Laufer JS et al. (1982) Worm Breeder's Gazette "no 5-methylcytosine in Ce DNA."

Laufer JS, Powers S, Wood WB

[Worm Breeder's Gazette, 1982]

Methylation of DNA has been implicated in gene regulation. About 4% of the cytosine residues are methylated in mammalian DNAs, and some specific changes in methylation are correlated with changes in gene activity (e.g., McGee & Ginder, Nature 280, 419, 1979). On the other hand, it was recently reported that Drosophila DNA contains no 5- methylcytosine (News & Views, Nature 290, 363, 1981). We undertook to determine whether C. elegans DNA contains methylated bases by reversed-phase high pressure liquid chromatography of L1 DNA hydrolyzed to free bases with formic acid. We detected no 5- methylcytosine or N5-methyl adenine. By comparison with hydrolyzed calf thymus DNA run on the same column, we can place an upper limit for 5-methylcytosine in C. elegans DNA at about 0.01% of total nucleotides, corresponding to <0.04% of cytosine residues.

McLarnon, Caitlyn et al. (2011) International Worm Meeting "Oxidative stress induces loss of GFP labeled neurons and delayed development to adulthood in an unc-13 mutant."

Kohn, Rebecca E., Minniti, Alicia N., Frymoyer, Christopher, Inestrosa, Nibaldo C., McGee, Caitlin, Roth, Elizabeth, King, Lauren, Gurenlian, Laura, McLarnon, Caitlyn

[International Worm Meeting, 2011]

We are interested in understanding how changes in neurotransmitter release affect sensitivity of Caenorhabditis elegans to oxidative stress. Oxidative stress can occur when cells fail to neutralize reactive oxidative species. Nucleic acids, lipids, and proteins can be damaged in response to oxidative stress and affected neurons may undergo neurodegeneration. Activity in neurons can confer resistance to oxidative stress. Products of the unc-13 gene regulate neurotransmitter release from all neurons in C. elegans and defects in this gene result in decreased abundance of neurotransmitters in synapses and paralysis. When wild type C. elegans are exposed to oxidative stress, there is a delay in the development of worms to adulthood. Following exposure of unc-13 mutants to the chemical paraquat, which induces oxidative stress, we found that unc-13 worms develop to adulthood later than wild type. Using strains with GFP labeled neurons, we examined the number of GABAergic, dopaminergic, and serotonergic neurons in worms with a normal functioning nervous system and with a mutation in unc-13 following exposure to paraquat. There was a decrease in the number of GFP labeled neurons and more neurons were missing in the unc-13 strain. These findings suggest that unc-13 mutants with decreased neurotransmitter release have increased sensitivity to oxidative stress. We plan to perform experiments to determine whether the GFP labeled neurons are undergoing apoptosis or necrosis.

Preston S et al. (2017) FASEB J "Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain."

Sternberg PW, Ansell BRE, Andrews KT, Nowell C, Chang BCH, Hofmann A, Crawford S, Korhonen PK, Baell J, Gijs MAM, Fisher GM, Young ND, Preston S, Mouchiroud L, Gasser RB, Jabbar A, Auwerx J, Davis RA, McGee SL, Cornaglia M

[FASEB J, 2017]

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mito-ribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Matthew McGee et al. (2007) International Worm Meeting "KASH and SUN proteins bridge the nuclear envelope to transfer force from the cytoskeleton to the nucleoskeleton during nuclear positioning."

Matthew McGee, Daniel Starr

[International Worm Meeting, 2007]

Nuclear positioning, which includes both migration and anchorage of the nucleus, is an important process in C. elegans development. The outer nuclear membrane (ONM) KASH proteins UNC-83 and ANC-1 are required for nuclear migration and anchorage, respectively. The inner nuclear membrane (INM) SUN protein UNC-84 is required for both nuclear migration and anchorage. The UNC-83 and ANC-1 KASH domains are thought to interact with the UNC-84 SUN domain in the perinuclear space. This allows force to be transferred from the cytoskeleton to the nucleoskeleton during nuclear positioning. KASH protein localization is dependent on SUN proteins, but SUN protein localization is not dependent on KASH proteins. Using a membrane-bound yeast two-hybrid system (Igor Stagljar, Dualsystems Biotech), we have shown that the KASH domain directly interacts with two separable domains of UNC-84, the SUN and linker domains. This interaction is required for nuclear migration and anchorage. Using a molecular genetics approach, we found that the UNC-84 SUN domain is required for KASH protein localization and function in vivo. We also demonstrated that point mutations in the KASH domain disrupt the function but not the localization of UNC-83. This suggests a stronger interaction, presumably to allow a large transfer of force during nuclear positioning, is required to move or anchor the nucleus than is required to simply localize the KASH proteins to the ONM (McGee, et al. Mol Biol Cell. 2006:1790-801). This KASH and SUN interaction appears to be conserved across eukaryotes. To identify residues in the linker domain that are required for interaction with the UNC-83 KASH domain, we are performing a mutational analysis of the UNC-84 linker domain in the membrane-bound two-hybrid system. We are also using this two-hybrid system in a screen to test whether there are unidentified binding partners with the SUN domain of UNC-84. In order to test the conservation of function between human and C. elegans KASH and SUN domains, we are testing whether the human domains in the C. elegans KASH and SUN proteins localize and are functional. We have found that a human KASH domain in UNC-83 is able to partially rescue UNC-83 function in migrating hyp-7 nuclei and localize to the nuclear envelope. We have also found that this hyp-7 rescue is dependent on the UNC-84 SUN domain. This suggests that the human KASH domain is able to interact with the UNC-84 SUN domain in vivo. Further experiments are testing the functional rescue of human SUN domains in UNC-84.

