[
International Worm Meeting,
2017]
Infertility affects 6.7 million women between the ages of 15 and 44 in the US. Our approach to solving the infertility problem is multidisciplinary; drawing from cell biology, chemistry, and physics to define how the transition metal zinc, maintains healthy female eggs. Zinc is critical in regulating mammalian egg development through large-scale, dynamic zinc fluxes. To expand on these findings, we utilize the model system Caenorhabditis elegans to further uncover zinc's role in egg viability maintenance. Many regulatory factors involved in the reproductive tract formation in C.elegans are conserved in higher organisms. Therefore, C.elegans are an excellent model to study zinc. We aim to uncover if zinc fluxes are conserved and characterize how zinc affects egg viability. We hypothesize that zinc fluxes are conserved and that proper zinc levels maintains oocyte viability. We sequestered zinc utilizing the chemical TPEN in the worm's growth environment. Results demonstrated that zinc insufficient (ZI) hermaphrodites yielded smaller broods and contained fewer unfertilized oocytes compared to controls, whether self-mated or not. By time-lapse confocal imaging, we discovered that zygotes exposed to low TPEN concentrations (10 uM) displayed improper polar body extrusion, hyperploidy, lagging chromosomes and improper cytoplasmic cleavage. Combined, these results suggest that zinc insufficiency impacts unfertilized eggs within the reproductive tract and not sperm from hermaphrodites or males. Moreover, zygotes remain sensitive after fertilization to changes in cytoplasmic zinc concentrations. Because we determined that zinc insufficiency impacts the maturing egg, we next performed X-Ray Fluorescence Microscopy (XFM) at Argonne National Laboratory to determine total zinc content during oocyte maturation. Total zinc is all of the zinc in the cell, bound and unbound. We discovered that total zinc content sharply increases after fertilization up to the zygote stage, and decreases after meiosis. This indicates that large-scale zinc fluxes in C.elegans occur similarly as mammals. We similarly examined labile zinc dynamics during oocyte maturation. Labile zinc is zinc that is accessible to our probes. Utilizing the zinc sensor ZincBy-1, we determined that after fertilization, labile zinc steadily increases in the cytoplasm up to Anaphase II. Then, labile zinc content declines steadily and remains at low levels through 2-cell. Labile zinc from the cytoplasm relocates to the extra embryonic matrix (EEM), and is visible beginning at pronuclear migration. Future experiments will allow us to understand the mechanism behind zinc fluctuations within maturing oocytes. Work with C.elegans can provide insight for the conserved mechanisms for zinc action during fertilization and proper egg formation. We will then be closer to understanding human infertility issues we face today.
[
FEBS Lett,
2002]
6-Photocholesterol, a new photoactivatable analog of cholesterol in which a diazirine functionality replaces the 5,6-double bond in the steroid nucleus, was used recently to identify cholesterol-binding proteins in neuroendocrine cells [Thiele, C., Hannah, M.J., Farenholz, F. and Huttner, W.B. (2000) Nat. Cell Biol. 2, 42-49], to track the distribution and transport of cholesterol in Caenorhabditis elegans [Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzchalia, T.V. (2001) Mol. Biol. Cell 12, 1725-1736], and to probe lipid-protein interactions in oligodendrocytes [Simons, M., Kramer, E.M., Thiele, C., Stoffel, W. and Trotter, J. (2000) J. Cell Biol. 151, 143-154]. To determine whether 6-photocholesterol is a faithful mimetic of cholesterol we analyzed the ability of this probe, under conditions in which it is not photoactivated to a carbene, to substitute for cholesterol in two unrelated assays: (1) to condense 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine monomolecular films and (2) to mediate the fusion of two alphaviruses (Semliki Forest and Sindbis) with liposomes. The results suggest that this analog is a suitable photoprobe of cholesterol.
Yu, Yawei, L'Etoile, Noelle D., Brueggemann, Chantal, Chen, Tsung-Yu, Zhang, Xiao-Dong, Altshuler-Keylin, Svetlana, O'Halloran, Damien M.
[
International Worm Meeting,
2011]
The Caenorhabditis elegans AWC neurons are responsible for sensation of a range of attractive volatile odors (Bargmann et al., 1993). Prolonged odor exposure in the absence of food leads to reversible decreases (adaptation) in the animal's attraction to that odor (Colbert and Bargmann, 1995; L'Etoile et al., 2002; Nuttley et al., 2002; Kaye et al., 2009; O'Halloran et al., 2009). It has been shown previously that the odor specificity of adaptation is determined by the feeding status of the animal (Colbert and Bargmann, 1997). That is, if a well-fed worm is exposed to benzaldehyde for a sustained period, it will adapt to both benzaldehyde and isoamyl alcohol (both sensed by AWC), this process is termed cross adaptation. In contrast, an unfed (starved) worm will adapt to benzaldehyde and its response to isoamyl alcohol will remain intact. The TAX-4 and TAX-2 cyclic nucleotide-gated (CNG) channel subunits are required for AWC-mediated olfactory responses (Coburn and Bargmann, 1996; Komatsu et al., 1999). TAX-4 is an a subunit that can form homomeric channels while TAX-2 is a b subunit that requires TAX-4 to form a functional channel (Coburn and Bargmann, 1996; Komatsu et al., 1999). C. elegans encodes two additional predicted a subunits, CNG-1 and CNG-3 (Cho et al., 2004a; 2004b; Coburn thesis, 1996). Here we report that CNG-1 is required in AWC to promote short-term (30-mins) cross adaptation between benzaldehyde and isoamyl alcohol. The ability of food to induce this cross adaptation is also dependent on the ASI sensory neurons. We also demonstrate that CNG-3 is required in AWC for adaptation to short-term (30-mins) exposures of odor. By using FRET, Bio-molecular Fluorescent Complementation assays, and genetically encoded calcium imaging we find that TAX-2::TAX-4::CNG-3 channels may promote short-term adaptation in AWC. Our data suggests the TAX-2::TAX-4::CNG-3 channel adopts a 3a:1b stoichiometry. By examining the electrophysiology of the CNG subunits in AWC, we also demonstrate that fast closing dynamics appear critical for proper short-term adaptation responses. Taken together our findings provide more understanding into the mechanisms and circuitry of how CNG channels shape olfactory plasticity.