Yasumi Ohshima et al. (2006) East Asia C. elegans Meeting "Food, feeding, digestion and defecation of the worm"

Yasumi Ohshima, Yusuke Kaku, Yuta Yoshidome, Yuji Osako, Katsuhiro Kunigo, Akiko Nakasone, Kohji Miyahara, Tomonori Nakamura

[East Asia C. elegans Meeting, 2006]

Food or nutrition is vital for the life of C. elegans as for other animals, and should affect growth, body size and activity of the worm. In the laboratory, worms usually feed on OP50, a uracil-requiring strain of E. coli, which is a good food for worms in the sense that it supports rapid growth and production of many progeny. However, E. coli is an enteric bacterium that may not be abundant in soil which is said to be the natural habitat of the worm. Avery & Shtonda (2003) identified from soil samples more than 10 bacterial species that supported growth of the worm. Yet, the entire image of the natural food of the worm is not clear. Although the mechanisms of feeding and defecation of E. coli have been extensively studied, much is still to be solved, for example, the mechanisms of food recognition, rate and target of defecation. Furthermore, virtually nothing is known on the mechanisms of digestion of food and absorption of nutrients in the gut of the worm. In mammals, many factors which seem to be involved in digestion or absorption are known, but there is little genetics in any animal. Thus, we have begun studies on some of these, and plan to ask further, as follows. 1) Heat-killed E. coli is not likely to support growth of C. elegans. 2) We are studying whether selected microorganisms are good food or not. For example, yeast S. cerevisiae probably does not support growth to adults. 3) We are trying to identify live or dead microorganisms in the gut of marked worms that were put into a soil sample, by DNA sequencing. 4) Several artificial microspheres of various size or chemical nature are used to get clues to food recognition, feeding and defecation. 5) We are trying to analyze the time course of feeding, digestion and defecation of GFP-marked E. coli. 6) We have found many C. elegans genes possibly involved in digestion or absorption based on amino acid sequence homology with those in mammals. We have obtained KO mutants for some of them from Dr. S. Mitani for reverse genetics. 7) We have begun isolation of mutants on digestion or absorption. The screening at present uses GFP-marked E. coli as food. Two candidate mutants were obtained that retain more GFP fluorescence in the gut without little change in the pumping cycle.

Calero M et al. (2001) J Biol Chem "Yop1p, the yeast homolog of the polyposis locus protein 1, interacts with Yip1p and negatively regulates cell growth."

Calero M, Collins RN, Whittaker GR

[J Biol Chem, 2001]

Rab proteins are small GTPases that are essential elements of the protein transport machinery of eukaryotic cells. Each round of membrane transport requires a cycle of Rab protein nucleotide binding and hydrolysis. We have recently characterized a protein, Yip1p, which appears to play a role in Rab-mediated membrane transport in Saccharomyces cerevisiae. In this study, we report the identification of a Yip1p-associated protein, Yop1p. Yop1p is a membrane protein with a hydrophilic region at its N terminus through which it interacts specifically with the cytosolic domain of Yip1p. Yop1p could also be coprecipitated with Rab proteins from total cellular lysates. The TB2 gene is the human homolog of Yop1p (Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levey, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H. M., Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) Science 253, 661-665). Our data demonstrate that Yop1p negatively regulates cell growth. Disruption of YOP1 has no apparent effect on cell viability, while overexpression results in cell death, accumulation of internal cell membranes, and a block in membrane traffic. These results suggest that Yop1p acts in conjunction with Yip1p to mediate a common step in membrane traffic.

Ashley, Nicholas et al. (2019) MicroPubl Biol

Ashley, Nicholas, Holgado, Andrea M.

[MicroPubl Biol, 2019]