Marina Meyerzon et al. (2008) Development & Evolution Meeting "The Conventional Kinesin Light Chain, KLC-2, Functions in Nuclear Migration"

Daniel Starr, Marina Meyerzon

[Development & Evolution Meeting, 2008]

Nuclear migration and anchorage are essential for a number of cellular and developmental events, such as fertilization, cell migration, establishment of polarity, and cell division. Defects in nuclear migration events have been linked to the human diseases Emery-Dreifuss muscular dystrophy and lissencephaly. In wild type, embryos hyp7 precursor nuclei migrate contra laterally across the dorsal midline. In unc-83 and unc-84 mutants, nuclear migration fails and nuclei are positioned in the dorsal cord. In unc-83(e1408) null animals, we observe 11.8 plus or minus 0.1 nuclei in the dorsal cord. UNC-83 and UNC-84 form a bridge across the nuclear envelope, transferring force required for nuclear migration from the cytoskeleton to the nuclear lamina. The C-terminal KASH domain of UNC-83 interacts with the SUN domain of UNC-84 in the perinuclear space, while the N-terminal region of the protein extends into the cytosol (McGee et al. 2006). To elucidate the role of the cytosolic portion of UNC-83, I performed a yeast-two-hybrid screen. The screen identified 45 potential interacting partners, including KLC-2, which was pulled out twelve times. KLC-2 interacts with the kinesin heavy chain protein UNC-116 to form a homologue of the mammalian conventional kinesin (Sakamoto et al. 2005). The null allele klc-2(km28) results in L1 arrest. A hypomorphic allele (km11), which consists of a large deletion in the endogenous klc-2 locus and an insertion of a partial second copy of the gene, is viable but has 7.3 plus or minus 0.2 hyp7 nuclei in the dorsal cord. Furthermore, rare escaper (km28) animals that could be analyzed for hyp7 migration prior to arrest have 10.4 plus or minus 0.7 nuclei in the dorsal cord. Immunofluorescence with a rabbit polyclonal antibody to KLC-2, generously provided by Frank McNally (Yang et al. 2005), indicates that KLC-2 is expressed in wild type and the km11 mutant hyp7 cells in a pattern indicative of exclusion from the nucleus and at least some co-expression with UNC-83. Expression in km11 mutants appears fainter and more punctate compared to wild type. Amino acids 137-362 of UNC-83 were sufficient to interact with KLC-2 using a directed yeast-two-hybrid approach. These data suggest a model in which UNC-83 functions as a cargo specific adapter to recruit kinesin to the nuclear envelope, where it generates the major force required to move the nucleus. Future work to test this model includes characterization of the km11 allele as well as the construction of a KLC-2-KASH construct, which we predict will target to the nuclear membrane and rescue nuclear migration in unc-83 mutants.

Erin C. Tapley et al. (2007) International Worm Meeting "Characterizing the UNC-83/UNC-84 interaction in vitro.."

Erin C. Tapley, Daniel A. Starr

[International Worm Meeting, 2007]

