Moorthy S et al. (1997) International C. elegans Meeting "THE SPECTRIN-BASED MEMBRANE SKELETON IN C.ELEGANS"

Bennett V, Moorthy S, Chen LS

[International C. elegans Meeting, 1997]

The spectrin-based membrane cytoskeleton was first characterized in human erythrocytes where it associates with integral membrane proteins to form a meshwork on the cytoplasmic surface of the plasma membrane, providing structural support for the lipid bilayer. Proteins closely related to spectrin and many accessory proteins of the erythrocyte membrane skeleton are associated with the membranes of most vertebrate and non-vertebrate cell. Here we report the preliminary characterization of the spectrin-based membrane cytoskeleton in C.elegans. Through ACeDB we have identified a highly conserved nematode homologue of betaG-spectrin, the major beta spectrin isoform in vertebrates. Using an affinity-purified antibody against the C. elegans protein, we demonstrate that spectrin is expressed at high levels throughout the nematode and is probably associated with the plasma membranes of all its cell types. We are currently screening several genetically mapped candidate let alleles. Meanwhile, to circumvent the probable lethal effect of a spectrin null, we used antisense RNA experiments to raise 'transgenic' lines which are variably deficient in betaG spectrin. The resulting 'phenotypes' range from embryonic or L1 larval arrest to dpy and/or unc 'carrier' adults with multiple lesions. The nature and characteristics of the 'phenotypes' suggest that spectrin is an essential protein that functions in multiple cell types and during multiple stages of development. Using an entire metazoan animal, we present the hypothesis that the spectrin-based membrane skeleton plays the role of a ubiquitous membrane structure that interacts with a variety of integral membrane proteins, and provides membranes with the mechanical stability and long-range order required to perform their diverse functions.

Moorthy S et al. (1996) East Coast Worm Meeting "A NOVEL C. ELEGANS ADDUCIN: A WHOLE NEW CAN OF WORMS"

Bennett V, Chen LS, Moorthy S

[East Coast Worm Meeting, 1996]

Vertebrate adducin is an actin-binding protein which can promote assembly of a spectrin-actin complex and can also act as a barbed-end actin capping protein. Biochemical and biophysical characterization in our lab has shown that active adducin is probably a tetramer consisting of two alpha subunits tightly complexed with either beta or gamma subunits. Adducin binds calmodulin and is an in vivo substrate for protein kinases C and A, all of which modulate adducin's spectrin-actin binding activity in different ways in in vitro assays. Nevertheless, vertebrate adducin has never been definitively linked to any known mutant phenotype. However mutations in a Drosophila adducin homologue, hu-li tai shao (hts), result in a female sterile phenotype in which egg chambers have fewer nurse cells, with abnormal actin distribution in the ring canals. Here we report preliminary results of our attempt to develop C. elegans as a genetic model system for adducin. We made a database search for C. elegans adducin and originally identified two partial cDNAs from Yuji Kohara's EST sequences. The first clone, yk26f2, which we call add-1 (C. elegans Adducin 1), maps to cosmid F39C12 on the left arm of LGX, close to lon-2. The 659 residue predicted protein shows significant homology to vertebrate adducin and hts in the head, neck and tail regions which suggests it may behave like the "canonical" adducins. Interestingly though, yk120b7(add-2) which lies on Cosmid F57F5 on the right arm of LGV, shows a novel structure. The C-terminal 261 residues show homology to the adducin "head" which normally lies at the N-terminus of other adducins. Instead Cad-2 has a novel 350 residue N-terminus that shows no homology in a BLAST search to known proteins. Cad-2 may use its adducin "head" to interact with Cad-1 in a heteromeric manner similar to that demonstrated for vertebrate adducins. But it also raises the exciting prospect that the adducin "head" domain may actually be a component of multiple proteins with different functions, in a manner analogous to that demonstrated for the kinesin and myosin head domains. We aim to resolve these possibilities using C. elegans as a model system.

Moorthy S et al. (2000) J Cell Biol "Caenorhabditis elegans beta-G spectrin is dispensable for establishment of epithelial polarity, but essential for muscular and neuronal function."

Bennett V, Chen LS, Moorthy S

[J Cell Biol, 2000]

The Caenorhabditis elegans genome encodes one alpha spectrin subunit, a beta spectrin subunit (beta-G), and a beta-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans alpha spectrin is reproduced by tandem depletion of both beta-G and beta-H spectrins. We propose that alpha spectrin combines with the beta-G and beta-H subunits to form alpha/beta-G and alpha/beta-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode beta-G spectrin and vertebrate beta spectrins exhibit three striking parallels including: (1) beta spectrins are associated with the sites of cell-cell contact in epithelial tissues; (2) the highest levels of beta-G spectrin occur in the nervous system; and (3) beta spectrin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode beta-G spectrin associates with plasma membranes at sites of cell-cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode beta-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, beta-G spectrin is not associated with internal membranes and depletion of beta-G spectrin was not associated with any detectable defects in secretion. Instead beta-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans beta-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.

S Moorthy et al. (1999) International C. elegans Meeting "The C. elegans Spectrin Based Membrane Skeleton"

V Bennett, L Chen, S Moorthy

[International C. elegans Meeting, 1999]

The spectrin based membrane cytoskeleton was first characterized in red cells where it forms a visualizable polygonal network on the cytoplasmic surface of cell membranes. Its principle component is the heteromeric protein spectrin, made up of alpha & beta subunits. It provides mechanical support & flexibility to the membrane, enabling survival of red cells in circulation. Proteins related to spectrin are expressed in almost all metazoan cells. We identified a C. elegans homolog of betaG spectrin on LGV (wm97e421). Injection of its ssRNA into syncytial gonads of hermaphrodites resulted in a variety of phenotypes in progeny, from arrested larvae to dpy unc adults, but near normal protein levels. However using dsRNA mediated interference (RNAi) we almost completely eliminate the protein and then 90% of progeny arrest as uncoordinated L1 larvae with neuronal defects. Hammarlund et al previously reported unc-70 alleles with similar phenotypes and proposed that unc-70 was a regulator of synaptic transmission (wcwm98p129). They now report that unc-70 is the C. elegans betaG spectrin and we concur. Our own results show that UNC70 is highly expressed in synapse rich structures of the nervous system, though it expresses throughout development and associates with membranes of most cell types. Hence unc-70 probably has multiple roles during development. C. elegans betaH sma-1 and alpha imo-1 spectrins have been previously identified (cgc3094, wcwm98p30). SMA1 and UNC70 have mutually exclusive subcellular localizations in gut and hypodermal cells (J. Austin pers. comm., our data). Using RNAi we found sma-1(RNAi) worms are viable and small with head morphogenetic defects; unc-70(RNAi) worms arrest as early L1 larvae of normal morphology; imo-1(RNAi) worms arrest as early L1 larvae that are shorter with head morphogenetic defects; and finally sma-1(RNAi);unc-70(RNAi) double mutant animals are morphologically indistinguishable from imo-1(RNAi) progeny, even though the latter have normal levels of UNC70 protein. These data suggest SMA1 and UNC70 play complementary roles in development and IMO1 is required for each alpha-beta spectrin complex.

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.

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.