Paul D Hartley et al. (2001) International C. elegans Meeting "The spe-5 Gene Encodes a Vacuolar (H+)-ATPase Beta Subunit that is Required for Spermatogenesis"

Guang-dan Zhu, Steven W L'Hernault, Paul D Hartley

[International C. elegans Meeting, 2001]

Spermatogenesis in all animals requires the orderly segregation of cytoplasm during a series of asymmetric cellular divisions. During C. elegans spermatogenesis, retention of essential components for spermiogenesis occurs, in part, because many reside within the fibrous body-membranous organelle (FB-MO) complexes. The FB-MO complexes segregate to spermatids during their formation and much of the work in our laboratory is focused on the morphogenesis and function of these Golgi-derived organelles. Prior work indicated that spe-5 mutants contain defective FB-MO complexes (K. Machaca and S. W. L'Hernault Genetics 146: 567-581), and we have cloned this gene in order to understand its molecular nature. spe-5 was previously mapped to a 0.5 cM region of chromosome I between unc-38 and dpy-5 , and single nucleotide polymorphism (SNP) mapping was used to narrow this interval. Four classes of homozygous recombinant worms were created from mates between unc-38 spe-5 dpy-5 Bristol hermaphrodites and wild type Hawaiian (strain CB4856) males. The recombinant strains were mapped against five SNPs found between N2 and CB4856 to place spe-5 within a ~65 kb region covered by one cosmid and part of a YAC. Differential display data (M. Jiang, 2001, et. al. PNAS USA 98: 218-223) suggested that only one sperm-specific ORF was located in this interval, and all six spe-5 alleles contained mutations in this gene. Five of the six spe-5 alleles were EMS induced, and the sixth is a Tc1 insertion that arose spontaneously in a mut-3 mutator background. The sequence of the spe-5 gene reveals that it encodes a vacuolar (H+)-ATPase beta subunit. Vacuolar ATPases are >750 kDa proton pumping "machines" composed of at least 13 different subunits. They are found in a wide range of organisms, and utilize the energy produced by ATP hydrolysis to acidify the lumen of membranous organelles (review by, T. H. Stevens and M. Forgac Annu. Rev. Cell Dev. Biol. 13: 779-808). A pH-sensitive fluorescent probe has shown that wild-type MOs become acidic during spermatid formation (K.L. Hill and S. W. L'Hernault, unpublished observations), and we presently are determining if spe-5 spermatids have acidified MOs. While there appears to be another C. elegans beta subunit that is expressed somatically, there are no other sperm-specific V-ATPase subunits. Prior data suggests that a pH change accompanies spermatid maturation (Ward et al., 1983, Dev. Biol . 98: 70-79) and we are exploring if the spermatid vacuolar ATPase participates in this process.

Paul D Hartley et al. (2002) East Coast Worm Meeting "Two vacuolar ATPase B subunits exist in the nematode genome, and spe-5 encodes one that is required for spermatogenesis"

Steven W L'Hernault, Paul D Hartley

[East Coast Worm Meeting, 2002]

Mature spermatozoa in all animals are streamlined, specialized cells that contain only the components required to find, approach, and fertilize oocytes. In C. elegans, asymmetric segregation of cytoplasm occurs at the end of spermatogenesis when all the essential components are placed into haploid spermatids budding from a residual body. The residual body contains unnecessary components and will eventually be reabsorbed. A specialized, Golgi-derived organelle called the fibrous body-membranous organelle (FB-MO) contains proteins necessary for functional spermatozoa. All FB-MOs segregate into spermatids that are budding from a residual body, and mutants affecting the morphogenesis or proper segregation of FB-MOs have been identified. spe-5 mutants rarely form spermatids and instead form terminal spermatocytes that have completed meiosis. These spermatocytes have an abnormal cratered appearance caused by the partial distension of the MO double membrane from the FB. The spe-5 gene encodes a B subunit of the vacuolar (H+)-ATPase, which is a multisubunit machine that hydrolyzes ATP to pump protons across a membrane and establish a proton gradient. Experiments using pH-sensitive indicators have shown that the MOs in spermatids have a low luminal pH (K. HILL and S. W. L'HERNAULT, unpubl.), so V-ATPases and therefore SPE-5 may be located on the MOs. Antibodies and GFP fusion constructs will be used to confirm the localization of SPE-5. A gene located on the X chromosome encodes another B subunit with 85% amino acid identity to SPE-5, and it is not yet known if it can functionally replace spe-5. The existence of two B subunits in the genome suggests that while both likely contribute to the activity of the V-ATPase, they may differ from each other by specialized functions. This hypothesis initially will be addressed by a two-hybrid assay that may reveal different protein binding specificities.

Wissmann AK et al. (1994) Worm Breeder's Gazette "Characterization of the let-502 Gene"

Mains PE, Wissmann AK, McGhee JD

[Worm Breeder's Gazette, 1994]

Characterization of the let-502 gene Andreas Wissmann, James D. McGhee and Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Mains PE (1994) Worm Breeder's Gazette "Interactions Between mei-1 and the unc-116 Kinesin"

Mains PE

[Worm Breeder's Gazette, 1994]

Interactions Between mei-l and the unc-116 Kinesin Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada

Sommer RJ et al. (1994) Worm Breeder's Gazette "Evolution of Vulva-Formation: Part II: Species With a Central Vulva"

Sommer RJ, Sternberg PW

[Worm Breeder's Gazette, 1994]

Evolution of vulva-formation: Part II: Species with a central vulva Ralf J. Sommer & Paul W. Sternberg, California Institute of Technology, Division of Biology 156-29, Pasadena, CA 91125

Li WQ et al. (1992) Worm Breeder's Gazette "Characterization of the Axonal Guidance and Outgrowth gene Unc-33"

Shaw JE, Li WQ, Herman RK

[Worm Breeder's Gazette, 1992]

Characterization of the axonal guidance and outgrowth gene unc-33 W. Li, R. K. Herman and J. E. Shaw Department of Genetics and Cell biology, University of Minnesota, St Paul, MN 55108

Sommer RJ et al. (1994) Worm Breeder's Gazette "Evolution of Vulva Formation: Part IV: Variation in AC Position Can Cause a Shift of Vulva Formation Towards P(4-6).p."

Sternberg PW, Sommer RJ

[Worm Breeder's Gazette, 1994]

Evolution of vulva formation: Part IV: Variation in AC position can cause a shift of vulva formation towards p(4- 6).p Ralf J. Sommer & Paul W. Sternberg, HHMI & California Institute of Technology, Division of Biology 156-29, Pasadena, CA

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.