Smejkal Gary B et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Pressure mediated scheme for isolating native proteins from Caenorhabditis elegans under mild nondenaturing conditions"

Anderson Jobriah, Smejkal Gary B, Morris Krystalynne, Thomas W Kelley, Kuo Winston P, Strader Thomas E

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

The tough exterior cuticle of Caenorhabditis elegans makes this nematode very resistant to lysis and impedes proteomic and glycoproteomic analyses. So resilient is this organism, that specimens sent into space were the only survivors of the disastrous crash of the space shuttle Columbia [1]. Proteomic analysis is particularly challenging when preservation of the native conformation and biological activity of proteins is required, thus prohibiting the use of denaturing chaotropes and detergents to enhance lysis. The dilemma is that in mild physiological buffers, nematodes can withstand hydrostatic pressure hydrostatic pressure up to 20,000 psi with a 2.3% survival rate [2]. While bead beating and sonication are commonly used to disrupt nematodes, heat generated by these methods causes denaturation of proteins, and subsequently, their loss through aggregation and precipitation. By comparison, pressure cycling technology (PCT) generates minimal heat by rapidly oscillating between high and atmospheric pressure. The ProteoSOLVE CE Native Kit was developed specifically to maximize protein yields from live nematodes under mild nondenaturing conditions. In the optimized protocol, live nematodes collected from culture are concentrated on a filter and the resulting paste is mixed with the CE SiC abrasives and transferred to a PULSE Tube. The amalgam is frozen solid by placing the tube on dry ice for ten minutes, then ground with the serrated Shredder ram insert until completely extruded through perforations of the lysis disk. The Native Reagent is added, followed by 40 pressure cycles at 35,000 psi. Protein yields were six times greater with the Shredder ram than with high pressure alone, and an order of magnitude higher when the Shredder ram was used in combination with freezing and abrasives. [1] Szewczyk, N.J. et al. (2005). Astrobiology, 5, 690-705. [2] Smejkal, G.B. et al. (2008). Plant and Animal Genome XVII Conference, San Diego, CA.

Hewitt, J.E. et al. (2017) International Worm Meeting "Functional assay identifies drugs that improve muscle strength in dystrophin-deficient Caenorhabditis elegans."

Hewitt, J.E., Pollard, A.K., Vanapalli, S.A., Szewczyk, N.J.

[International Worm Meeting, 2017]

The most common type of muscular dystrophy, Duchenne muscular dystrophy (DMD), affects 1 of every 3500 live male births. Currently, the disease is poorly understood and there is no cure-only treatments such as prednisone to control symptoms. Previous research with DMD has utilized a number of model organisms, including mice, dogs, and nematodes. Each organism has its challenges; for mice and dogs, challenges include limited screening ability and ethical issues. While C. elegans seems like an ideal platform for testing compounds and improving understanding of mechanisms relating to dystrophin, researchers have not been able to obtain a DMD-like phenotype in dystrophin mutants cultured in standard environments unless using a strain with a secondary mutation. This background mutation raises some questions about the similarity of the muscle degeneration mechanism to that of humans with DMD. Thus, there is a great need to establish an assay that can detect muscular degradation and measure the effects of drugs in dystrophin-deficient mutants. For this reason we have developed nematode strength as a phenotype of interest in strains with muscular mutations. For this study, we utilized two dystrophin deficient strains: dys-1(cx18), the most commonly utilized mutant in C. elegans DMD studies, and dys-1(eg33). Both strains are reported as having nonsense mutations, although the stability and interactions of the resulting proteins are unknown. Using our NemaFlex platform, we measured the strength of the nematodes on days 3, 5, and 7 of life. While the cx18 allele did not confer discernible differences from the wild-type control at any time point, the eg33 allele was associated with weakness on days 5 and 7 when post-developmental muscle degeneration becomes more apparent. We then checked if prednisone or melatonin had any effect on the strengths of the worms. As expected, treated wild-type animals and the cx18 animals were not discernible from their non-treated counterpart, whereas the eg33 mutants improved their strength significantly under all treatments. Furthermore, some treatments returned the strength of the eg33 mutants to wild-type levels. These results suggest that there is an interesting difference between the two mutants that requires further investigation. The ability to screen drugs for the effect on nematode strength offers a couple of promising outcomes. Using this assay, we may be able to discover new pharmacological compounds that improve strength, offering more options for treating DMD in humans. Moreover, combining our assay with analyses of cellular components in the nematode could offer additional insight on the genetic mechanisms behind muscular dystrophy.

