Wortham, Tesheka S. et al. (2009) International Worm Meeting "Molecular Genetic Analysis of unc-100, a Gene Important for Normal Myofibril Organization in C. elegans."

L'Hernault, Steven W., Benian, Guy M., McClinic, Karissa N., Wortham, Tesheka S., Mercer, Kristina B., Qadota, Hiroshi

[International Worm Meeting, 2009]

Mutations in the gene unc-100 result in animals showing reduced motility and disorganization of myofibrils. unc-100 was identified in a motility-requiring selection and polarized light screen for defects in muscle function and structure (Zengel & Epstein, 1980). Currently, there are two alleles available for our characterization of unc-100, namely su115 (from the CGC) and su149 (kindly provided by Pam Hoppe). By polarized light microscopy, pharyngeal muscle appears normal, but the body wall muscle cells have myofibrils with an overall reduction in birefringence and disorganization in the patterning of A and I-bands. Interestingly, the polarized light defect of su115, but not su149, is suppressed by growth at 15 deg C. Through the use of a "swimming" assay, we determined that both alleles are slower moving than wild type, with su149 showing the most dramatic defect-su149 L4 or adults are ~65% slower than wild type. Brood sizes of each mutant allele are reduced by 50% as compared to wild type. The somatic gonad shows an abnormal morphology that is probably due to a defect in distal tip cell migration, which could explain the observed reduction in brood sizes. Staining of unc-100 mutant muscle with antibodies to a variety of known sarcomeric proteins revealed disruption of thick filaments, M-lines and dense bodies; this disruption is more severe in the muscle of su149 animals. Electron microscopy of each mutant allele shows missing M-lines, broken dense bodies that are often detached from the cell membrane, and a lack of defined A- and I-bands. Although the current genetic map position on WormBase for unc-100 is I:22.75+/-1.190 cM, we used three-factor and SNP mapping to place unc-100 on chromosome I between 1.87 and 2.26 cM which contains 17 overlapping cosmids. In transgenic rescue experiments, injecting the cosmids in sets of three, we were able to obtain rescue of unc-100 with the three cosmids, ZK524, T28F4 and C26C6. We are currently performing single cosmid rescue experiments to determine which of the three cosmids unc-100 lies on. We will then conduct RNAi with each gene predicted on the single cosmid to determine if we can phenocopy unc-100. We also plan to determine the mutation sites of both alleles and perform transgenic rescue using a single predicted gene. Once the unc-100 gene has been identified, we will develop anti-UNC-100 antibodies to localize UNC-100 protein in muscle, and use the protein to screen an extensive yeast 2-hybrid bookshelf of M-line, dense body and thick filament components, to find interacting proteins.

Furukawa, Miho et al. (2009) International Worm Meeting "Twitchin Kinase Interacts with a Conserved MAP Kinase Activated Protein Kinase in C. elegans Muscle."

Wortham, Tesheka S., Furukawa, Miho, Qadota, Hiroshi, Benian, Guy M.

[International Worm Meeting, 2009]

Sarcomeres of probably all animals contain a number of extraordinarily large polypeptides (700 kDa - 4 MDa) composed of multiple Ig and Fn3 domains, one or two protein kinase domains, and in some cases, elastic regions. C. elegans muscle contains three such giant proteins: twitchin (754 kDa) in the polar regions of the A-band, UNC-89 (up to 900 kDa) in the middle of the A-band, and TTN-1 (2.2 MDa) in the I-band. Twitchin is encoded by unc-22, which has a characteristic mutant phenotype: "twitching" of the worm''s surface that originates from the underlying muscle. When wild type animals are exposed to the acetylcholine agonist nicotine, they become paralyzed, whereas, when unc-22 mutants twitch violently. The protein kinase domain shows protein kinase activity in vitro and is autoinhibited by the 60 residues C-terminal of the catalytic core. Recent molecular dynamics simulations and AFM experiments suggest that this normally autoinhibited kinase is activated by small forces that occur during muscle contraction/relaxation cycles (Greene et al. 2008). Despite this information, substrates or binding partners for the twitchin kinase domain were unknown. We screened a yeast 2-hybrid library using as bait, Ig25-Fn31-kinase of twitchin, and recovered clones representing K08F8.1. K08F8.1 encodes a protein homologous to mammalian MAP kinase activated protein kinase 2. There are, in fact, 3 MAPKAP kinase 2 homologs in C. elegans: K08F8.1, MAK-2 and MNK-1. Homology among the human and worm proteins resides only within the kinase domains; in this region, K08F8.1 is 53% identical to human MAPKAP kinase 2. SAGE data on WormBase indicates that only K08F8.1 is highly expressed in body wall muscle. Yeast 2-hybrid domain mapping shows that the twitchin / K08F8.1 interaction: (1) requires only the kinase domain of twitchin; and (2) residues 60-405 (essentially the kinase domain) of K08F8.1 are minimally required. Comparable regions of TTN-1 and UNC-89 do not interact with K08F8.1, and preliminarily, MAK-2 and MNK-1 fail to interact with twitchin. Luckily, two independently generated intragenic deletions of K08F8.1, ok2987 and tm3455, are available. By swimming assay, the motility of ok2987 is 14% lower than wild type. Significantly, both ok2987 and tm3455 are hypersensitive to nicotine (they become paralyzed at a faster rate than wild type). The abnormal response to nicotine suggests that K08F8.1 acts in the same pathway as twitchin.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Miller, Rachel K. et al. (2009) International Worm Meeting "CSN-5, a Component of the COP9 Signalosome Complex, Regulates the Levels of UNC-96 and UNC-98, two Components of M-lines in C. elegans muscle."

