Ortiz, Nathaniel et al. (2021) International Worm Meeting "A model for partial depletion of disease gene homologs reveals dose-dependent effects of the Kabuki Syndrome-related factors SET-16/KMT2D and UTX-1/KDM6A"

Ortiz, Nathaniel, Heiman, Maxwell, Bodamer, Olaf, Lee, Eunjung, Choi, Jaejoon

[International Worm Meeting, 2021]

Human genetic disorders often involve haploinsufficiency, in which disruption of just one copy of a gene causes disease symptoms. For example, the developmental disorder Kabuki Syndrome (KS) is caused by disruption of a single copy of the genes that encode the histone modifying factors KMT2D and KDM6A. By contrast, in laboratory organisms, loss-of-function mutations are typically recessive. For example, null alleles of the C. elegans KS gene homologs, SET-16 and UTX-1 respectively, appear outwardly normal as heterozygotes (50% gene dose) but are lethal as homozygotes (0% gene dose). We reasoned that more severe depletion of SET-16 and UTX-1 - for example, to 10% or 20% of normal levels - might provide a better model of the cellular defects caused by haploinsufficiency in KS. In order to systematically manipulate protein levels, we turned to the auxin-inducible degron (AID) system. We inserted tandem GFP and AID tags at the endogenous SET-16 and UTX-1 genes. Consistent with previous reports, we find that SET-16 and UTX-1 are both expressed in all, or nearly all, cells beginning in mature gametes and continuing through adulthood. We find that titrating auxin levels causes a dose-dependent depletion of SET-16 or UTX-1 as assessed by GFP intensity, using either a somatic- or germline-specific TIR1 ubiquitin ligase. For example, in somatic cells of animals treated with 0 mM, 0.01 mM, 0.1 mM, or 1.0 mM auxin, we observed relative GFP intensities of 100%, 74%, 24%, and 1% for SET-16 and 100%, 14%, 3%, and 0% for UTX-1. To determine how partial depletion affects cellular phenotypes, we performed transcriptional profiling of SET-16 or UTX-1 somatic depletion strains grown under each auxin condition, in triplicate. We identified ~3000 and ~1500 genes whose expression levels are significantly correlated to the extent of SET-16 or UTX-1 depletion, respectively, across conditions. Overall, the gene lists were highly concordant, for example, 95 of the 100 genes whose expression is most highly correlated with SET-16 levels are also significantly correlated with UTX-1 levels. Intriguingly, 24 of the top 100 affected genes are members of the pals gene family, which has been implicated in gene silencing and pathogen-driven stress responses. Overall, this study provides a general strategy for partial depletion of disease-relevant genes that may better reflect the cellular changes that occur in human disorders involving genetic insufficiency.

Wang C et al. (2018) Nat Commun "Author Correction: Small-molecule TFEB pathway agonists that ameliorate metabolic syndrome in mice and extend C. elegans lifespan."

Zhao T, Oswald NW, Li Y, Lin R, Wang C, Jaramillo J, Zhou A, McMillan EA, Douglas PM, MacMillan JB, Huang G, Luo M, Gao J, Mendiratta S, Lin Z, Wang Z, Niederstrasser H, Posner BA, Brekken RA, White MA

[Nat Commun, 2018]

The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected inboth the PDF and HTML versions of the Article.

Shindou, Tomomi et al. (2015) International Worm Meeting "Electrophysiological properties of ASE neurons."

Wickens, Jeffery, MARUYAMA, Ichiro, Shindou, Tomomi, Shindou, Mayumi, Murayama, Takashi

[International Worm Meeting, 2015]