Zhang L et al. (2010) Biology of the C. elegans Male, Madison, WI "Genetic Basis of Development and Mating Behavior Evolution in C. elegans"

Zhang L, Garcia L

[Biology of the C. elegans Male, Madison, WI, 2010]

A chemically defined axenic medium, C. elegans maintenance medium (CeMM), has been established for liquid culturing of C. elegans (Lu & Goetsch, 1993). Since organisms must coordinate behavioral and physiological response to changing environmental conditions, we are interested in how CeMM, as an environmental condition, affects development and mating behavior in C. elegans. Worms grown on CeMM plates take longer to develop and exhibit a prolonged reproductive period with a decreased brood size. For N2 animals, L1 to L4 larval development extends for seven days. Adult hermaphrodites would then lay eggs for approximately 10 days, which is similar to a previous report (Szewczyk et al., 2006). Compared with N2, CB4846, a Hawaiian strain, grows much slower and after 16 days, has fewer progeny (about 10 worms in the 1st generation). Interestingly, unlike N2, the 1st generation of CB4856 progeny developmentally arrests in L1, suggesting that the 1st generation of CB4856 could not utilize or synthesize some critical factors that N2 can. We have crossed N2 and CB4856, and obtained 200 recombinant inbred lines, which will be used to map the genes responsible for the viability difference between the two strains. We have also begun to study how CeMM affects reproductive behavior in the male. The male does not successfully copulate in CeMM. We found that a possible reason for reduced mating efficiency was that neuron-muscular circuits induced spicule protraction had a reduced responsive to acetycholine agonists such as arecoline. We are in the process of figuring out how genes control the development and mating behavior of N2 strain in CeMM, and what is the relationship between development and mating behavior in the experimental evolution history. To do this, we are screening for EMS generated N2 mutant hermaphrodites that grow faster and are more fertile in CeMM. We will serially select and then EMS-mutate hermaphrodites to gradually breed lines that become more adapted and fit for growth in CeMM, we will then ask if male mating, a behavior not directly selected for also adapts in the axenic medium. Lu N.C. & Goetsch K.M., 1993. Carbohydrate requirement of Caenorhabditis elegans and the final development of a chemically defined medium. Nematologica 39:303-311. Szewczyk N.J. et al., 2006. Delayed development and lifespan extension as features of metabolic lifestyle alteration in C. elegans under dietary restriction. J Exp Biol 209:4129-4139.

Zhang, Liusuo et al. (2011) International Worm Meeting "A recessive gene controls adaptation to a chemically defined medium in C. elegans."

Zhang, Liusuo, Garcia, L. Rene

[International Worm Meeting, 2011]

