Berg, Maureen et al. (2015) International Worm Meeting "The C. elegans gut microbiota and its significance to its host."

Ho, Joshua, Parke, Caitlin, Alvarez-Cohen, Lisa, Shapira, Michael, Stenuit, Ben, Berg, Maureen

[International Worm Meeting, 2015]

The animal intestine is home to an abundance of microorganisms that play diverse roles in host health and disease. Characterizing the gut microbiota has become an active area of research, but the relative contribution of factors shaping microbiota composition is still unclear. Gut microbiota composition may be determined by environmental availability or shaped by host factors, enabling the selection of potentially beneficial taxa. Thus, it is of interest to understand the significance of those selected microbes, or those that are lost due to aberrant assembly, for their host. Using the genetically tractable Caenorhabditis elegans as a model host and 16S rDNA 'deep sequencing,' we are studying the assembly of the gut microbiota starting from diverse environmental communities. Comparisons of worm microbiotas to microbiotas in their soil environment revealed that worm microbiotas resembled each other, even when assembled from different microbial environments. Our results support a dominant contribution of the host niche in shaping the gut microbiota. Among the recurring taxa in the gut microbiota, we found Enterobacter and Pseudomonas, both of which provide services to their hosts. One resident, P. mendocina, is able to provide protection against the gram-negative pathogen P. aeruginosa by priming the p38-oxidative stress pathway, while E. cloacae can extend survival of worms infected with the gram-positive pathogen, Enterococcus faecalis. Assessing these interactions between gut isolates and their host will fill in a gap of our understanding of C. elegans interactions with microbes, and will help establish C. elegans as a model for studying host-microbiota interactions.

Berg, Maureen et al. (2015) International Worm Meeting "Assembly of the Caenorhabditis elegans gut microbiota is a deterministic process shaped by the host."

Shapira, Michael, Alvarez-Cohen, Lisa, Wang, Andrew, Ho, Joshua, Stenuit, Ben, Knight, Matthew, Berg, Maureen, Parke, Catelin

[International Worm Meeting, 2015]

Animals harbor gut microbiotas that play critical roles in the maintenance of host health, from digestion to immunity. Characterizing the gut microbiota has become an active area of research, but the relative contribution of factors shaping microbiota composition is still unclear. C. elegans could provide a useful model to study such questions, utilizing genetically homogenous populations to discern common patterns in microbiota composition beyond individual variation. However, to date, very little is known about the natural C. elegans microbiota. To fill this gap, we grew worms of the N2 wildtype strain in the lab in natural-like mini-environments of different composted soils, and used 16S rDNA deep sequencing to characterize the assembled microbiota of adult worms. Comparisons of worm microbiotas to microbiotas in their soil environment revealed that worm microbiotas resembled each other even when assembled from different microbial environments. This enabled defining a core gut microbiota that accounted for ca. 50% of gut microbes, and was primarily composed of members of the Enterobacteriaceae, Pseudomonadaceae, and Xanthomonadaceae families. Beyond the shared core, two distinct types of worm microbiotas were identified, distinguished by varying abundance of core taxa as well as the presence of auxiliary taxa. Ecological analyses indicated that the assembly rules for worm microbiotas differed from those in their soil habitat, and pointed at the importance of competitive interactions between gut-residing taxa. Our results support a dominant contribution of the host niche in shaping the gut microbiota, and suggest contributions from inter-microbial competition within the gut. The data presented provides a framework to better understand the worm natural history and its interactions with microbes.

Okano H et al. (2000) Japanese Worm Meeting "The SWI/SNF complex is required for asymmetric cell division in C. elegans"

Kouike H, Sawa H, Okano H

[Japanese Worm Meeting, 2000]

In C. elegans, the polarities of many cells undergoing asymmetric divisions are regulated by LIN-44/Wnt and LIN-17/Frizzled. To identify more genes required for asymmetric division, we are screening for mutants affecting the asymmetric division of the T cell in the tail that is regulated by lin-17 and lin-44. We have so far identified 17 new mutants corresponding to 13 different genes. Lineage analyses have shown that the T cell division can be symmetric in mutants of at least 7 of the genes (psa-1 through psa-7). Using temperature-sensitiveness of the psa-1 and psa-4 mutant, we found that these genes are required during the mitosis of the T cell. We have cloned psa-1 and psa-4 to found that they encode homologs of SWI3 and SWI2 of yeast, respectively. These proteins are known to be components of the complex called SWI/SNF. The complex can remodel chromatin structure by destabilizing histone-DNA interaction. These results suggest that the chromatin structure is altered by the SWI/SNF complex in one of the daughter cells during the T cell division. In budding yeast, the SWI/SNF complex is required for the asymmetric expression of HO endonuclease in the mother but not in the daughter cell. HO catalyzes switching of the mating types in the mother cell. Therefore, our findings demonstrate that at least some aspects of the mechanisms of asymmetric cell division are conserved between yeast and a multicellular organism.

