GOPAL, S. et al. (2017) International Worm Meeting "Proteoglycans in stem cell development."

Pocock, R., GOPAL, S.

[International Worm Meeting, 2017]

Proteoglycans are composed of a core protein and multiple glycosaminoglycan chains. At least 14 proteoglycans are encoded in the mammalian genome whereas Caenorhabditis elegans carry four. The proteoglycans present in C. elegans include, a syndecan (sdn-1), a perlecan (unc-52) and two glypicans (lon-2, gpn-1). These glycosylated proteins are involved in several cellular functions, including cell-cell/cell-matrix attachment, cancer and neuronal development. Besides being signaling molecules, proteoglycans are involved in the activation of other signaling molecules such as Wnts (Munoz et al., 2006) or Notch (Pisconti et al. 2010). In addition to developmental defects in C. elegans, the loss of the proteoglycan sdn-1 leads to reduced brood size, suggesting a function for SDN-1 in germline development. The germline houses the only stem cell population in C. elegans and our data suggest that proteoglycans play a role in stem cell proliferation and differentiation. In C. elegans, stem cells are regulated by a network of RNA-regulatory molecules and the Notch signaling pathway components to control differentiation and the production of eggs and sperm. Earlier studies in mammals also implicated syndecan-3 along with Notch in cell differentiation, though the underlying mechanism is poorly understood. In addition, syndecan-2, -3 and -4 were shown as regulators of stem cell development. We have recently identified a role for syndecans in the control of transient calcium changes in C. elegans. This syndecan-mediated calcium regulation is achieved through the mechanically sensitive, transient receptor potential (TRP) channels. Transient calcium is an important factor in stem cell development where calcium fluxes impact the cell cycle. We have also identified germline specific defects in syndecan mutant worms, which is controlled by syndecan expression at specific domains. We hypothesize that syndecan is involved in germline maintenance by regulation of transient calcium through TRP channels. Our study currently focuses on the role of syndecans in stem cell development and differentiation through various factors including notch signaling, calcium metabolism and extracellular mechanical forces.

Gopal S et al. (2016) Worm "Redefining the role of syndecans in C. elegans biology."

Couchman J, Pocock R, Gopal S

[Worm, 2016]

Cytosolic calcium is an important factor during fertilization, development and differentiation. Hence, the control of cytosolic calcium levels has been studied extensively for several decades. Numerous calcium channels have been identified and their mechanism of action elucidated. However, the mode of calcium channel regulation remains elusive. Here we discuss our recent findings regarding the role of syndecans in the regulation of cytosolic calcium levels. Syndecans are transmembrane proteoglycans present in both vertebrates and invertebrates that interact with extracellular ligands resulting in the activation of several downstream signaling pathways. We identified a previously unappreciated role of syndecans in cytosolic calcium regulation in mammals that is conserved in C. elegans. We concluded that calcium regulation is the basic, evolutionarily conserved role for syndecans, which enables them to be integral for multiple cellular functions.

Gopal S et al. (2017) Dev Biol "Automated three-dimensional reconstruction of the Caenorhabditis elegans germline."

Boag P, Pocock R, Gopal S

[Dev Biol, 2017]

The Caenorhabditis elegans germline is widely used as a model to study stem cell development, chromosome dynamics and apoptosis. Major readouts of germline phenotypes such as cell counting and protein expression profiling are routinely analysed manually and in a two-dimensional manner. The major disadvantages of the existing approaches are 1) they are time-consuming and laborious and 2) there is an inability to study the effects of genetic mutations in three dimensions. Here, we demonstrate a rapid, automated method for analysing the three-dimensional distribution of proteins, germline nuclei and cytoskeletal structures in the C. elegans germline. Using this method, we have revealed previously unappreciated germline organisation and cytoskeletal structures that will have a major impact on the characterisation of germline phenotypes. To conclude, our new method dramatically enhances the efficiency and resolution of C. elegans germline analysis and may be applied to other cellular structures.