A pair of ASE chemosensory neurons, ASEL and ASER, are major salt sensors, and play critical roles in chemotaxis to NaCl. Calcium imaging has previously revealed that ASEL and ASER are activated by an increase and decrease in NaCl concentrations, respectively (Suzuki et al., 2008; Ortiz et al., 2009). These asymmetric responses by ASEL and ASER to changes of NaCl concentrations are crucial to efficient chemotaxis of C. elegans toward higher concentrations of NaCl. While Goodman et al. (1998) reported in situ whole-cell patch-clump recording of ASER, electrophysiological characterisation of ASE neurons is still required to understand how the neurons respond to the NaCl concentration changes.Toward the goal, we have investigated electrophysiological properties of ASE neurons in wild-type C. elegans by in vivo whole-cell patch-clamp recordings, and have found that both of ASE neurons showed resting membrane potentials of approximately -60 mV and membrane resistances of about 2 Gomega. In both of ASE neurons, voltage responses to current injections showed solitary action potentials. Depolarization of wild-type ASEL was observed when a puff of 150 mM NaCl was applied to the animal's nose in bath solution containing 50 mM NaCl. On the other hand, a puff of NaCl-free buffer induced ASER depolarization. These results are consistent with those of calcium imaging. To understand roles of the action potentials in ASE, we are currently trying to analyse electrophysiological properties of ASE neurons in various mutants.References1. Suzuki et al., Nature 454: 114-118 (2008)2. Ortiz et al., Current Biology 19: 996-1004 (2009)3. Goodman et al., Neuron 20: 763-772 (1998).

Epstein HF et al. (1988) J Cell Biol "Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures."

Epstein HF, Berliner GC, Ortiz I, Casey DL

[J Cell Biol, 1988]

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.

Epstein HF et al. (1985) J Cell Biol "Myosin and paramyosin are organized about a newly identified core structure."

Berliner GC, Miller DM, Epstein HF, Ortiz I

[J Cell Biol, 1985]

Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D.M. III, I. Ortiz, G.C. Berliner, and H.F. Epstein, 1983, Cell, 34: 477-490). Biochemical and electron microscope studies of KCl-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.

Gibson, Sophia B et al. (2021) MicroPubl Biol

Harper, Clare S, Gibson, Sophia B, Lackner, Laura L, Andersen, Erik C

[MicroPubl Biol, 2021]

Parasitic nematode infections continue to have an enormous impact on human and livestock health worldwide (Hotez et al., 2014; Kaplan & Vidyashankar, 2012). A limited arsenal of anthelmintic drugs are available to combat these infections. One of the most widely used classes is benzimidazoles (BZ), and resistance against this class is widespread (Kaplan & Vidyashankar, 2012). Previous studies to understand parasitic nematode resistance using the free-living model organism Caenorhabditis elegans showed that variation in the C. elegans beta-tubulin gene ben-1, an ortholog of beta-tubulins in parasitic nematodes, confers resistance to BZ drugs (Dilks et al., 2020; Driscoll et al., 1989; Hahnel et al., 2018). The most common missense mutation resistance alleles are F167Y, E198A, and F200Y (Avramenko et al., 2019; Mohammedsalih et al., 2020). Although computational models have predicted that these amino acids are involved in the binding of BZ compounds to beta-tubulins, the binding remains to be investigated empirically at the structural level because nematode-specific beta-tubulin structures have not been created (Aguayo-Ortiz et al., 2013; Hahnel et al., 2018). To better understand the mechanisms of resistance, we sought to obtain those crystallographic structures.

Smith, Heidi K. et al. (2009) International Worm Meeting "Functional Analysis of Asymmetrically-Expressed GCY Proteins in C. elegans."

Hobert, Oliver, Ortiz, Christopher O., Smith, Heidi K.

[International Worm Meeting, 2009]

Chemosensation allows animals to evaluate their environment, detect food, other animals, and dangerous toxins while responding with appropriate behaviors essential to the animal''s survival. A robust chemosensory system can be generated even from a seemingly simple nervous system, such as that of C. elegans, which can detect and respond to a vast number of chemical cues. One important, but poorly understood, strategy used by C. elegans is to "lateralize" the function of some of its sensory neurons, such as the ASE neurons, thus increasing the discriminatory power of a system comprised of relatively few elements. We have found that the ASE neurons respond to several salt cues and these responses are asymmetric in terms of whether the left or the right ASE neuron responds to a specific salt cue (Ortiz et al., submitted). Previous work in our lab has furthermore identified a number of guanylyl cyclase genes as having a role in the chemotaxis asymmetry of ASE (Ortiz et al., submitted). Mutant analysis has revealed that individual gcy genes are specifically required for sensing particular ions. Such specificity could be conferred through either the protein''s receptor family ligand-binding region (RFLBR) in its extracellular domain or by the protein''s guanylyl cyclase (GC) domain in its intracellular region. We seek to identify the molecular mechanisms by which these asymmetrically expressed receptor type guanylyl cyclases confer the specificity that underlies this lateralization. In order to test these predictions, intra- or extra-cellular domains of individual GCY proteins were swapped, chimeric proteins were introduced into mutant background animals in a cell-specific manner, and then rescue of chemotaxis defects were tested. The individual GCY protein domains that confer the cellular specificity of ASE neurons, which enables them to mediate responses only to particular cues are identified by evaluating assay output. Results from such experiments allow for the characterization of the domain(s) essential for specificity. By identifying these molecular mechanisms, key predictions of the role that these proteins play in ASE neurons, putatively functioning either as chemoreceptors or, alternatively, as signal transducers, can begin to be tested. We will pursue further strategies for elucidating these molecular mechanisms as part of an overall effort in exploring the relationship between individual genes, their patterns of expression in specific cellular contexts, and the chemosensory behaviors exhibited by C. elegans in response to salt cues found in its environment.