Williams, Kyle C. et al. (2011) International Worm Meeting "Essential Functions of IFA-2 Domains in C. elegans Fibrous Organelles."

Plenefisch, John, Williams, Kyle C.

[International Worm Meeting, 2011]

The C. elegans fibrous organelle (FO) complex consists of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments (IFs). FOs are found most prominently in the epidermis where they overlie body wall muscle and serve to transmit force from the muscle to the cuticle. IFA-2, one of four epidermally expressed IFs whose loss results in epidermal fragility and failure of muscle-cuticle force transmission, localizes to FOs. Unlike IFB-1 and IFA-3 that are required embryonically, IFA-2, although expressed in the embryo, is not essential for FO function until postembryonic stages. This distinctive phenotype allows us to specifically explore the functions of IFA-2 domains, and map their interactions with other FO associated proteins. All IFs contain three domains: a central rod domain and globular head and tail domains. The rod is essential for assembly of IFs into mature filaments while less is known about the functions of the head and tail domains. Roles of the head and tail domains of IFA-2 were examined by expressing GFP-tagged IFA-2 variants in transgenic animals and examining their phenotype and localization in wild-type and null backgrounds. Mutant variants included IFA-2 deleted for the head domain (DH), deleted for the tail domain (DT), deleted for both (RO), and variants that contained only the head domain (HO) or tail domain (TO). The expression and function of DH is virtually indistinguishable from full-length IFA-2. DT results in a lowered incorporation of IFA-2 into FOs and is unable to rescue a null allele of ifa-2, suggesting the tail domain is essential to IFA-2 function. Though both HO and TO are expressed well in the hypodermis at all stages, incorporation into FOs is variable between individual animals. High levels of early-stage HO incorporation into FOs correlate with a dominant-negative muscle detachment phenotype, while low-level incorporation results in healthy animals. This suggests the head domain interacts with FO components essential to tissue integrity. To determine the protein components of the FOs that interact with the head and tail domains, yeast two-hybrid and co-localization experiments are in progress. This work should help to elucidate the role of IFA-2 in FO function and reveal the underlying defects leading to tissue fragility.

Cao, Jianfeng et al. (2021) International Worm Meeting "Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation"

Yan, Hong, Zhao, Zhongying, Tang, Chao, Chan, Lu-Yan, Guan, Guoye, Cao, Jianfeng, Wong, Ming-Kin, Ho, Vincy Wing Sze

[International Worm Meeting, 2021]

The invariant development and transparent body of the nematode Caenorhabditis elegans enables complete delineation of cell lineages throughout development. Despite extensive studies of cell division, cell migration and cell fate differentiation, cell morphology during development has not yet been systematically characterized in any metazoan, including C. elegans. This knowledge gap substantially hampers many studies in both developmental and cell biology. Here we report an automatic pipeline, CShaper, which combines automated segmentation of fluorescently labeled membranes with automated cell lineage tracing. We apply this pipeline to quantify morphological parameters of densely packed cells in 17 developing C. elegans embryos. Consequently, we generate a time-lapse 3D atlas of cell morphology for the C. elegans embryo from the 4- to 350-cell stages, including cell shape, volume, surface area, migration, nucleus position and cell-cell contact with resolved cell identities. We anticipate that CShaper and the morphological atlas will stimulate and enhance further studies in the fields of developmental biology, cell biology and biomechanics (Cao†, Guan†, Ho†, et al. Nat. Commun., 2020, 11: 6254).

Michael Dybbs et al. (2001) International C. elegans Meeting "A screen for new Hic mutants"

Josh Kaplan, Michael Dybbs

[International C. elegans Meeting, 2001]

ABSTRACT International Worm Meeting 2001 A screen for new Hic mutants Michael Dybbs and Joshua Kaplan 361 LSA, Dept. of Mol. and Cell Biology, University of California, Berkeley, CA 94720 Previous studies by our lab and others have shown that release of acetylcholine at the C. elegans neuromuscular junction (NMJ) is regulated by a G-protein signaling network. These studies have shown that serotoninergic signaling through goa-1 G a o and dgk-1 DAG kinase inhibits acetylcholine release. An opposing muscarinic signaling pathway through egl-30 G a q and egl-8 PLC b enhances acetylcholine release via the production of diacylglycerol ( DAG). Two RGS proteins, egl-10 RGS and eat-16 RGS, further regulate this pathway by stimulating the GTPase activity of goa- 1 G a o ( eat-16 RGS) and egl-30 G a q ( egl-10 RGS). Mutations in the inhibitory pathway ( goa-1 , dgk-1 ) make animals hypersensitive to paralysis by the acetylcholine esterase inhibitor, aldicarb (Hic) Mutations in the muscarinic pathway ( egl-30 , egl-8 ) make animals resistant to aldicarb (Ric). In order to identify additional molecules which negatively regulate release, we are conducting a broad screen for Hic mutants. Aldicarb sensitivity could be a result of either mutations that effect presynaptic neurotransmitter release or postsynaptic response to neurotransmitter in the muscle. We are distinguishing these effects using levamisole, a cholinergic agonist that acts directly on the acetylcholine receptors in the muscle. We are focusing on mutants that display a wild-type muscle sensitivity and are thus acting presynaptically. Mutants isolated in our pilots screens will be described.

