worm. Using strains with germline expression of GFP::ubiquitin, we have observed ubiquitinated vesicles that cluster around the sperm DNA after fertilization. We propose that these vesicles are sperm-derived based on three observations: 1) they are not seen in oocytes, 2) they are closely associated with the sperm DNA, and 3) they stain positive with a sperm-specific antibody (see below for more details). Using Mitotracker tagging of sperm, we show that the sperm mitochondria are closely associated with the ubiquitinated vesicles but are not ubiquitinated themselves. The sperm vesicle-mitochondria conglomerate remains compact and close to the sperm DNA throughout the maternal meiotic divisions. Starting around prophase of the first mitosis, the sperm vesicles and mitochondria begin to disperse and their numbers diminish. By the 8-cell stage, we no longer detect either. In order to learn more about the nature of the ubiquitinated vesicles, we have done additional labelling experiments. They stain with wheat germ agglutinin, indicating that they are indeed membrane bound vesicles. They do not stain with mitotracker or lysotracker. They stain positively with the 1CB4 antibody that recognizes the sperm-specific organelle, MO. 1CB4 stains the ubiquitinated vesicles, but not the sperm mitochondria in the embryo. Thus, the vesicles seen in the fertilized embryo may be derived from MO structures in the sperm. Linkage specific antibodies to polyubiquitin show that the vesicles harbor K63, but not K48, linked polyubiquitin chains. K63 chains can sometimes trigger autophagy suggesting that autophagic destruction may be involved in the elimination of the sperm-derived vesicles and possibly also the nearby sperm mitochondria. This interesting association between sperm mitochondria and these unusual ubiquitinated vesicles may reveal a novel mechanism for the elimination of paternal mitochondria from the fertilized egg. 1- Tsang and Lemire, (2002) Bioch. Cell Biol. 80 645-654; 2- Sutovsky et al., (2000) Biol Reprod. 63 582-90.

M Zhen et al. (1999) International C. elegans Meeting "A C. elegans liprin protein SYD-2 regulates presynaptic active zones"

M Zhen, Y Jin

[International C. elegans Meeting, 1999]

Chemical synapses allow information flow between neurons and their target cells. At synaptic junctions, specialized cellular structures are developed in both pre- and post-synaptic cells to facilitate synaptic transmission. Presynaptic structure is characterized by the accumulation of synaptic vesicles and an electron-dense membrane structure called the active zone. To elucidate the molecular mechanisms underlying the presynaptic architecture, we carried out a genetic screen for mutants with abnormal presynaptic morphology of GABAergic motoneurons using the P unc-25 -SNB-1::GFP marker 1,2 . From 5,000 haploid genomes, we identified 12 new genes, called syd s ( sy napse d efective), which are required for expression and localization of the SNB-1::GFP marker 1 . We report here the cloning and functional analysis of the syd-2 gene. In syd-2 mutants axonal outgrowth is normal but the SNB-1::GFP marker and other presynaptic proteins are diffusely localized at the nerve termini, suggesting that syd-2 specifically affects synapse formation. syd-2 encodes a C. elegans liprin. Liprins have been shown to interact with the second phosphatase domain of LAR-type receptor-tyrosine phosphatases (RPTPs) and localize RPTPs to focal adhesions in mammalian cell cultures 3 . SYD-2 protein is expressed in the presynaptic region independent of synaptic vesicles, indicating that it might be a structural component in the presynaptic termini. We analyzed the ultrastructure of chemical synapses in syd-2 animals, and found that the total synaptic vesicle number per synapse was not altered in syd-2 mutants, whereas the length of active zones was significantly increased. We propose that SYD-2 plays a role in synapse formation by serving as an anchor protein to regulate active zone formation. SYD-2 might regulate a tyrosine phosphorylation signaling cascade at synaptic junctions by recruiting RPTPs. 1. Zhen and Jin (1997) 11th International Worm Meeting Abstract 682. 2. Nonet (1999) J. Neurosci. Methods (in press) 3. Serra-Pages et al. (1998) J. Biol. Chem. 273: 15611-15620.

