V J Butler et al. (2008) European Worm Meeting "Use of Microarray to identify cyclosporin A and a novel dimedone compound EM234 effects on C. elegans at a genomic level."

V J Butler, A P Page

[European Worm Meeting, 2008]

C. elegans is extensively used as an animal model to study changes in gene. expression. Very little is known about mechanisms of action caused by drugs. and toxins in C.elegans.. Preliminary studies using Cyclosporin A (CsA) an immunosuppressant, was. shown to cause moulting, cuticle and gut structural defects suggesting. inhibition of protein folding, possibly through members of the immunophilin. family which consist of the cyclophilins (CYN) and the FK506-binding. protein families both possess peptidyl prolyl cis-trans isomerase (PPIases). activity. A number of novel dimedone derived compounds have been. identified as human CYN-A inhibitors and tested against C. elegans (Yang et. al, 2007). Initial results using a genomic approach of microarray analysis. and comparative real time PCR suggest that CsA and the novel dimedone. compound EM234 appear to affect cuticle synthesis in C. elegans down. regulating gene expression of essential collagen genes; dpy-17, dpy-14, sqt-. 3 in addition to hbl-1 a transcription factor which plays a role in. embryonic viability, locomotion, egg laying, body morphology, and. hypodermal differentiation. Further investigation using comparative real. time PCR was carried out on collagen associated genes col-129, cut-3 and. cht-1, which scored a P value of <0.01 to verify the microarray results.. cyn-7 gene expression was also tested as this gene was shown to be down. regulated and its encoded protein is similar to CYN-3 which is an analogue. of human Cyn-A through which CsA acts. cyn-7 was also shown to be required. for embryonic viability in one set of RNAi experiments (Maeda et al 2001).. Dimedone treated worms showed an overall down regulation in expression of. the collagen associated genes indicating that the dimedone based compound. may be acting through similar pathways as CsA.. Yang.Y et al. 2007. Biochem Biophys Res Commun. 363. 1013-1019.. Maeda I, et al. 2001. Current Bio. 11, 171-176.

Butler JA et al. (1994) Worm Breeder's Gazette "Looking For Extracellular Matrix Proteinases in C. elegans"

Kramer JM, Butler JA

[Worm Breeder's Gazette, 1994]

LOOKING FOR EXTRACELLULAR MATRIX PROTEINASES IN C. ELEGANS. James A. Butler and James M. Kramer, Northwestern University Medical School. Department of CMS Biology, Chicago IL 60611

Wang, A. et al. (2019) International Worm Meeting "Progranulin Polymorphisms in Aging and Stress Resistance."

Kao, A, Cortopassi, W, Butler, V, Jacobson, M, Wang, A.

[International Worm Meeting, 2019]

The cysteine-rich protein progranulin (PGRN) has been implicated in the pathobiology of several diseases including neurodegeneration and lysosomal storage related diseases (Baker, Nature 2006; Cruts, Nature 2006; Neumann, Science 2006; Smith, Am J Hum Genet 2012). Progranulin can be processed into bioactive granulin peptides, the number of which vary from species to species. C. elegans progranulin contains 3 granulins. Despite the variance in number of domains, the granulins share a stable, compact structure of beta-pleated sheets connected by disulfide bonds(Palfree, Plos One 2015). Studies have shown that loss of progranulin leads to features of accelerated aging, such as lipofuscin deposits and decreased lysosome protease activity (Ahmed, The American Journal of Pathology 2010; Ward, Science Translational Medicine 2017). In killifish, progranulin polymorphisms segregate with a short-lived aging phenotype (H151Q, C449W, R158H) (Valenzano, Cell 2015). The cysteine to tryptophan polymorphism is particularly interesting due to the cysteine's structural contribution to disulfide bonds. In addition, the histidine to glutamine polymorphism could potentially alter pH sensitivity. We have shown that pgrn-1(-) animals demonstrate a robust, stress-resistant phenotype that interestingly, can be decoupled from lifespan extension (Butler, J Neurosci 2019; Meredith, Plos Genetics 2013). Furthermore, progranulin acts in the lysosome and directly increases the activity of cathepsin-D (CTSD), a crucial lysosomal protease (Butler, Human Molecular Genetics 2018). We hypothesize that while complete absence of progranulin and granulin does not affect lifespan, that the polymorphisms in the granulin domains will impact aging and lifespan as well as stress response via regulation of lysosomal protease activity. To address this, we have created single copy insertions using MosSCI of these progranulin SNPs into C. elegans. We plan to test lifespan of these worms. We will also measure markers of aging and test stress response. To better understand potential molecular mechanisms, we will use molecular modeling to elucidate changes to progranulin and CTSD/ASP-3 interactions and test these in vivo. We anticipate that certain progranulin polymorphisms will increase lifespan and healthspan. We also predict that stress resistance will improve. We anticipate these will be a result of progranulin polymorphisms enhancing proteostasis via improved lysosomal protease activity.