Dasgupta, Krishnakali et al. (2009) International Worm Meeting "Understanding the role of PKC-1 signaling pathway in regulating Dense Core Vesicle secretion."

Sieburth, Derek, Dasgupta, Krishnakali

[International Worm Meeting, 2009]

Synaptic vesicles (SV) and dense core vesicles (DCV) both exhibit calcium dependent neurotransmitter release. However, differences in their biogenesis, release properties, and recycling suggest that each type of organelle may use some unique proteins to carry out their functions. PKC-1, a protein kinase C ortholog in worms, is one such candidate for a unique DCV regulating molecule, since it has been shown to regulate neuropeptide secretion, while possibly not affecting SV release.(1) PKC-1 is most similar to the human eta and epsilon isoforms which are activated by the second messenger DAG via their C1 domains. In order to identify additional components that act in the PKC-1 pathway to regulate DCV secretion, we have used a constitutively active PKC-1 mutant which lacks the autoinhibitory domain and is predicted to be active in the absence of DAG. Expression of this transgene specifically in motor neurons causes hypersensitivity to an acetylcholinesterase inhibitor, aldicarb (Hic) and loopy locomotion. We screened for suppressors of either the loopy or Hic phenotype of animals expressing the constitutive PCK-1, and identified ~30 suppressors that restore movement or aldicarb response. We are in the process of mapping them and characterizing their effects on neuropeptide secretion. Presently, we have identified one aldciarb resistant candidate allele, vj3, which shows decreased uptake of NLP-21::YFP by coelomocytes and increased accumulation of DCVs in axons. However, vj3 mutants do not cause an accumulation of the SV marker at synapses, suggesting that vj3 mutants specifically reduce DCV secretion. vj3 maps to a small interval on LG I that does not contain any known neurotransmitter secretion genes. We are currently in the process of fine mapping, and tesing candidate genes for aldicarb resistant phenotypes by RNAi. 1.PKC-1 regulates secretion of neuropeptides; Derek Sieburth1, Jon M Madison & Joshua M Kaplan; Nature Neuroscience 10, 49 - 57 (2007).

Vidal-Gadea A G et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Biogenic amines mediate transition between crawling and swimming in C. elegans"

Brauner M, Gottschalk A, Erbguth K, Kressin L, Vidal-Gadea A G, Topper S, Young L, Pierce-Shimomura J T

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

A wide variety of animals must quickly adjust their pattern of locomotion to successfully navigate through different environmental niches. Selection and execution of the appropriate locomotory pattern is therefore paramount to survival. Although C. elegans is capable of performing many adaptive behaviors, it has been controversial whether forward crawling and swimming represent distinct gait-like forms of locomotion or the modulation of a single form of locomotion [1-3]. Biogenic amines have been shown to mediate the transition between gait-like forms of locomotion across taxa as diverse as sea slugs, leeches, lampreys and humans. We previously reported that C. elegans crawls and swims with distinct kinematics and different patterns of muscle activity [2]. We now combine quantitative behavioral analysis, optogenetic tools and neuronal ablation to show that C. elegans uses biogenic amines to switch between crawling and swimming in a gait-like manner. As in other invertebrates, we find that serotonin mediates the smooth transition from crawling to swimming in C. elegans. Serotonin is further required to inhibit motor behaviors (e.g. foraging and pharyngeal pumping) during swimming that normally only accompany crawling. Mirroring the role of dopamine in other invertebrates, C. elegans uses dopamine to successfully initiate and maintain crawling when emerging from liquid. Over 600 million years of separate evolution notwithstanding, the highly conserved role played by biogenic amines such as dopamine and serotonin across taxa attests to how vital their function is to adaptive strategies for locomotion. Korta J, Clark DA, Gabel CV, Mahadevan L, Samuel AD. J. Exp. Bio. 2007 210:2383-9.Pierce-Shimomura JT, Chen BL, Mun JJ, Ho R, Sarkis R, McIntire SL. PNAS. 2008 105:20982-7.Berri S, Boyle JH, Tassieri M, Hope IA, Cohen N. HSFP J. 2009 3:186-93.