Macroautophagy (hereon referred as Autophagy) is a cellular housekeeping mechanism that uses a double membrane to target and engulf cell products for the formation of autophagosomes. These double membrane organelles then fuse to lysosomes where cell products are degraded and recycled (Nakamura and Yoshimori, 2018). Reports show that autophagy plays an important role in pathogen defense, development, starvation adaptations, and aging (Mizushima et al., 2008). Identifying molecular mechanisms responsible for autophagy in mammalian cells has been possible as a result of studying model systems, such as Saccharomyces cerevisiae. Autophagy related genes (Atg) are evolutionarily conserved; therefore, research of autophagy in simpler organisms have informed the roles of Atg in mammalian cells (Ruck et al., 2011; Mercer et al., 2018; Tyler and Johnson, 2018). Analysis of autophagy mutants in C. elegans revealed that bec-1/Atg6/ Beclin 1 is essential for dauer development, a quiescent state that survives harsh conditions such as lack of nutrients, high nematode density, and high temperatures by inducing autophagy (Melndez et al., 2003; Melndez and Levine 2009). To further investigate the phenotypes associated with the bec-1 (ok691) mutation, we studied nematodes possessing a 3000 base pair deletion mutation (allele ok691) in the bec-1 locus (Figure 1A). Previous reports show that the bec-1 (ok691) mutation is lethal, however a small proportion of homozygous bec-1 (ok691) animals reach adulthood due to maternal effect, but do not reproduce due to sterility (Figure 1B, Melendez and Levine 2009). Our analysis of survival throughout adulthood shows that lifespan is significantly reduced in homozygous bec-1 (ok691) mutants (Figure 1C). These nematodes do not live longer than 8 days of adulthood and 50% of animals died at day 5 of adulthood. In contrast, heterozygous bec-1 (ok691) mutant lifespan is not significantly different from control animals, ruling out previously discovered haploid insufficiency effects of Beclin 1 in the lifespan of C. elegans (Sinha and Levine, 2008).

Jennifer L Watts et al. (2003) International Worm Meeting "Inhibition of germline proliferation by omega-6 fatty acids"

John Browse, Jennifer L Watts

[International Worm Meeting, 2003]

Alteration in the dietary content of fatty acids can lead to modulation of the structure/function of membrane-bound receptors, ion channels, enzyme activities, and cellular signaling [1]. We report that germline proliferation is acutely inhibited when C. elegans are propagated on plates containing 18- and 20-carbon omega-6 polyunsaturated fatty acids. These affects are specific and are not observed during propagation on monounsaturated or omega-3 polyunsaturated fatty acids. The omega-6 fatty acid-supplemented worms exhibit a range of Glp phenotypes that correlate with the actual amounts of fatty acid incorporated into bacterial and nematode membranes. Germline migration phenotypes are also common; supplemented worms often exhibit either stunted germlines with no turn or aberrant turns, and the anterior gonad is frequently more severely affected than the posterior gonad. The fatty acid-induced Glp phenotype is epistatic to the tumorous germline phenotype of gld-1. C. elegans strains with altered membrane polyunsaturated fatty acid composition due to mutations in genes encoding fatty acid desaturase and elongase activities [2] show increased or decreased sensitivity to dietary omega-6 fatty acids, depending on their ability to synthesize and metabolize these fatty acids. In addition, the pale egg (Peg) class of nrf mutants, which are resistant to fluoxetine (Prozac)-induced nose contraction [3], are resistant to the fatty acid-induced mitotic inhibition. These results imply that dietary fatty acids are incorporated and modified in the ER of intestinal cells and are subsequently transported by a specific pathway to the developing germline. We suggest that excess omega-6 fatty acids that reach the germline interfere with signaling events that direct mitosis and gonad migration. 1.Nakamura, M.T., et al., Lipids, 2001. 36(9): p. 961-4; 2.Watts, J.L. and J. Browse, Proc Natl Acad Sci U S A, 2002. 99(9): p. 5854-9; 3.Choy, R.K. and J.H. Thomas, Mol Cell, 1999. 4(2): p. 143-52.

Ashley, Nicholas et al. (2019) MicroPubl Biol

Holgado, Andrea M., Ashley, Nicholas

[MicroPubl Biol, 2019]