A chemically defined axenic medium, C. elegans maintenance medium (CeMM), has been established for culturing of C. elegans (Lu & Goetsch, 1993). Worms grown on CeMM plates take longer to develop and exhibit a prolonged reproductive period with a decreased brood size (Szewczyk et al., 2006). For N2 animals, about 10% of them develop into adults at day 8 after hatching, whereas the others develop into adults within the following 10 days. Compared to the lab-bred N2 strain, CB4856, a wild Hawaiian strain, takes greater than 16 days to reach adults. We are interested in determining genetic and molecular basis of developmental evolution that promotes animal cells to adapt to alternative nutritional environments. By screening known mutants which are possibly involved in feeding and growth, we observed that hermaphrodites contain mutations in fat-3, lev-11, ser-7, tph-1 and unc-73 grow much faster than N2 worms in CeMM (30-80% mutants reach adults between 5 and 7 days), while daf-2, daf-19, che-2, mdt-15, osm-9, unc-26, unc-32 and unc-42 mutants grow much slower or developmentally arrest in L1 (the majority could not develop into adults by two weeks). Through screening for EMS generated N2 mutant hermaphrodites that grow faster in CeMM, we identified the rg1402, rg1003 and rg804 alleles, which cause 90%, 90% and 75% of the worms to develop into adults on the synthetic medium by day 5, respectively. In addition, we have screened another 10 alleles which could develop to adults between day 5 and 6. Two alleles which cause the worms develop into adults at day 4 were identified through EMS mutagenesis of rg1003. We will serially select and then EMS-mutate hermaphrodites to gradually breed lines that become more adapted and fit for growth in CeMM. Outcrossing experiments demonstrated that enhanced adaptation to CeMM caused by rg1402, rg1003 and rg804 are inherited as a single-locus recessive trait. We are using classical mapping together with whole genome sequence of rg1003 to determine the molecular identity of the recessive allele. The characterization of the fast growth mutants in CeMM will greatly facilitate to systematically analyze the profound impact of nutrition on animal physiology, energy homeostasis and metabolism diseases. Lu N.C. & Goetsch K.M., 1993. Carbohydrate requirement of Caenorhabditis elegans and the final development of a chemically defined medium. Nematologica 39:303-311. Szewczyk N.J. et al., 2006. Delayed development and lifespan extension as features of metabolic lifestyle alteration in C. elegans under dietary restriction. J Exp Biol 209:4129-4139.

Pollard, A.K. et al. (2017) International Worm Meeting "Worms in space for outreach on Earth."

Gaffney, C.J., Szewczyk, N.J., Pollard, A.K., Phillips, B.E., Etheridge, T.

[International Worm Meeting, 2017]

Muscle atrophy is one of the main (mal)adaptations associated with prolonged spaceflight. We have previously established C. elegans as a model for studying spaceflight induced alterations in muscle with past experiments demonstrating reproducible expression changes in cytoskeletal and metabolic proteins and their encoding mRNAs. We are currently preparing a European Space Agency sponsored spaceflight experiment that aims to manipulate the stability of integrin based muscle attachment complexes and insulin-like signalling to determine if either or both of these predicted molecular mechanisms causally regulate the gene and protein expression changes observed in response to spaceflight. As spaceflight, particularly launches, captures the public's imagination, we have planned outreach activities to capitalize on this existing interest in spaceflight and to raise the profile of space-based life science research in the United Kingdom (UK). We have divided our outreach messages into three main themes. Firstly, we will describe the advantages of using C. elegans as a model to study biology and highlight some of the impacts on human health that have arisen from studies of the worm. Secondly, we will discuss the use of C. elegans as a model to study space physiology with a focus on how our past and current experiments were physically conducted. Thirdly, we will raise awareness of how the findings from our studies are being translated into new knowledge of the regulation of human muscle both on Earth and in space. We will present examples of how each message will be presented in a poster format for use at our outreach activities and provide electronic versions of these posters for other educators who may wish to use them. Three distinct dissemination routes will be used to maximise the impact of our outreach. Firstly, we will engage with the UK media to produce a video documentary of our experiment to provide the public with deeper understanding of how space experiments are actually conducted. Secondly, outreach days will be run at museums in each of the capital cities in the UK. Thirdly, we will run a number of hands-on workshops at schools in the middle and South West of England. For the outreach days we will employ the three posters described above and an extended version of our school workshop activities. The workshop activities will cover all three of our outreach messages, using wild-type worms and those with mutations relevant to our spaceflight studies (e.g., unc-112 and cha-1), and giving students the opportunity to handle spaceflight experimental hardware. We will discuss the details of our workshop activities for other educators who wish to run similar activities.