Maike Thamsen et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Peroxiredoxin-2 Plays a Crucial Role in the Oxidative Stress Resistance of C. elegans"

Maike Thamsen, Caroline Kumsta, Ursula Jakob

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Aging is a complex physiological process and numerous aging theories have been proposed. One of the leading models is the free radical theory of aging, which suggests that the accumulation of reactive oxygen species (ROS) is causally linked to aging and cell death. Aging cells have been found to accumulate proteins with oxidative modifications, including side chain carbonylation and thiol modifications. It is this oxidative damage to specific cellular proteins that is often proposed to constitute one of the major mechanisms that link oxidative stress to age-associated loss of critical physiological functions. We have now started to monitor and visualize the effects of oxidative stress treatment of C. elegans using 2D gel electrophoresis. This analysis revealed a number of proteins that show substantially increased levels of oxidative protein modifications. One of these proteins is the highly abundant PRDX-2, a 2-Cys Peroxiredoxin, which is responsible for the detoxification of peroxides. We found that short-term exposure of synchronized L4 larvae to sublethal concentrations of HO leads to the rapid over-oxidation of PRDX-2''s catalytic cysteine. This reversible overoxidation leads to the inactivation of PRDX-2''s peroxidase activity and apparently turns PRDX-2 into a molecular chaperone. Prdx-2 knockout worms show substantial delays in oxidative stress recovery and reveal a significantly shortened lifespan at 15degC. To dissect the peroxidase function of PRDX-2 from its chaperone activity, we began to analyze the role of PRDX-2 in sestrin knockout worms. Sestrins are a class of proteins, which have recently been shown in human colon carcinoma (RKO) cells to selectively reduce the overoxidized form of 2-Cys peroxiredoxins. Preliminary evidence suggests that sestrin knockout worms accumulate PRDX-2 in the overoxidized form, indicating that sestrin might be the dedicated PRXD-2 sulfinic acid reductase in C. elegans. Interestingly, both prdx-2 and sestrin knockout worms reveal similar phenotypes, indicating that it is indeed the peroxidase activity of PRDX-2 that plays the major lifespan determining role in C. elegans.

Tiffany Timbers et al. (2008) C.elegans Neuronal Development Meeting "Long-term Mechanosensory Habituation is Dependent upon CMK-1 and CRH-1 in Caenorhabditis elegans"

Tiffany Timbers, Catharine Rankin

[C.elegans Neuronal Development Meeting, 2008]

The transcription factor CREB plays a role in memory in a number of species; in C. elegans behavioural analysis of the crh-1(tz2) deletion mutant (homologous to mammalian CREB) has shown that this gene is necessary for long-term memory for mechanosensory habituation. crh-1(tz2) mutants displayed no long-term memory 24 hours after training. CRH-1 activity is dependent upon its level of phosphorylation by a variety of kinases. We have identified CMK-1 (homologous to mammalian CaMK1) as a possible activator of CRH-1 in the pathway that mediates long-term memory for habituation. cmk-1(ok287) deletion mutants phenocopy crh-1(tx2) mutants as they also display no long-term memory for habituation. Further evidence of CMK-1 acting as the upstream kinase in CRH-1 activation comes from Kimura and colleagues'' who demonstrated that CMK-1 and CRH-1 have overlapping expression patterns, that CMK-1 is capable of activating CRH-1 in vivo, and that cmk-1(ok287) mutants have greatly decreased levels of activated CRH-1. They also identified CKK-1 (homologous to mammalian CaMKK) as the upstream activating kinase of CMK-1. Interestingly, we have tested ckk-1(ok1033) mutants and found that they show normal long-term memory for habituation. This suggests that a novel kinase may act as the upstream kinase activating CMK-1 in the pathway that mediates long-term memory. Long-term memory for habituation in C. elegans is associated with a decrease in the area of GLR-1 synaptic clusters 24 hours after training (measured by confocal imaging of glr-1::gfp) (Rose et al., 2003). crh-1(tz2) mutant worms expressing this GLR-1::GFP transgene do not show a decrease in the area of GLR-1::GFP clusters after long-term memory training. This suggests that the decrease observed in wild-type worms is dependent upon CRH-1 activity. The direct mechanism by which CRH-1 does this is under investigation. We thank Dr. Yoshishige Kimura for the crh-1(tz2) strain, aswell as Dr. Anne Hart and Dr. Joshua Kaplan for glr-1 strains. This work was supported by operating grants from NSERC and CIHR and by Graduate Fellowships from NSERC, MSFHR and CIHR to TAT.