Black, Christopher et al. (2017) International Worm Meeting "Sex-specific kinetochore features in sperm meiosis."

Black, Christopher, Cota, Vanessa, Cintra, Thais, Wu, Jui-Ching, Chu, Diana

[International Worm Meeting, 2017]

Chromosome segregation in sperm has unique features that make it different from oocyte meiosis. Oocytes undergo two rounds of asymmetrical chromosome segregation without centrosomes, while sperm have symmetrical divisions and utilize centrosomes to organize microtubule attachment to chromosomes. This attachment is mediated by a protein complex called the kinetochore, but the role of the kinetochore during sperm meiosis remains poorly understood. We report here that sperm-specific phosphatases GSP-3/4 may regulate kinetochore-microtubule connection to organize sister chromatid orientation from segregating to the same pole in meiosis I to opposite poles in meiosis II. gsp-3/4 mutant sperm exhibit delayed resolution of a univalent lagging X chromosome and sisters fail to separate. Therefore, I hypothesize that sperm uniquely require kinetochore-microtubule attachment/detachment and GSP-3/4 regulate detachment. Interestingly, GSP-3/4 localize in a kinetochore-like pattern, which suggests these phosphatases may regulate kinetochore function. Sperm also have distinct outer kinetochore localization patterns (KNL-1, KNL-3, and NDC-80), compared to oocytes. For example, similar to oocyte meiosis, KNL-1 and KNL-3 localize in a cup shape on chromosomes, but NDC-80 surrounds chromosomes only in sperm. A bigger difference between oocyte and sperm meiosis is kinetochore presence at anaphase. In oocytes, immunofluorescence results depicted the kinetochore disassembles from chromosomes during anaphase I. However, the kinetochore is retained during anaphase in sperm suggesting kinetochore importance for chromosome segregation. The question still remains, do kinetochores mediate the connection between chromosomes and microtubules in sperm? Recent super-resolution images of immunostained sperm in anaphase I show there is a gap between chromosomes and microtubules where NDC-80 localizes, indicating the kinetochore functions in sperm meiosis to connect chromosomes to microtubules during anaphase separation. Further, live imaging of H2B labeled DNA and GFP labeled tubulin show that sperm meiosis exhibits pole-chromosome shortening (anaphase A) in anaphase I, which contrast to oocyte meiosis and mitosis, which rely on anaphase B mechanisms. This result taken together with the gsp-3/4 mutant phenotypes of delayed lagging X resolution and sister separation failure, support GSP-3/4 role in detaching microtubules from kinetochores. GSP-3/4-regulated detachment would allow X resolution and sisters to shift orientation towards opposite poles. Microtubule detachment from kinetochores may be unique in sperm to reconcile the combination of centrosome organized microtubules and chromosomes going through two rounds of segregation.

Jason R. Pfeiffer et al. (2007) International Worm Meeting "The Na+/H+ exchanger, nhx-2, influences lipid deposition and metabolic gene expression."

David Johnson, Keith Nehrke, Jason R. Pfeiffer

[International Worm Meeting, 2007]

RNAi-mediated knockdown of the intestinal Na&#43/H&#43 exchanger, nhx-2, leads to a complex phenotype, resulting in worms with an acidified intestinal intracellular pH (pHi) that develop slowly into small and clear adults. Our initial observations suggested that normal fat metabolism may require NHX-2 and we hypothesized that nhx-2(RNAi) acts as a caloric restriction mimetic. To further test this hypothesis, we stained lipid directly with Sudan Black in fixed worms and also with Nile Red by feeding. Knockdown of nhx-2 caused a loss of intestinal staining in both the Sudan Black stained and the Nile Red fed worms. The Sudan Black staining also revealed a marked expansion of hypodermal lipid droplets in nhx-2(RNAi) animals. The droplets appeared early during larval development and apparently occurred only at the anterior and posterior aspects of hyp7. Similar hypodermal lipid droplets have been described previously in insulin signaling mutants. To determine if this lipid droplet phenotype is also characteristic of dietary restriction (DR) we stained a common DR model, the eat-2(ad1113) mutant, with Sudan Black. The pattern of Sudan Black staining was essentially the same as that for N2 worms, suggesting that the nhx-2(RNAi) phenotype differs at least in this aspect from a well-established model of DR. In an effort to better understand the molecular aspects of the complex metabolic phenotype caused by nhx-2(RNAi), we performed semi-quantitative RT-PCR using a metabolic primer set directed at one-hundred and fifty target genes (courtesy of Dr. Mark van Gilst). The most profound and significant changes occurred in several transcripts coding for components of the fatty acid beta-oxidation pathways in both mitochondria and peroxisomes. We are currently generating reagents to examine cell-specific changes in metabolic gene expression in hyp7 and in the intestine, where the effects of nhx-2(RNAi) are apparently functionally dichotomous.