Smith BS et al. (1990) Cryobiology "Cryopreservation of the entomogenous nematode parasite Steinernema feltiae (= Neoaplectana carpocapsae)."

Hodgson-Smith A, Popiel I, James ER, Smith BS, Minter DM

[Cryobiology, 1990]

Cryopreservation studies were conducted with the J1 juvenile and third stage infective juvenile (IJ) larval stages of the entomogenous parasitic nematode Steinernema feltiae (=Neoaplectana carpocapsae). The main parameters evaluated were (i) tolerance of the organisms to the cryoprotectants methanol, ethanediol, glycerol, and dimethyl sulfoxide; (ii) the incubation temperature and exposure period in cryoprotectant; and (iii) slow cooling (circa 1C min-1) vs rapid cooling (circa 5,100C min-1). The J1 stage was sensitive to all cryoprotectants at >20% (v/v). This sensitivity increased at 22 and 37C. Exsheathed IJ stage organisms tolerated exposure to 50% (v/v) ethanediol or Me2SO and 60% (v/v) methanol or glycerol. A proportion (3.4%) of J1s preincubated in 20% (v/v) methanol for 10 min at 0C survived slow cooling to -35C followed by plunge into liquid nitrogen. Preincubation in 60% (v/v) Me2SO for 45 sec at 0C followed by rapid cooling yielded a higher proportion (12.3%0 of surviving J1s. The infective IJ stage only survived rapid cooling. Preincubation for 20 min at 0C in either 60% (v/v) methanol or 45% glycerol, followed by rapid cooling at 5,100C min-1, yielded 30-34% viable IJs following thawing. These larvae were capable of further growth and development in vitro. The results suggest that the organisms are vitrified during the rapid cooling step. For routine maintenance of strains of S. feltiae and related

Kane PM (2006) Microbiol Mol Biol Rev "The where, when, and how of organelle acidification by the yeast vacuolar H+-ATPase."

Kane PM

[Microbiol Mol Biol Rev, 2006]

All eukaryotic cells contain multiple acidic organelles, and V-ATPases are central players in organelle acidification. Not only is the structure of V-ATPases highly conserved among eukaryotes, but there are also many regulatory mechanisms that are similar between fungi and higher eukaryotes. These mechanisms allow cells both to regulate the pHs of different compartments and to respond to changing extracellular conditions. The Saccharomyces cerevisiae V-ATPase has emerged as an important model for V-ATPase structure and function in all eukaryotic cells. This review discusses current knowledge of the structure, function, and regulation of the V-ATPase in S. cerevisiae and also examines the relationship between biosynthesis and transport of V-ATPase and compartment-specific regulation of acidification.

Goddard JM et al. (1986) Proc Natl Acad Sci U S A "Isolation and characterization of Caenorhabditis elegans DNA sequences homologous to the v-abl oncogene."

Goddard JM, Capecchi MR, Weiland JJ

[Proc Natl Acad Sci U S A, 1986]

DNA sequences homologous to the v-abl oncogene were isolated from a Caenorhabditis elegans genomic library by their ability to hybridize with a v-src probe. The DNA sequence of 2465 nucleotides of one clone was determined. This region corresponds to the 5' protein kinase domain of v-abl plus approximately equal to 375 base pairs toward the 3' end. Four potential introns were identified. The homology between the deduced amino acid sequence of the C. elegans clone and that of the 1.2-kilobase-pair protein kinase region of v-abl is 62%. The tyrosine residue corresponding to the tyrosine that is phosphorylated in the v-src protein is conserved in the C. elegans sequence. When 95 amino acids around this tyrosine were compared with the corresponding sequences of Drosophila c-abl, v-abl, and v-src, the identities were 83%, 79%, and 56%, respectively. Hybridization of the cloned DNA with C. elegans poly(A)+ RNA revealed a major transcript of 4.4 kilobases.

Ernstrom GG et al. (2012) Genetics "V-ATPase V1 sector is required for corpse clearance and neurotransmission in Caenorhabditis elegans."