Nuclear migration and positioning is important to a wide variety of eukaryotes. For example, mammalian skeletal muscles must specialize nuclei at the neuromuscular junction, and in the developing human brain nuclei must migrate toward the cortex. Using C. elegans, we have identified two proteins that function in nuclear migration: UNC-83 and UNC-84. UNC-84 is a member of a family of proteins that share a conserved C-terminal SUN domain and that localize to the inner nuclear membrane (INM). UNC-83 is a member of a family of proteins that share a conserved C-terminal KASH domain and that localize to the outer nuclear membrane (ONM). Using genetic methods, we have shown that both the SUN and KASH domains are essential for nuclear migration in vivo and that a stronger SUN-KASH interaction is required for nuclear migration than for nuclear localization. Membrane bound yeast two hybrid (MBYTH) assays show that the SUN domain of UNC-84 directly interacts with the KASH domain of UNC-83 in the perinuclear space and that two independent domains, SUN and linker, of UNC-84 interact with UNC-83s KASH domain (for further details see McGee et. al 2006). We propose that the SUN-KASH interaction creates a structural bridge across the nuclear envelope that allows the transfer of force from the nuclear lamina to the cytoskeleton during migration. To further characterize the KASH-SUN and KASH-SUN-linker interactions biochemically, we have expressed and purified several MBP-6His fusion proteins from E. coli. Previous transmembrane (TM) prediction programs suggest that UNC-84 has one TM domain located at residues 512-532, which is consistent with our model that the N-terminus of UNC-84 is nucleoplasmic and the C-terminus resides in the perinuclear space. The largest fusion protein, which contains H568-V1111 is insoluble. TopPred2, a TM prediction program, suggests that UNC-84 may have as many as five TM domains. Three of these TM domains reside within the previously described linker domain: 685-705, 719-739, and 763-783. Previous data from our MBYTH suggest that the region between 742-780 is important for membrane targeting. Smaller fusion proteins, D739-V1111 and R783-V1111, which lack either one or both of the last two predicted TMs have also been expressed and purified. Both R783-V1111 and the SUN domain fusion protein T931-V1111 are completely soluble. Soluble SUN-linker and SUN fusion proteins will be used to test the interaction with a custom synthesized UNC-83 KASH peptide in vitro. As the linker domain may be vital for force transfer, it is essential to narrow down its functional domain. To do this, we first need to clearly establish where UNC-84s TM domains are located. Protease protection assays using Promegas TNT &#174; T7 Quick for PCR , S35 methionine and canine microsomes are currently being performed.

Knight JK et al. (1996) West Coast Worm Meeting "GENETIC CONTROL OF GASTRULATION INITIATION: PRELIMINARY CHARACTERIZATION OF A MATERNAL-EFFECT MUTANT"

Wood WB, Knight JK

[West Coast Worm Meeting, 1996]

The genetic control of gastrulation is not well understood in C. elegans. We have isolated a temperature-sensitive mutation, ct226ts, which results in failure to initiate gastrulation. The mutation, tentatively defining a gene which we propose to call gad-1 (gastrulation defective), exhibits a strict maternal effect. At 16 degrees C, homozygous worms grow slightly slower than wild-type, are variably defective in egg-laying and gonad placement, and lay 24% dead eggs. At 25 degrees C, 100% of embryos laid by a homozygous hermaphrodite arrest with a clear gastrulation defect: the E blastomeres do not migrate into the embryo, and the embryo does not undergo gastrulation. Analysis of ct226 mutant embryos with the 4D microscope has shown that the 2E4 division is premature and in the a-p rather than the d-v direction. The 4E8 division is also early and in an aberrant plane, and all 8 E cells remain on the ventral surface of the embryo. Gastrulation initiation appears to be completely blocked; there is no delayed migration of the gut precursors, nor do the mesoderm or germ-line precursors migrate into the interior of the embryo. We have determined that the temperature sensitive period for ct226 extends from oogenesis to the 28- to 44-cell stage. This is consistent with the gene being required for the initiation of gastrulation. Despite lack of morphogenesis, terminally arrested defective embryos have differentiated (though misplaced) muscle and hypodermal cells, as assayed with monoclonal antibodies MHCA (5.6.1) and MH27, respectively. Gut granules are also present, although clearly mislocalized in a cluster at the very posterior of the embryo. We have mapped ct226 to the center of LG V, between unc-83 and dpy-11. This region is uncovered by the deficiency nDf18, but not by the deficiency sDf26. About 90% of embryos laid by ct226/nDf18 hermaphrodites at 16 degrees C are inviable, a substantially higher penetrance than for embryos from ct226 homozygotes, suggesting that ct226 is a hypomorphic allele. The Gad phenotypes of the inviable embryos from ct226/nDf18 and ct226/ct226 hermaphrodites are similar. All of the lethals in this immediate region, (let-439, let-473, and let-337) complement ct226. Thanks in part to deficiency endpoint mapping of nDf18 by C. Malone and A. McGee in the Han lab, we have narrowed the location of ct226 on the physical map to a small region between the left endpoint of nDf18 and dpy-11. We are currently injecting appropriate cosmids in an attempt to rescue ct226 homozygous mutant animals. It is noteworthy that zen-1 (Ferguson et. al WBG 13(5):66)and end-1(Zhu et. al 1995 Worm Mtg. abstract 33), the two non-maternal-effect gastrulation defective mutants isolated to date, have delayed E cell migration, but are able to complete gastrulation in most cases. In contrast, previously identified maternal-effect gastrulation defective mutants (1, reviewed in 2) arrest with mislocalized gut granules and only partial or no gastrulation. emb-16(g19ts) and ct226 display the most severe phenotype, with very early Ea and Ep divisions and no E cell migration. Interestingly, these two characteristics are also shared by embryos in which embryonic transcription has been blocked by ama-1 antisense RNA (Powell-Coffman, Knight and Wood WBG 14(1):70). Our immediate goal is to clone and molecularly characterize gad-1. We also intend to search for embryonically transcribed genes required for gastrulation in the hopes of understanding the functional relationship between maternally and embryonically expressed genes in the control of this process. 1. Denich, K.T.R., Schierenberg, E., Isnenghi, E., and Cassada, R. (1984). Roux's Arch. Dev. Biol. 193, 164-179. 2. Bucher, E.A. and Seydoux, G. (1994). Seminars Dev. Biol. 5, 121-130.