Christopher J Cronin et al. (2005) International Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

William R Schafer, Zhaoyang Feng, Kuang-man Huang, Paul W Sternberg, Christopher J Cronin

[International Worm Meeting, 2005]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see An imaging system for standardized quantitative analysis of C. elegans behavior, Zhaoyang Feng, et al, BMC Bioinformatics 2004, 5:115, An automated system for measuring parameters of nematode sinusoidal movement, Christopher J Cronin, et al, BMC Genetics 2005, 6:5, and previous meeting abstracts). We have consolidated our development efforts and merged our independent system designs to create a single, unified behavior analysis system. The combined system provides researchers with all of the quantification tools and database functionality of the individual UCSD and Caltech systems, but also establishes a common, open software and hardware foundation for continued development by Caltech, UCSD and the community. Current efforts include expanding system capabilities for the automated analysis of mating behavior with the challenge of tracking multiple moving individuals, both in contact with and separated from each other.

Yoshiko Iizumi et al. (2010) East Asia Worm Meeting "High-molecular weight polyphenols from black tea increase lifespan in Caenorhabditis elegans via the forkhead transcription factor DAF-16"

Tetsuo Ozawa, Yoshiko Iizumi, Osamu Numata, Ryusuke Niwa

[East Asia Worm Meeting, 2010]

Tea is the most popular beverage all over the world. It has been reported that teas have beneficial physiological effects in several aspects, including antioxidation, antiobesity, acceleration of lipid metabolisms, anti-carcinoma activity and antimutagencity. However, it is still unclear which chemical components in tea are critical for these beneficial effects. Our group has recently reported that high-molecular weight polyphenols from oolong tea and black tea, designated MAF (mitochondrial activation factor) increases mitochondrial membrane potential and cellular ATP level. To investigate the physiological effect of the polyphenols MAF application to organisms in vivo, we chose the nematode Caenorhabditis elegans as a model organism. C. elegans is a well-established model for aging studies. Several studies provide evidence that C. elegans responds upon exposure to various natural compounds, including low-molecular weight polyphenols such as resveratrol, with increased stress resistance and/or extended lifespan. Here we demonstrates that MAF exposure leaded to increase mean lifespan of wild type N2 worms up to 20%. The MAF-mediated longevity was much effected as compared to the increased lifespan induced by resveratrol. Furthermore, this longevity required the forkhead transcription factor DAF-16, which is the key regulator for the insulin-dependent longevity in C. elegans. In conclusion, MAF from black tea has robust and reproducible benefits during aging via a part of the conventional longevity pathway.

Ademola A. Adenle et al. (2007) International Worm Meeting "The function of latrophilin in mediating the toxicity of black widow spider venom in C. elegans."

David De Pomerai, Ademola A. Adenle, David R. Bell

[International Worm Meeting, 2007]