Karen Curto et al. (2007) International Worm Meeting "Regulation of protein degradation in muscle: CaMKII and calcineurin as potential regulators of MAP kinase signaling."

Karen Curto, Nate Szewczyk, Neil Umbreit, Lew Jacobson

[International Worm Meeting, 2007]

Multiple receptor signals modulate protein degradation in C. elegans muscle through multiple mechanisms. Activation of EGL-15 FGF receptor promotes activation of MPK-1 MAP kinase, which in turn triggers non-proteasome dependent protein degradation (Szewczyk & Jacobson, 2003). This is opposed by signal from the DAF-2 insulin/IGF receptor (Szewczyk & Jacobson, 2007). In a seemingly independent mechanism, influx of calcium upon activation of nicotinic acetylcholine receptor inhibits proteasome-mediated muscle protein degradation (Szewczyk et al., 2000). We have now found that UNC-43 calcium/calmodulin-dependent protein kinase II (CaMKII) also has a role in controlling muscle protein degradation. Gain-of-function mutation unc-43(n498) activates CaMKII (without increasing its phosphorylation) and promotes progressive protein degradation. The CaMKII-gf mutation causes an increase in phospho-MAPK, whereas transgene-activated MAPK does not increase phospho-CaMKII. A mutant (egl-19gf) with elevated intramuscular calcium also contains increased levels of both phospho-CaMKII and phospho-MAPK, suggesting that elevated calcium activates CaMKII, which in turn activates MAPK by an unknown mechanism. Conversely, we find that reduction-of-function mutant unc-43(e408) is resistant to protein degradation that occurs in wild-type after either hyperactivation of the FGFR pathway or inhibition of the IGFR pathway. The amount of total MPK-1 protein is increased in unc-43rf mutants, possibly implying that the worm attempts to compensate for low CaMKII signal by up-regulating MAPK. Our current hypothesis is that UNC-43 CaMKII acts as a calcium-responsive positive modulator (sensitivity control?) of the FGFR-Ras-Raf-MEK-MAPK cascade. Reduction-of-function mutations in either tax-6 or cnb-1, which encode the two subunits of the calcium/calmodulin-dependent protein phosphatase calcineurin, result in decreased protein levels in muscle, suggesting that calcineurin may act to oppose muscle protein degradation. Neither reduction- nor gain-of-function mutations in tax-6 or cnb-1 alter the levels of total or phospho-CaMKII; thus, calcineurin may act downstream of or in parallel to CaMKII to provide yet another mechanism by which calcium regulates muscle protein degradation. Supported by NSF grant MCB-0542355.

Hewitt, J.E. et al. (2019) International Worm Meeting "Robust phenotyping of a C. elegans model of muscular dystrophy reveals clinically relevant phenotypes and drug responses."

Vanapalli, S.A., Gaffney, C.J., Etheridge, T., Pollard, A.K., Lesanpezeshki, L., Hewitt, J.E., Deane, C.S., Szewczyk, N.J.

[International Worm Meeting, 2019]

The most common type of muscular dystrophy, Duchenne muscular dystrophy (DMD), affects 1 of every 3500 live male births. Currently, the disease is poorly understood and there is no cure-only treatments such as prednisone to control symptoms. The disease is caused by a mutation in the dystrophin gene; The C. elegans homologous gene, dys-1, gives the opportunity for testing compounds and improving understanding of mechanisms of DMD in worms. To facilitate this work we have been examining new phenotypes of dys-1 and their response to drug treatment. For this study, we utilized dys-1(eg33), which is reported as having a nonsense mutation. Using our NemaFlex platform we found dys-1 animals were significantly weaker than their wild-type counterparts and were also deficient in thrashing. We also find that dys-1 mutants display severe mitochondrial fragmentation, levamisole resistance, depolarization of the mitochondrial membrane, and an abnormally high basal oxygen consumption rate (OCR). We then tested the effects of prednisone and melatonin on dys-1 mutants and found an improvement in muscle strength, thrashing rate, and mitochondrial network integrity under drug treatments. Prednisone also returns basal OCR to wild-type-like levels, but it does not correct the depolarization of the mitochondrial membrane. We are currently phenotyping the impact of treatment with prednisone, melatonin, and serotonin by assaying in three unique mechanical environments: crawling (via NemaFlex), swimming (via C. elegans Swim Test Software - CeleST), and burrowing (via a novel, hydrogel burrowing assay). Using this platform, we have found that it is possible to uncover phenotypes that are not corrected by drug treatment, suggesting that our multi-environment phenotyping is of value both for drug discovery and mechanistic work with dys-1.