Knight JK et al. (1996) Worm Breeder's Gazette "Preliminary characterization of a maternal-effect mutant defective in gastrulation initiation."

Knight JK, Wood WB

[Worm Breeder's Gazette, 1996]

The genetic control of gastrulation is not well understood in C. elegans. We have isolated a temperature-sensitive mutation, ct226ts, which results in failure to initiate gastrulation. The mutation, tentatively defining a gene which we propose to call gad-1 (gastrulation defective), exhibits a strict maternal effect. At 16C, homozygous worms grow slightly slower than wild-type, are variably defective in egg-laying and gonad placement, and lay 24% dead eggs. At 25C, 100% of embryos laid by a homozygous hermaphrodite arrest with a clear gastrulation defect: the E blastomeres do not migrate into the embryo, and the embryo does not undergo gastrulation. Analysis of ct226 mutant embryos with the 4D microscope has shown that the 2E4 division is premature and in the a-p rather than the d-v direction. The 4E8 division is also early and in an aberrant plane, and all 8 E cells remain on the ventral surface of the embryo. Gastrulation initiation appears to be completely blocked; there is no delayed migration of the gut precursors, nor do the mesoderm or germ-line precursors migrate into the interior of the embryo. Despite lack of morphogenesis, terminally arrested defective embryos have differentiated (though misplaced) muscle and hypodermal cells, as assayed with monoclonal antibodies MHCA (5.6.1) and MH27, respectively. Gut granules are also present, although clearly mislocalized in a cluster at the very posterior of the embryo. We have mapped ct226 to the center of LG V, between unc-83 and dpy-11. This region is uncovered by the deficiency nDf18, but not by the deficiency sDf26. Embryos laid by ct226/nDf18 hermaphrodites at 16C are inviable about 90% of the time, a substantially higher penetrance than for embryos from ct226 homozygotes, suggesting that ct226 is a hypomorphic allele. The Gad phenotypes of the inviable embryos from ct226/nDf18 and ct226/ct226 hermaphrodites are similar. None of the lethals in this immediate region, (let-439, let-473, and let-337) fails to complement ct226. Thanks in part to deficiency endpoint mapping of nDf18 by C. Malone and A. McGee in the Han lab, we have narrowed the location of ct226 on the physical map to a small region between the left endpoint of nDf18 and dpy-11. We are currently injecting appropriate cosmids in an attempt to rescue ct226 homozygous mutant animals. Previously described mutations in at least seven other genes cause gastrulation defects; five of these show maternal effects (emb-5, emb-13, emb-16, emb-23, and emb-31) and two do not [zen-1 (Ferguson et. al WBG 13(5):66) and end-1(Zhu et. al 1995 Worm Mtg. abstract 33)]. It is noteworthy that the non-maternal-effect mutants isolated to date have delayed E cell migration, but are able to complete gastrulation in most cases, while all the maternal-effect mutants arrest with mislocalized gut granules and only partial or no gastrulation (1, reviewed in 2). emb-16(g19ts) and ct226 display the most severe phenotype, with very early Ea and Ep divisions and no E cell migration. Interestingly, these two characteristics are also shared by embryos in which embryonic transcription has been blocked by ama-1 antisense RNA (Powell-Coffman, Knight and Wood WBG 14(1):70). We intend to combine our characterization of ct226 with a search for embryonically transcribed genes required for gastrulation in the hopes of understanding the functional relationship between maternally and embryonically expressed genes in the control of gastrulation. 1. Denich, K.T.R., Schierenberg, E., Isnenghi, E., and Cassada, R. (1984). Rouxs Arch. Dev. Biol. 193, 164-179. 2. Bucher, E.A. and Seydoux, G. (1994). Seminars Dev. Biol. 5, 121-130.