Mercer, Kristina B., Benian, Guy M., Miller, Rachel K., Wortham, Tesheka S., Stark, Thomas J., Qadota, Hiroshi

[International Worm Meeting, 2009]

UNC-98 and UNC-96 are two small proteins that localize to the M-lines in body wall muscle. Mutants of each gene have a similar and characteristic phenotype: by polarized light microscopy, each shows disorganization of myofibrils and birefringent needles at the ends of the muscle cells. There is genetic and biochemical evidence that UNC-98 and UNC-96 interact with each other (Mercer et al. 2006), and with paramyosin (Mercer et al. 2006; Miller et al. 2008). unc-96 mutants contain discrete accumulations of UNC-98 protein, and unc-98 mutants contain accumulations of UNC-96 protein. Moreover, in both unc-98 and unc-96 mutants, paramyosin is localized both normally to A-bands and abnormally in accumulations. By western blot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. To explain this and other data, we have proposed a model in which UNC-98 and UNC-96 act as chaperones to promote the incorporation of paramyosin into thick filaments (Miller et al. 2008). We now report that, unexpectedly, both UNC-98 and UNC-96 interact with CSN-5, a component of the conserved COP9 signalosome which has been implicated in a wide variety of functions usually linked to ubiquitin-mediated proteolysis. The interactions were initially found by screening of a yeast 2-hybrid library, and then confirmed by biochemical methods. Anti-CSN-5 antibody co-localized with paramyosin at A-bands in wild type, and co-localized with accumulations of paramyosin in unc-98, unc-96, and unc-15 mutants. Double knock down of csn-5 and the homologous csn-6 could slightly suppress the unc-96 mutant phenotype. In the double knock down of csn-5 and csn-6, the levels of UNC-98 protein were greatly increased and the levels of UNC-96 protein were slightly reduced, suggesting that CSN-5 promotes the degradation of UNC-98 and that CSN-5 stabilizes UNC-96. In unc-15 and unc-96 mutants, CSN-5 protein was reduced, implying the existence of feed back regulation from myofibril proteins to CSN-5 protein levels. Our results are the first to implicate CSN-5 or the COP9 signalosome in myofibrillar organization or function.

Vandecandelaere I et al. (2017) PLoS One "Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus."

Deforce D, Coenye T, Van Nieuwerburgh F, Vandecandelaere I

[PLoS One, 2017]

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.

Vandecandelaere I et al. (2014) Pathog Dis "Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms."

Coenye T, Vandecandelaere I, Depuydt P, Nelis HJ

[Pathog Dis, 2014]

Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.

Kamp F et al. (2010) EMBO J "Inhibition of mitochondrial fusion by -synuclein is rescued by PINK1, Parkin and DJ-1."

Haass C, Hegermann J, Giese A, Eimer S, Kamp F, Lutz AK, Nuscher B, Wender N, Brunner B, Winklhofer KF, Exner N, Beyer K, Bartels T

[EMBO J, 2010]

Aggregation of -synuclein (S) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of S is largely unknown. We demonstrate with in vitro vesicle fusion experiments that S has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, S binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous S. In contrast, siRNA-mediated downregulation of S results in elongated mitochondria in cell culture. S can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, S prevents fusion of differently labelled mitochondrial populations. Thus, S inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of S is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin 1-79 or by DJ-1 C106A.

El Mouridi, Sonia et al. (2021) MicroPubl Biol

AlHarbi, Sarah, El Mouridi, Sonia, Frkjr-Jensen, Christian

[MicroPubl Biol, 2021]

For El Mouridi, S; AlHarbi, S; Frkjr-Jensen, C (2021). A histamine-gated channel is an efficient negative selection marker for C. elegans transgenesis. microPublication Biology. 10.17912/micropub.biology.000349.

Nam K et al. (1997) Mol Cell Biol "Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation."

Nam K, Boeke JD, Devine SE, Trambley J, Lee G

[Mol Cell Biol, 1997]

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.