Chung, George et al. (2011) International Worm Meeting "A member of the DOG-1 family is essential for normal cell division."

Rose, Ann, Chung, George

[International Worm Meeting, 2011]

The Rose laboratory has been studying genes encoding members of a helicase subfamily that includes dog-1 (1,2 and see poster by Jones & Rose, this meeting) and rtel-1 (3,4). A third member of this family has orthology to the yeast gene, CHL1. In the absence of the gene function, worms grow to become sterile adults (Ste) with a movement defect (Unc). Using the D-neuron-specific GFP marker (unc-47::gfp, constructed by the Jorgensen lab), we visualized these neurons with fluorescence microscopy. Both newly hatched wild-type and mutant animals showed 6 DD neurons derived from embryogenesis. Additional 14 D neurons (13 VD and DVB) developed in the wild-type animals prior to adulthood. In mutants, however, we observed an average of only 12 D neurons rather than the expected 20. In order to determine if other cell types were affected in a similar manner, we used a seam cell marker (scm::gfp, constructed by the Rothman lab) to visualize the presence of seam cells during L4. The homozygous mutant worms only had an average of 10 seams cells per row, fewer than the 16 in wild-type worms. Furthermore, many mutant animals did not have mature oocytes. In animals that did have mature oocytes, DAPI staining revealed diakinetic chromosome spots in excess of the expected six. Taken together, this gene is requireed for cell division. (1)Youds, J.L., O'Neil, N.J. & Rose, A.M. Homologous recombination is required for genome stability in the absence of DOG-1 in Caenorhabditis elegans. Genetics 173, 697-708 (2006). (2)Youds, J.L. et al. DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions in interstrand cross-link repair. Mol. Cell. Biol 28, 1470-1479 (2008). (3)Barber, L.J. et al. RTEL1 maintains genomic stability by suppressing homologous recombination. Cell 135, 261-271 (2008). (4)Youds, J.L. et al. RTEL-1 enforces meiotic crossover interference and homeostasis. Science 327, 1254-1258 (2010).

Janelle J Bruinsma et al. (2005) International Worm Meeting "Mechanisms Regulating Zinc-Homeostasis in C. elegans"

Janelle J Bruinsma, Kerry Kornfeld

[International Worm Meeting, 2005]