Macroautophagy (hereon referred as Autophagy) is a cellular housekeeping mechanism that uses a double membrane to target and engulf cell products forthe formation of autophagosomes. These double membrane organelles then fuse to lysosomes where cell products aredegraded and recycled (Nakamura and Yoshimori, 2018). The loss of autophagy function in the motor cortex has been associated with progression of neurodegenerative symptoms in Parkinsons disease (Kaila and Lang 2015; Fahn et al. 2004). To better understand the role of Beclin 1 and autophagy in neurons, we studied locomotion in C. elegansmutants for bec-1. Locomotion in both homozygous (bec-1 -/-) and heterozygous bec-1(ok691) (bec-1 +/-) mutants showed significant defects (Figure 1). Homozygous bec-1(ok691)mutants backwards locomotion rates ranged between 3-8 body bends/minute at ages 1-7 days of adulthood and heterozygous bec-1(ok691)mutants were 6-9 body bends/minute at comparable ages. These defects in locomotion suggest a role of autophagy controlling behaviors that are neuronal dependent.Previous research has shown that loss of motor function could be a result of neurodegeneration (Fahn et al., 2004; Kalia and Lang, 2015). Additionally, C. elegans studies demonstrated that mutations in bec-1associate with degeneration of muscle cells and aging (Herndon et al., 2002). Together, our findings and previous reportsindicate that autophagy gene product, BEC-1, may play an important role in motor neuron physiology, survival, or both. Jia and colleagues have observed collections of visible protein aggregates in nematodes that elicit locomotion defects (Jia et al., 2007). This conclusion should be considered as preliminary as we have not verified by an alternative line of investigation (e.g., a second allele or transgene rescue) that the observed phenotypes are specific to bec-1(ok691).However, our study is also consistent with previous findings in Parkinsons disease patients where accumulation of protein aggregates was associated with loss of autophagy and accelerated onsets symptoms of decreased motor function (Ermine et al., 2018; Fahn et al., 2004).

Blackwell, Alexander et al. (2021) International Worm Meeting "Studying chromatin regulation at single cell resolution during C. elegans postembryonic development"

Gaarenstroom, Tessa, Van den Heuvel, Sander, Blackwell, Alexander

[International Worm Meeting, 2021]

Despite containing identical genomes, developing cells differentiate into a plethora of diverse cell types. Distinct patterns of gene expression now form the basis for classifying different cell fates. How the combinatorial activity of transcription factors, chromatin regulators and histone modifications achieve the proper spatiotemporal patterns of gene expression is a major question in developmental biology. Biologists increasingly appreciate the need to investigate gene expression regulation at the single-cell level because much heterogeneity and complexity is lost when averaging across populations of cells. However, profiling chromatin at the single cell level is challenging due to limited input material. Chromatin immunocleavage with sequencing (ChIC-seq) is an efficient method to study chromatin modifications from low input samples. ChIC-seq utilises antibody targeted micrococcal nucleases, leading to controlled, binding-dependent enzymatic digestion of DNA. This releases short fragments which become preferentially incorporated during library preparation and enables high resolution mapping of genomic positions. Crucially, the absence of crosslinking and immunoprecipitation steps, required in less sensitive techniques such as ChIP-seq, leads to minimal material loss. Recently, ChIC-seq was used to profile histone modifications in single human cells [1]. Adapting ChIC-seq to profile histone modifications in C. elegans will provide a powerful tool for studying the epigenetic regulation of development. Here, we present progress in optimising ChIC-seq for profiling chromatin modifications at single-cell level across a developmental time-course in C. elegans. Specifically, we combine Cre/Lox lineage tracing with cell isolation and FACS procedures in order to isolate postembryonic mesoderm cells. Following prolonged quiescence, the mesoblast precursor resumes proliferation and produces fourteen muscle cells, two scavenger cells, and two migratory bipotent myoblasts over 24-hours. By profiling chromatin modifications at high temporal resolution, we aim to reveal regulatory processes controlling cellular proliferation and differentiation. This work will shed light on how epigenetic modifications contribute to cellular decision making in a living animal. [1] Ku WL, Nakamura K, Gao W, Cui K, Hu G, Tang Q, Ni B, Zhao K. (2019) Single-cell chromatin immunocleavage sequencing (scChIC-seq) to profile histone modification. Nature Methods, vol. 16, pages 323-325.