Gopal S et al. (2021) Nat Commun "A somatic proteoglycan controls Notch-directed germ cell fate."

Ng L, Amran A, Pocock R, Elton A, Gopal S

[Nat Commun, 2021]

Communication between the soma and germline optimizes germ cell fate programs. Notch receptors are key determinants of germ cell fate but how somatic signals direct Notch-dependent germ cell behavior is undefined. Here we demonstrate that SDN-1 (syndecan-1), a somatic transmembrane proteoglycan, controls expression of the GLP-1 (germline proliferation-1) Notch receptor in the Caenorhabditis elegans germline. We find that SDN-1 control of a somatic TRP calcium channel governs calcium-dependent binding of an AP-2 transcription factor (APTF-2) to the glp-1 promoter. Hence, SDN-1 signaling promotes GLP-1 expression and mitotic germ cell fate. Together, these data reveal SDN-1 as a putative communication nexus between the germline and its somatic environment to control germ cell fate decisions.

Gopal S et al. (2018) J Vis Exp "Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton."

Pocock R, Gopal S

[J Vis Exp, 2018]

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Gopal, S. et al. (2019) International Worm Meeting "Syndecan controls germline development by regulating Notch Receptor transcription."

Pocock, R., Kimble, J., Elton, A., Bulow, H., Crittenden, S., GOPAL, S.

[International Worm Meeting, 2019]

Syndecans are transmembrane proteoglycans that control cell behavior through regulation of growth factor signaling, cell adhesion and cytoskeletal organization. In C. elegans, loss of sdn-1leads to reduced progeny, suggesting a function for SDN-1 in germline development. The germline houses the only stem cell population in C. elegansand our data show that sdn-1mutant hermaphrodites have reduced stem cell number. We previously showed that the absence of syndecan causes elevated cytosolic Ca2+levels through dysregulation of TRP Ca2+channels. SDN-1 also performs this function in the germline as removal of the TRP-2 channel restores wild-type germline stem cell number to sdn-1mutant animals. To examine the downstream effect of the SDN-1/TRP-2 axis, we analyzed the transcriptome of sdn-1mutant germlines and found that multiple stem cell proliferation and meiosis promoting genes are downregulated in the absence of sdn-1. In particular, the glp-1 mRNA was reduced. GLP-1 is a Notch Receptor that is crucial for germline development. We found that downregulation of glp-1expression was reversed in sdn-1; trp-2double mutant animals. Finally, our recent data suggest that SDN-1 controls glp-1expression through a highly conserved AP transcription factor binding motif. This identifies a novel regulatory mechanism that controls Notch Receptor expression in the germline.

Gopal S et al. (2015) J Cell Biol "Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels."

Gopal S, Okina E, Park PW, Pataki C, Pedersen ME, Stevens T, Couchman JR, Multhaupt HA, Sogaard P, Xian X, Pocock R, Griesbeck O

[J Cell Biol, 2015]

Transmembrane heparan sulfate proteoglycans regulate multiple aspects of cell behavior, but the molecular basis of their signaling is unresolved. The major family of transmembrane proteoglycans is the syndecans, present in virtually all nucleated cells, but with mostly unknown functions. Here, we show that syndecans regulate transient receptor potential canonical (TRPCs) channels to control cytosolic calcium equilibria and consequent cell behavior. In fibroblasts, ligand interactions with heparan sulfate of syndecan-4 recruit cytoplasmic protein kinase C to target serine714 of TRPC7 with subsequent control of the cytoskeleton and the myofibroblast phenotype. In epidermal keratinocytes a syndecan-TRPC4 complex controls adhesion, adherens junction composition, and early differentiation in vivo and in vitro. In Caenorhabditis elegans, the TRPC orthologues TRP-1 and -2 genetically complement the loss of syndecan by suppressing neuronal guidance and locomotory defects related to increases in neuronal calcium levels. The widespread and conserved syndecan-TRPC axis therefore fine tunes cytoskeletal organization and cell behavior.

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.