Zinc is an essential trace element that organisms extract from nutrient sources. Since high or low concentrations of Zn2+ can be toxic, organisms strive to maintain optimal cellular Zn2+ levels regardless of dietary availability. The mechanisms controlling Zn2+-homeostasis are poorly characterized in any organism; however, studies in yeast and some other organisms have implicated ZIP and CDF family members in regulating zinc transport across biological membranes. We previously identified a mutation in the gene encoding C. elegans CDF-1 in a genetic screen for suppressors of constitutively active LET-60 Ras. CDF-1 is most similar to mammalian ZnT-1; ZnT-1 activity increases Zn2+ efflux in cultured cells. cdf-1(lf) mutants are hypersensitive to supplemental Zn2+ but not Cd2+, Co2+, or Cu2+, suggesting that CDF-1 is specifically involved in Zn2+ metabolism. In a similar genetic screen, Yoder et al. (2003) identified a mutation in the sur-7 gene that also encodes a CDF protein. sur-7(lf) mutants are also hypersensitive to supplemental Zn2+, indicating that this conserved family of transmembrane proteins regulates cellular Zn2+ levels. Other mutants that affect Zn2+-homeostasis in C. elegans have not been reported. To further investigate the mechanisms regulating Zn2+-metabolism, we conducted a genetic screen to isolate mutants that are resistant to toxicity caused by culturing worms in a high concentration of Zn2+. In appropriate media conditions, supplemental Zn2+ causes dose-dependent larval lethality that is 100% penetrant. Using these culture conditions, we screened for chemically induced mutants that display resistance to supplemental Zn2+. We screened about 280,000 haploid genomes and isolated 19 mutants. These mutations appear to represent at least four genes. These mutations may reduce the concentration of cytosolic Zn2+ and affect genes involved in cytosolic Zn2+ sensing or influx. To develop appropriate conditions for both Zn2+-restriction and -supplementation, we recently began culturing worms in liquid C. elegans Maintenance Medium (CeMM), a chemically defined medium (Szewczyk et al., 2003). CeMM normally contains Zn2+ at 150M, added in the form of ZnCl2. We found that wild-type worms cultured in CeMM lacking the ZnCl2 arrest as early larvae. Wild-type animals similarly arrest as L1s when cultured in CeMM with high concentrations of supplemental Zn2+. We will describe our progress in characterizing the response of wild-type, cdf-1(lf), and sur-7(lf) animals when cultured in CeMM containing a large range of Zn2+ concentrations.

Yang Zhao et al. (2005) International Worm Meeting "Worms in space"

Yang Zhao, Robert Johnsen, Ann Rose, David Baillie

[International Worm Meeting, 2005]

It is well known that radiation causes mutational damage, which can result in susceptibility to cancer, radiation-sickness, and death at high dosages. However, little is known about the biological effects of long-term exposure to radiation in space because of the technical difficulties involved in placing a multicellular model organism in a space environment for long time periods. One possibility for such study is use of the easy-maintainable, self-fertilizing hermaphroditic nematode, Caenorhabditis elegans. C. elegans has many characteristics that make it an excellent model system for use in space. Maintenance of C. elegans is relatively easy: it is small and can be fed/cultured in a chemically defined axenic media (CeMM)[1,2]. Worms will survive for several months in CeMM, and can be maintained indefinitely by passage to fresh media. Samples can be isolated in space for relatively large scale mutational analysis on Earth. The wide variety of research resources available for biological analysis in C. elegans provides a promising backdrop for the development of the system to study the effects of traveling and living in space. The ICE-First, 1st International Caenorhabditis elegans Experiment, was organised by Michel Viso (Centre National detudes Spatiales) in collaboration with investigators from Canada, France, Japan and the US, aimed to use C. elegans as a model system for biological studies in space. The launch occurred on April 19, 2004 and the landing on April 30, 2004. The goal of the Canada group project was to measure the mutational effects of the flight and stay on the Space Station. Two strains were examined: CC1 (widetype) obtained from C. Conley, NASA[2], and BC2200 (dpy-18; unc-46/eT1) from D. Baillie, SFU[3,4,5]. Both strains were grown in CeMM and several approaches were taken to measure potential mutational changes, including whole genome microarray analysis, deletion of poly-G stretches, occurrence of unc-22 mutations, lethal mutations, and alterations in telomere length. The worms from the flight are being analysed and compared to ground and laboratory controls for genetic alterations. No significant difference in the mutation rate was detected between the flight samples and the control. However, our studies demonstrate the advantage of the system in the potential to develop a biological integrating dosimeter for long term space travel. Funded by the Canadian Space Agency (CSA). References 1 Lu NC, Goetsch KM. Nematologica 1993;39: 303331 2 Szewczyk NJ et al. BMC Biotechnol 2003 3(1): 19 3 Rosenbluth RE et al. Genetics 1981 99(3-4): 415-28 4 Rosenbluth RE et al. Mutation Research 1983 110(1): 39-48 5 Johnsen and Baillie Genetics 1991 129(3):735-52