Kenji Sugioka et al. (2008) Development & Evolution Meeting "Wnts Regulate Spindle Asymmetry to Produce Asymmetry of Cell Fates"

Kenji Sugioka, Hitoshi Sawa

[Development & Evolution Meeting, 2008]

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, the T and EMS cells divide asymmetrically in a Wnt-dependent manner. After the cells are polarized by Wnt proteins secreted from the posteriorly located cells, WRM-1/ beta-catenin localizes asymmetrically to the posterior nucleus at telophase to regulate asymmetric fates of the daughter cells (Takeshita and Sawa 2005, Nakamura et. al. 2005). But the mechanism for asymmetric WRM-1 nuclear localization is largely unknown. To gain insights into this mechanism, we carefully analyzed behaviour of spindle by observing GFP::beta-tubulin signal during the division of the EMS cells. We found that the numbers of astral microtubules were asymmetric during telophase; higher at the anterior spindle pole than the posterior one. This spindle asymmetry was regulated by MOM-2/Wnt, MOM-5/Frizzled and GSK-3/Gsk3beta. To know the significance of this phenomenon in asymmetric division, we tracked the cell fates of EMS daughters after observing spindle in mom-2 RNAi embryos. As reported previously, mom-2 RNAi embryos showed 29% gutless phenotype due to the fate transformation of posterior daughter E to the anterior daughter MS. We found strong correlation between symmetric spindle in EMS and the gutless phenotype in mom-2 embryos, suggesting that the asymmetry in spindle is required for the asymmetric cell fate decision. In addition, we found that zen-4/kinesin mutants showed symmetric nuclear localization of WRM-1::GFP at telophase during the postembryonic T cell division. Although ZEN-4 and CYK-4 form a complex to regulate cytokinesis, WRM-1 localization was symmetric only in zen-4 but not in cyk-4 mutants, suggesting that ZEN-4 regulates WRM-1 localization in a CYK-4 independent manner. These results raise a model that ZEN-4/kinesin transports WRM-1 away from the nuclei along spindle to accelerate WRM-1 exports from the nuclei. Asymmetry in spindle make this process to occur preferentially in the anterior nucleus to generate WRM-1 asymmetry. Our results suggest that Wnts control beta-catenin nuclear localization through regulating spindle.

S Ahmed et al. (1999) International C. elegans Meeting "Two C. elegans mutants with defects in telomere replication"

S Ahmed, J Hodgkin

[International C. elegans Meeting, 1999]

The germline is an immortal cell lineage which is passed from one generation to the next indefinitely. To identify genes which are required for germline immortality, we looked for C. elegans mutants with mortal germlines - worms which could reproduce for several healthy generations but eventually become sterile. A number of mortal germline (mrt) mutants were identified which become effectively sterile between generations F4 and F16. Two have been studied in detail: mrt-1 and mrt-2 accumulate end-to-end chromosome fusions in later generations and exhibit telomere shortening, indicating that both mutants become sterile as a result of defects in telomere replication. Studies in yeast and mouse suggest that the late-onset chromosome fusion phenotype observed in mrt-1 and mrt-2 probably results from a loss of in vivo activity of the telomere replication enzyme telomerase (1, 2). mrt-1 , mrt-2 and the mrt-1,mrt-2 double mutant all have the same telomere shortening rates, indicating that MRT-1 and MRT-2 act in the same telomere maintenance pathway. However, mrt-2 has additional phenotypes - it is hypersensitive to X-rays and has mutator activity. mrt-2 mapped near a candidate gene Y41C4A.o that encodes a homolog of the S. pombe RAD1 checkpoint protein which when mutated results in X-ray hypersensitivity (3). The mrt-2 allele has a splice site mutation in Y41C4A.o which eliminates most of this highly conserved gene product. In addition, high copy number arrays of Y41C4A.o produce a phenocopy of mrt-2 X-ray hypersensitivity - possibly via cosuppression silencing of this gene in the germline. Therefore the MRT-2 checkpoint protein may be required for proper processing of both abnormal double-strand breaks caused by X-rays and normal double-strand breaks present at telomeres. These results are consistent with the notion that telomeres are replicated as a special kind of DNA damage. 1. Nakamura TM, et al. (1998), Science 282: 493-496. 2. Blasco MA, et al. (1997), Cell 91: 25-34. 3. Sunnerhagen P, et al. (1990), Mol Cell Biol. 10: 3750-3760.