Black widow spider venom (BWSV) is known to cause neurotransmitter release from nerve terminals due to its content of high molecular weight proteins, the latrotoxins (LTX), that are known to bind with high affinity to three neural proteins in mammals. We have established C.elegans as a model organism to study the function of the binding protein, latrophilin (a member of the class B family of G-protein coupled receptors), and its role in regulating neurotransmitter release by latrotoxins, by showing that latrophilin is required for lethality of BWSV by RNAi experiments. However, a latrophilin-knockout worm is required for determining the function of the latrophilin gene. The lat-1(ok1465) allele has a deletion of the lat-1 gene, and we know that ~97% of lat-1(ok1465) homozygous worms arrest or die before adulthood, with only 3 adult offspring per animal; moreover, there is strong interaction between the lat-1 allele and various G-protein mutants. However, it is unclear if this lethality is due to the deletion in the lat-1 gene, or to adventitious mutations introduced during the process of mutagenesis to create this allele. When transgenic worms were created with the B0457 cosmid (containing the full sequence of the lat-1 gene), they rescued the lethality of lat-1(ok1465) worms, whilst concurrent injections with the unc-54::C marker plasmid yielded six surviving adults from greater than 80 transgenic embryos, showing that the rescue was not due to the unc-54::C. Our results show that the lat-1(ok1465) allele causes developmental lethality and also an increase in defecation cycle timing, and that the B0457 cosmid can rescue both these defects. This is strong evidence that the lat-1 gene is responsible for these defects. Current work aims to rescue the lat-1(ok1465) defects with a lat-1 cDNA construct, and to determine the role of this gene in mediating the toxicity of Black Widow Spider venom.

Almotayri, A. et al. (2019) International Worm Meeting "Culinary spices reduce the body fat content of Caenorhabditis elegans."

Thomas, J., Almotayri, A., Jois, M., Munasinghe, M.

[International Worm Meeting, 2019]

Oxidative stress and inflammation are critical components in the development of obesity. Culinary herbs and spices, abundant in anti-oxidant and anti-inflammatory phytochemicals, may be useful in the prevention of obesity. In the present study, we report strong anti-obesity properties of culinary spices. Wild-type Bristol N2 strain (Caenorhabditis Genetics Centre) were grown on NGM plates seeded with E. coli (OP50). Worms were exposed to extract (1mg of dry powder per 1ml of NGM) of red chilli, black pepper, ginger, and turmeric. Fat content was determined on day 5 (adult stage) by staining with Oil-Red O. Compared to control group, chilli, ginger and pepper reduced the fat content of the worms by 92, 89 and 57% respectively. Turmeric had no effect on the fat content of C elegans. In conclusion, culinary herbs and spices may be useful in the prevention of obesity and C. elegans is a useful model for screening the anti-obesity properties of culinary spices.

Pena-Gonzalez, I. et al. (2013) International Worm Meeting "Suppressors of TDP-1 toxicity in Caenorhadbitis elegans."

Link, CD., Pena-Gonzalez, I.

[International Worm Meeting, 2013]

Suppressors of TDP-1 toxicity in Caenorhadbitis elegans Ilana Pena-Gonzalez1 and Christopher D Link1, 2 1 Integrative Physiology, University of Colorado, Boulder, CO 80309, USA 2 Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309, USA RNA binding protein TDP-43 forms damaging aggregates in multiple neurodegenerative diseases. We have found that the deletion of tdp-1, the C. elegans ortholog of TDP-43, leads to increased accumulation of double stranded RNA. TDP-1 is not believed to bind to dsRNA itself; however the marked increase in dsRNA accumulation in worms deleted for tdp-1 implies TDP-1 normally participates in limiting the stability and structure of dsRNA. Although worms deleted for TDP-1 do not have a severe phenotype, overexpressed nuclear TDP-1 is toxic in worms. One possible explanation for this is that too much TDP-1 leads to excessive disruption of structured RNAs needed for normal RNA metabolism. Making use of this overexpression model we have established a heat shock induction system to help identify components associated with TDP-1's prevention of dsRNA accumulation. Identifying mutations that suppress the neurotoxic effects of overexpressed TDP-1 could help identify others factors contributing to the complex mechanism of disassembling dsRNA. A mutagenesis screen has identified four mutant strains that partially suppress the toxicity of overexpressed TDP-1. We have also tested suppressors of other toxic proteins. Further identifying the pieces of the mechanism regulating dsRNA breakdown could help better explain the pathogenic pathway of TDP-1 and consequently TDP-43 in disease.