Ambrose R Kidd III et al. (2004) Mid-west Worm Meeting "SYS-1, an atypical beta-catenin, acts with POP-1/TCF to control cell fates in C. elegans"

Judith Kimble, Jennifer A Miskowski, Ambrose R Kidd III

[Mid-west Worm Meeting, 2004]

POP-1/TCF controls numerous asymmetric cell divisions during C. elegans development. Our focus has been on an asymmetric division that generates the proximal-distal axis of the gonad. Both POP-1 and other classical Wnt/MAPK signaling components promote the distal fate in daughters of somatic gonadal progenitor cells (1). Mutations in the sys-1 (for sy mmetrical s isters) gene affect Z1/Z4 asymmetry in a similar way and interact genetically with pop-1 (1,2). We now have five sys-1 mutations. Two sys-1 deletions are embryonic lethal, which appears to be the null phenotype. Three other sys-1 mutations lead to sterility with defects in Z1/Z4 asymmetry; their molecular lesions include a missense mutation, an intronic change, and a silent mutation. The sys-1 promoter drives GFP in many cells throughout development. Given its genetic interaction with pop-1, its effect on Z1/Z4 asymmetry, its broad expression pattern and embryonic lethal null phenotype, we suggest that SYS-1 may control many asymmetric divisions during C. elegans development. A major clue to the mechanism by which SYS-1 controls fates comes from its molecular identification as a divergent b -catenin. SYS-1 contains at least three ARM repeats, a motif typical of b -catenin/Armadillo, and rescues a bar-1 null mutant when driven by a bar-1/ b -catenin promoter. Furthermore, by two-hybrid assay, SYS-1 binds POP-1 in a region that includes the b -catenin binding domain. Finally, a lack of SYS-1 does not affect POP-1 nuclear asymmetry (3). We suggest that SYS-1 works with POP-1 to control cell fates. Our favorite model is that SYS-1 acts with POP-1 as a transcriptional coactivator to control target genes. Predictions from this model are being tested and will be presented. --------------------- 1. Siegfried and Kimble (2002) Development 129, 443-453. 2. Miskowski et al. (2001) Developmental Biology 230, 61-73. 3. Siegfried et al. (2004) Genetics 166, 171-186.

AG Davies et al. (1999) International C. elegans Meeting "Functional Overlap between the mec-8 Gene and Five sym Genes in C. elegans"

JE Shaw, CA Spike, AG Davies, RK Herman

[International C. elegans Meeting, 1999]

Inactivating mutations in a majority of the 19,000 protein-coding genes of C. elegans may result in wild-type or nearly wild-type phenotypes. Some of these mutationally-silent genes may encode essential functions redundantly. In an attempt to identify such genes, we have screened for mutations that are synthetic lethal with a mec-8 loss-of-function mutation. We have so far identified five independent mutations in four genes, sym-1 through sym-4 , where sym stands for sy nthetic lethal with m ec-8 . The phenotype conferred by each sym mutation by itself is essentially wild-type. Mutation in sym-1 or sym-2 in combination with any mec-8 loss of function mutation leads to embryonic arrest at the twofold stage of elongation, with high penetrance. Mutation in sym-3 or sym-4 in combination with any mec-8 loss-of-function mutation gives arrested hatchees with bulbous noses. Earlier work showed that mec-8 encodes a regulator of alternative RNA splicing and that mec-8 null mutants have defects in sensory neurons but are generally viable and fertile. The mec-8; sym-1 arrested embryos exhibit severe defects in attachment of body muscle to extracellular cuticle. sym-1 can encode a protein containing a signal sequence and 15 contiguous leucine-rich repeats. Our one sym-1 mutant allele is a nonsense mutation in the middle of the coding sequence. A fusion of sym-1 and the gene for green fluorescent protein rescued the synthetic lethality of mec-8; sym-1 mutants. The fusion protein was secreted from the apical hypodermal surface of the embryo. We propose that SYM-1 helps to attach body muscle to extracellular cuticle. Neither northern blots nor cDNAs gave any evidence for alternative splicing of sym-1 transcripts. We favor the idea that sym-1 provides a function that is redundant with that of a gene whose transcripts are processed by MEC-8. RNA-mediated interference experiments indicated that a close relative of sym-1 functionally overlaps both sym-1 and mec-8 in affecting muscle attachment. We speculate that sym-2 may act in the same pathway as sym-1 and that sym-3 and sym-4 act in an unrelated pathway.