Rodriguez, Carlos et al. (2021) International Worm Meeting "Knockdown of Bora homolog spat-1 results in crossover and synapsis defects in C. elegans meiotic prophase"

Carlton, Peter, Sato-Carlton, Aya, Rodriguez, Carlos

[International Worm Meeting, 2021]

Chromosomes that have undergone crossing over in meiotic prophase must maintain sister chromatid cohesion somewhere along their length between the first and second meiotic divisions. To accomplish this, the holocentric organism Caenorhabditis elegans creates two chromosome domains of unequal length termed the short arm and long arm, which become the first and second site of cohesion loss at meiosis I and II. The mechanisms that confer distinct functions to the short and long arm domains remain poorly understood. Previously we and others have shown that phosphorylation of SYP-1, a central element of the synaptonemal complex (SC), at Thr452 provides a binding site for a Polo-like kinase PLK-2, and phosphorylated SYP-1 and PLK-2 cooperatively localize to the short arm to guide downstream factors triggering cohesin degradation at the short arm (Sato-Carlton et al. 2017; Brandt et al. 2020). Previous studies have shown that Polo kinase is activated via phosphorylation of its activation loop by Aurora kinase, and this interaction is promoted by Bora/SPAT-1 in mitosis (Tavernier et al. 2015). To understand the mode of Polo-like kinase regulation during meiotic prophase, we examined the effect of spat-1 knockdown during oogenesis. We found that PLK-2 failed to spread to short arms but instead was confined at crossover designation sites in spat-1 RNAi gonads. In addition, we found that homologous chromosome synapsis and crossover formation are impaired in spat-1 RNAi animals. Interestingly, excess crossover designation, ranging from 6 to 13 sites per nucleus, was found in spat-1 RNAi animals. Computational tracing of three-dimensional chromosome images revealed the presence of multiple crossover designation sites on the same chromosome, suggesting that crossover interference is impaired. These observations are reminiscent of plk-2 mutant phenotypes, and suggest the possibility that SPAT-1 regulates PLK-2 during meiotic prophase. Brandt J. N., K. A. Hussey, and Y. Kim, 2020 Spatial and temporal control of targeting Polo-like kinase during meiotic prophase. J. Cell Biol. 219 Sato-Carlton A, Nakamura-Tabuchi C, Chartrand SK, Uchino T, Carlton PM (2018) Phosphorylation of the synaptonemal complex protein SYP-1 promotes meiotic chromosome segregation. J Cell Biol 217:555-570 Tavernier N., A. Noatynska, C. Panbianco, L. Martino, L. Van Hove, et al., 2015 Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos. J. Cell Biol. 208: 661-669.

Sternberg, Paul et al. (2015) International Worm Meeting "WormBase 2015."

Berriman, Matt, Howe, Kevin, Kersey, Paul, Stein, Lincoln, Harris, Todd, Sternberg, Paul, Schedl, Tim

[International Worm Meeting, 2015]

WormBase has existed for 15 years and has evolved in many ways. The new website is fully operational and has made the process of adding new data types, displays, and tools easier. Behind the scenes we are piloting an overhaul of the underlying database infrastructure to allow us to handle the ever increasing data, have the website perform faster, and allow more frequent updates of information. This is a critical time for the project, as we face considerable pressure from two directions. The first is that our funders really want us to do more with less. We are responding to this by leading the way in making curation (the process of extracting information from papers and data sets into computable form) more efficient using a new version of Textpresso (to be released later this calendar year); by discussing with other model organism information resources ways to work together to be more efficient and inter-connected; and by seeking additional sources of funding. The second, delightful, pressure is an increase in data and results generated by the C. elegans and nematode communities. While we are handling this increase by changes in our software for curation, the database infrastructure, and the website, we do need your help. Many of you have helped us over the last few years to identify data in your papers or by sending us data directly. We now need you to help with a few types of information by submitting the data via specially designed, user-friendly forms that ensure good quality and the use of standard terminology. In particular, we have a large backlog of uncurated information associating alleles with phenotypes. We pledge to make this process as painless as possible, and to improve WormBase's description of phenotypes with your feedback, starting at this meeting at the WormBase booth, workshops and posters. With your help, continual improvement of our efficiency, and additional sources of funding, we are optimistic that we can do much more with even somewhat less effort.Consortium: Paul Davis, Michael Paulini, Gary Williams, Bruce Bolt, Thomas Down, Jane Lomax, Todd Harris, Sibyl Gao, Scott Cain, Xiaodong Wang, Karen Yook, Juancarlos Chan, Wen Chen, Chris Grove, Mary Ann Tuli, Kimberly Van Auken, D. Wang, Ranjana Kishore, Raymond Lee, John DeModena, James Done, Yuling Li, H.-M. Mueller, Cecilia Nakamura, Daniela Raciti, Gary Schindelman.