Hoppe PE et al. (1996) J Cell Biol "Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans."

Hoppe PE, Waterston RH

[J Cell Biol, 1996]

Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.

Andrew D Goldsmith et al. (2007) International Worm Meeting "The Mechanisms of Amino Acid Gustation."

Andrew D Goldsmith, Oliver Hobert

[International Worm Meeting, 2007]

Gustation of amino acids by C. elegans has been noted for over 30 years (Ward, 1973), but the mechanisms by which this occurs are largely unknown. There is some evidence (Bargmann and Horvitz, 1991) that the ASE gustatory neurons are required for sensation of at least some amino acids. We are currently examining a large panel of amino acids for sensation by C. elegans. We are determining to what extent ASE plays a role in amino acid sensation by assaying strains with genetic ablations of ASE for taxis in response to presentation of amino acids. As the ASE neurons have previously been shown to be functionally lateralized in regard to salt sensation (see abstract by Ortiz et al.), we are also in the process of assaying individuals with either ASEL or ASER ablations, as well as mutants that disrupt ASE asymmetry. Previous work (Pierce-Shimomura et al. 2001) has shown that C. elegans is able to discriminate between ions sensed by different neurons, and this discrimination is dependent on ASE asymmetry. We are examining whether individual amino acids can also be discriminated by worms and whether this feature depends on ASE asymmetry. We are also testing whether salts that can sensed by ASE can be efficiently discriminated from amino acids. Lastly, to elucidate the molecular mechanisms of gustatory signal transducion, we are making use of our analysis of the ASE transcriptome, determined by SAGE analysis (see Abstract by Etchberger et al.). This analysis has revealed a plethora of molecules that are candidate signaling components and we are attempting to verify their expression in ASE and test their involvement using mutant analysis.

Otsuka AJ et al. (1996) Midwest Worm Meeting "Characterization of a novel thick filament epitope recognized by monoclonal antibody LS25."

Cheung HT, Lee M, Otsuka AJ

[Midwest Worm Meeting, 1996]

In experiments directed toward producing a panel monoclonal antibodies to C. elegans ECM, we isolated a monoclonal antibody, designated LS25, which stained the A-band structures in many different muscles such as body-wall, pharynx, vulva, intestine, and anal sphincter. In immunoblot analysis, LS25 recognized a series of protein bands ranging from 30 to 90 kD. Because LS25 stained structures in many thick filament defect mutants such as unc-54 (e190), unc-15 (e73), and unc-22 (e66), it seemed unlikely that it recognized myosin, paramyosin, or twitchin. Using LS25 to screen a cDNA library, a cDNA clone containing ~2.3 kb DNA insertion fragment was obtained. The nucleotide sequence analysis of a portion of the clone revealed that it matched the egl-45 gene (Genbank Access #388573), and contained a region with amino acid sequence similarity to human trichohyalin. Trichohyalin is an intermediate filament binding protein found in hair follicles and cores. In contrast, we report the first finding that trichohyalin may be a component of the muscle thick filament. Based on our finding that EGL-45 may be a thick filament component, we analyzed the published N-terminal amino acid sequence of thick filament P28 core protein (Epstein et al., 1995) and discovered that it contained similarities to human trichohyalin. These results are consistent with the egl-45 phenotype and reveal relationships between the trichohyalin-related proteins, the muscle thick filament core, and the muscle mutants producing Egl phenotype. REFERENCE Epstein, H. F., G. Y. Lu, P. R. Deitiker, I. Ortiz, and M. F. Schmid, Journal of Structural Biology 115, 163-174 (1995).