Ramadin, V. et al. (2019) International Worm Meeting "The search for additional proteins that physically associate with the LIM-7 transcriptional complex."

Ramadin, V., Vallier, L. G.

[International Worm Meeting, 2019]

The LIM-Homeodomain (LIM-HD) family of transcription factors, which are conserved throughout evolution, are characterized by the presence of two specialized zinc fingers that are located N-terminally to a DNA-binding homeodomain. LIM-HD proteins have been well studied in the context of neuronal patterning in many phyla, in addition to being present and studied in a variety of non-neuronal tissues. The Vallier lab is interested in the LIM-HD protein, LIM-7, an essential transcription factor. lim-7 is expressed in many tissues including the reproductive system, nervous system, muscular system, and alimentary system. To begin to understand the role of lim-7 in these tissues, as well as its essential role for viability, the Vallier lab has been taking a multi-prolonged approach. Currently, 25 known proteins are known to physically interact or regulate lim-7 and an equal number are predicted to interact. We are interested to see if any of these known factors are acting in a combinatorial fashion in a transcriptional complex with LIM-7 in various tissues to specify tissue identity. As a first step toward this goal, we wanted to see if any additional proteins were associating with lim-7 physically. Therefore, we attempted an affinity pull down of protein lysates using a tagged lim-7 strain, to assess the presence of proteins that were associated with it by Coomassie gels, using Western Blots for loading controls and LIM-7 presence. Preliminary attempts have shown promising results. Additional affinity pull-down experiments to optimize conditions are in progress and will be analyzed by mass spectroscopy. Results will be reported.

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.

Inoue T et al. (2005) J Bioenerg Biomembr "Structure and regulation of the V-ATPases."

Qi J, Jefferies K, Wang Y, Forgac M, Inoue T, Hinton A

[J Bioenerg Biomembr, 2005]

The V-ATPases are ATP-dependent proton pumps present in both intracellular compartments and the plasma membrane. They function in such processes as membrane traffic, protein degradation, renal acidification, bone resorption and tumor metastasis. The V-ATPases are composed of a peripheral V(1) domain responsible for ATP hydrolysis and an integral V(0) domain that carries out proton transport. Our recent work has focused on structural analysis of the V-ATPase complex using both cysteine-mediated cross-linking and electron microscopy. For cross-linking studies, unique cysteine residues were introduced into structurally defined sites within the B and C subunits and used as points of attachment for the photoactivated cross-linking reagent MBP. Disulfide mediated cross-linking has also been used to define helical contact surfaces between subunits within the integral V(0) domain. With respect to regulation of V-ATPase activity, we have investigated the role that intracellular environment, luminal pH and a unique domain of the catalytic A subunit play in controlling reversible dissociation in vivo.

Millard, J. et al. (2017) International Worm Meeting "Exploring the effects of the V-ATPase inhibitor luteolin on development, germline apoptosis, and FB-MO acidification in C. elegans."

Henderson, M., Millard, J., Dexter, K.

[International Worm Meeting, 2017]

The Vacuolar-type Adenosine Triphosphatase (V-ATPase) is a large multi-subunit enzyme complex found within intracellular and plasma membranes of animals, fungi, and plants. Contemporary research efforts have elucidated the role of V-ATPases in neoplastic maladies. Increased V-ATPase expression has been shown to support neoplastic metabolism and migration. The extensive involvement of V-ATPases in pathological conditions qualifies it as a suitable target for pharmaceutical intervention. Luteolin is a flavone found in a variety of plants, and is frequently consumed as a dietary supplement. Luteolin has been demonstrated to inhibit V-ATPase activity in mammalian cell culture. During spermatogenesis in C. elegans, the fibrous body-membranous organelles (FB-MOs) act as secretory vesicles responsible for the delivery of proteins to the surface of the sperm prior to fertilization. V-ATPases function to acidify the FB-MOs, contributing to their maturation to spermatozoa. Successful spermatogenesis is contingent on the fidelity of the V-ATPase in this acidification process. This change in pH can be detected by the use of LysoSensor Blue (Molecular Probes) staining. Disruption or inhibition of the V-ATPase complex is reliably reported by the LysoSensor staining assay. This allows for screening of potential V-ATPase inhibiting compounds such as luteolin. The toxicity of luteolin to C. elegans was demonstrated by growth and development assays. Examination of the effects of luteolin on germline apoptosis were performed using the ced-1::gfp reporter strain and acridine orange staining. Successful inhibition of FB-MO acidification supports the role of the V-ATPase in spermiogenesis as well as the potential therapeutic role of luteolin. These methods also provide an efficient medium for investigation of other V-ATPase inhibitory compounds.