David R. Bell et al. (2006) European Worm Meeting "The Function of Latrophilin in C. elegans"

S. van der Poel, P.N.R. Usherwood, D. Kendall, J. Davy, A. Ademola, I.R. Duce, D. de Pomerai, David R. Bell, I. R. Mellor

[European Worm Meeting, 2006]

David R. Bell, A. Ademola, J. Davy, S. van der Poel, D. Kendall, P.N.R. Usherwood, I.R. Duce, I. R. Mellor and D. de Pomerai. Black Widow spider venom (BWSV) contains latrotoxins that induce catastrophic neurotransmitter release in mammals, and is known to bind with high affinity to three neural proteins in mammals, including latrophilin. We have investigated C. elegans as a model system for studying the function of these proteins, and their role in regulating neurotransmitter release by latrotoxins.. We have shown that latrophilin is required for the lethality of BWSV in C. elegans by RNAi experiments. We now present data on the characterisation of null mutants of the C. elegans latrophilin, lat-1, illustrating the function of this gene in C. elegans, and show that lat-1 nulls are resistant to emodepside. The expression of latrophilin-GFP constructs is examined, and compared with the phenotypes seen in null mutants. Genetic interactions between endogenous signaling pathways and the lat-1 nulls have been examined by the construction of double mutant worms, revealing novel facets of lat-1 function.

Steven Augustine et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "See spot run: a tracking system with a resolution of microns and milliseconds"

Steven Augustine, Shawn Lockery

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Quantitative, automated analyses of movement are essential to investigations of genetic and neuronal mechanisms of behavior in C. elegans. Current tracking systems are of two main types: those that record the worm's centroid and those that record the worm's centerline. Centroid-based systems require minimal image processing but do not preserve the worm's shape, making it difficult to detect subtle changes in waveform. Centerline-based systems can detect subtle changes in waveform, but they require extensive image processing and can be confused by closed postures such as omega turns and coils. Here we report a new tracking method that reports subtle waveform effects with minimal image processing. The new method adopts the simple expedient of tracking a tiny black spot painted on the worm. The spot is a mixture of black artists' pigment and diluted Vaseline. We refer to this as the spotted worm tracking system. The system utilizes two computers. One computer commands the stage to keep the spot near the center of the optical field and records a movie of the worm. The other computer reads a pair of displacement encoders attached to the microscope stage. A virtual clap-board is used to synchronize the movie with stage-displacement data for off-line processing. The position and velocity of the spot are reconstructed from the location of the spot in successive movie frames and the position of the stage at the time of frame acquisition. Operating at 30 frames/sec, the system has a maximum spatial precision (in um) of 3.5 and 1.9 in X and Y directions, respectively. Custom software for off-line processing can distinguish between forward and reverse locomotion without user input, thereby facilitating kinetic analyses of locomotion in wild-type and mutant worms. Utilizing the system's increased spatial and temporal resolution, we have observed "micropauses," a frequent yet previously undetected behavioral state of near-zero velocity with a mean dwell time of 0.15 sec. Micropauses are observed during transitions between forward and reverse locomotion, but also during apparently continuous bouts of forward or reverse locomotion. Thus the new system may contribute to revised definitions of fundamental locomotory states in C. elegans.

Lustigman S et al. (1998) East Coast Worm Meeting "C. elegans as a model for studying the role of proteins in the molting and development of human parasitic nematode, Onchocerca volvulus and development"

Hashmi S, Lustigman S

[East Coast Worm Meeting, 1998]

The nematode, Onchocerca volvulus causes Onchocerciasis or river blindness in human. The parasite is transmitted in human by bites of Simulium black flies. For the past few years, Lustigman's group initiated a search for the enzymes involved in the molting process of this nematode. Our emphasis is on the transition from L3 to L4, which can be obtain in vitro as well. The molting involves separation of the cuticle from the underlying epidermis. The formation of a new cuticle arises from the outermost surface of the epidermis, and the shedding of the old cuticle. Previously Lustigman et al shown the involvement of cysteine protease and their endogenous inhibitors as well as a lectin binding protein and other proteins with unknown function in the molting process of infective larvae L3 and successful development of O. volvulus L4. Many of these proteins relevant to the molting and development of O. volvulus have been cloned and characterized. We are using C. elegans as a model to study the function of these proteins. Such studies could provide means for preventing infection and diseased associated with O. volvulus infection.