Weimer R, Watanabe S, Pawar DR, Greenstein D, Ernstrom GG, Jorgensen EM, Hobson RJ

[Genetics, 2012]

The vacuolar-type ATPase (V-ATPase) is a proton pump composed of two sectors, the cytoplasmic V(1) sector that catalyzes ATP hydrolysis and the transmembrane V(o) sector responsible for proton translocation. The transmembrane V(o) complex directs the complex to different membranes, but also has been proposed to have roles independent of the V(1) sector. However, the roles of the V(1) sector have not been well characterized. In the nematode Caenorhabditis elegans there are two V(1) B-subunit genes; one of them, vha-12, is on the X chromosome, whereas spe-5 is on an autosome. vha-12 is broadly expressed in adults, and homozygotes for a weak allele in vha-12 are viable but are uncoordinated due to decreased neurotransmission. Analysis of a null mutation demonstrates that vha-12 is not required for oogenesis or spermatogenesis in the adult germ line, but it is required maternally for early embryonic development. Zygotic expression begins during embryonic morphogenesis, and homozygous null mutants arrest at the twofold stage. These mutant embryos exhibit a defect in the clearance of apoptotic cell corpses in vha-12 null mutants. These observations indicate that the V(1) sector, in addition to the V(o) sector, is required in exocytic and endocytic pathways.

Jennifer M Ross et al. (2002) Mid-west Worm Meeting "A role for the Polycomb Group in the development of the C. elegans male nervous system"

Jennifer M Ross, David A Zarkower

[Mid-west Worm Meeting, 2002]

Development of the C. elegans male-specific nervous system depends on interplay between two regulatory pathways, one involving the Hox genes mab-5 and egl-5, and the other involving the conserved sexual regulator mab-3. Males harboring Hox or mab-3 mutations lack V rays, sensory structures required for mating. The Hox proteins activate the bHLH gene lin-32 to specify the V rays, while the doublesex homolog MAB-3 synergizes with the Hox/lin-32 pathway to promote V ray differentiation. V rays can be restored in mab-3(-) males by overexpression of lin-32, suggesting that LIN-32 acts as a primary determinant of V ray fate, while MAB-3 potentiates LIN-32 activity.

Baird, Scott E. et al. (2011) International Worm Meeting "Ray Pattern Variation in C. elegans: Mapping a Major-Effect QTL on LGV."

Baird, Scott E., Bailey, Daniel

[International Worm Meeting, 2011]

Cryptic variation of ray pattern has been observed in recombinant-inbred lines (RIL) derived from crosses of N2 and CB4856 strains of C. elegans (Guess et al., 2007). The variant pattern consists of the anterior displacement of ray 3 into a position immediately adjacent to ray 2. QTL analyses of these RIL identified a major-effect QTL on the left arm of chromosome V (QTL-V). One RIL, QX34, possessed a recombinant chromosome V that contained CB4856 alleles from -12.72 to -7.93 cM, a region that precisely corresponded to QTL-V. When this chromosome (QX34-V) was crossed into an otherwise N2 background (strain PB2034), the variant ray pattern was observed at a frequency of 0.50. Based on genotypes of other RIL with recombination breakpoints in this region, it appeared that QTL-V resulted from allelic variation at two or more loci. To confirm this result, we are mapping the gene(s) in this region responsible for the observed variation in ray pattern. Initial experiments demonstrated that the CB4856 alleles responsible for QTL-V were recessive to the corresponding N2 alleles. From deletion heterozygotes, it was determined that at least one gene responsible for QTL-V lies to the right of -9.00 cM. However, deletion of a region from -12.06 to -8.15 did not uncover the ray pattern variant. Therefore, either allelic variation at two or more genes is required for QTL-V or the gene responsible for QTL-V resides between -8.15 and -7.93 cM. Attempts at recombination mapping of QTL-V are ongoing but have been hindered by an apparent suppression of recombination in PB2034/N2 heterozygotes.

Rane HS et al. (2014) Eukaryot Cell "The contribution of Candida albicans vacuolar ATPase subunit VB, encoded by VMA2, to stress response, autophagy, and virulence is independent of environmental pH."

Bernardo SM, Parra KJ, Hayek SR, Lee SA, Rane HS, Binder JL

[Eukaryot Cell, 2014]

Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the VB subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase Vo and V subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of V-ATPase to virulence is independent of host pH.