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[
J Gen Physiol,
2007]
MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O''Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43-50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O''Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039-1042). To gain insight into the structure-function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit-subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.
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[
J Biochem,
1985]
Myosin was purified rapidly from the nematode Caenorhabditis elegans by an improved method. Crude actomyosin was extracted from the worms at low ionic strength. Paramyosin was removed by repeating the precipitation of myosin filaments in the presence of Mg2+ and the dissolution of them in 0.6 M NaCl. Actin was removed by ultracentrifugation in the presence of Mg-ATP and finally by column chromatography on DEAE-cellulose. This method gave a good yield of myosin (20-30 mg from 50 g wet weight of worms), and its EDTA(K+)-ATPase activity was about 3-fold higher than that of myosin prepared by the method of Harris and Epstein (1979). ATP hydrolysis by nematode myosin showed an initial Pi-burst due to formation of the myosin-phosphate-ADP complex. Tryptophan fluorescence of myosin was enhanced about 8% by ATP. The relationship between the structure and function of myosin is discussed based on the above results and the amino acid sequences of myosins from rabbit skeletal muscle and Caenorhabditis elegans.
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[
MicroPubl Biol,
2020]
An environment is often represented by numerous sensory cues. In order to better survive, an animal often needs to detect and process sensory cues simultaneously to make an appropriate behavioral decision. The Caenorhabditis elegans (C. elegans) genome encodes homologues of a significant number of molecules expressed in mammalian brains, which allows characterizing of the molecular and circuit basis for multi-sensory behavior during decision-making (Bargmann, 1998). In addition, studies have demonstrated various genes and neurons in behavioral differences previously observed in sensory behavior and decision-making when comparing different organisms, such as, nematodes. These variations in behavior involve differences in neuronal signals and neurons. For example, catecholamine signaling, neuropeptide Y-like receptors and pheromone chemoreceptors (Debono and Bargmann, 1998, Srinivasan et al., 2008; Bendesky et al., 2010, Mcgraph et al., 2011). In this study, we investigate the behavioral differences across various species of nematodes, by examining multiple types of Caenorhabditis species and how they may differ in a multi-sensory behavioral assay (Harris et al., 2019). The multisensory behavioral assay involves examining how different species of nematodes that are exposed to conflicting cues, behave when compared to the standard wild type, C. elegans Bristol N2 worms. We use a multi-sensory behavior paradigm to address these questions to determine any difference in 2-nonanone-dependent food leaving, that assesses food leaving during exposure to the repellent 2-nonanone. Wild type N2 C. elegans were examined as the control in comparison to other Caenorhabditis species, including, Caenorhabditis remanei (C. remanei), Caenorhabditis nouraguensis (C. nouraguensis), and Caenorhabditis portoensis (C. portoensis).
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[
Dev Biol,
2005]
The nematode gonad is an exemplary system for the study of organogenesis and fundamental problems in developmental and cellular biology. Nematode gonads vary dramatically across species (Chitwood, B.G., Chitwood, M.B., 1950. Introduction to Nematology." University Park Press, Baltimore; Felix, M.A., Sternberg, P.W., 1996. Symmetry breakage in the development of one-armed gonads in nematodes. Development 122, 2129-2142). As such, comparative developmental biology of gonadogenesis offers the potential to investigate changes in developmental and cellular processes that result in novel organ morphologies and thus may give insights into how these changes can affect animal bauplane. Pristionchus pacificus is a free-living nematode that diverged from the model nematode Caenorhabditis elegans around 200-300 million years ago. The morphology and development of P. pacificus is highly homologous to that of C. elegans. However, many differences in morphology and the underlying molecular signaling networks are easy to identify, making P. pacificus ideal for a comparative approach. Here, we report a detailed description of the P. pacificus hermaphrodite gonad using electron and fluorescent microscopy that will provide a basis for both phenotypic studies of genetic mutations and in vivo molecular studies of cloned genes involved in P. pacificus gonad development. We report that the morphology of the P. pacificus gonad is distinct from that of C. elegans. Among these differences are germ line patterning differences, heterochronic differences, novel gonadal arm-migrations, novel cellular composition of some somatic tissues (e.g., the number of cells that comprise the sheath and different spermathecal regions are different), the absence of a somatic tissue (e.g., the spermathecal valve cells), a novel architecture for the sheath, and changes in the cellular and sub-cellular morphology of the individual sheath cells. Additionally, we report a set of cell ablations in P. pacificus that indicate extensive cell communication between the somatic gonadal tissues and the germ line. Individual ablation experiments in P. pacificus show significant differences in the effects of individual somatic tissues on germ line patterning in comparison to C. elegans.
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[
MicroPubl Biol,
2020]
In response to adverse environments C. elegans larvae may form stress-resistant and developmentally arrested dauer larvae. Entry into dauer interrupts developmental progression after the second larval molt (Cassada and Russell 1975), and the dauer formation decision must be coordinated with developmental pathways. Heterochronic genes control stage-specific cell fate decisions, and these genes interconnect with the dauer formation (daf) pathway in several ways. Dauer formation and daf genes affect heterochronic phenotypes, and conversely, heterochronic genes can impact dauer by controlling the timing of dauer formation, the morphology of dauer larvae, or the ability to enter dauer (Liu and Ambros 1989; Liu and Ambros 1991; Antebi et al. 1998; Tennessen et al. 2010; Karp and Ambros 2011; Karp and Ambros 2012). Here we find that the
lin-41 heterochronic gene regulates the dauer formation decision and dauer morphogenesis via its canonical target,
lin-29. The temperature-sensitive
daf-7(
e1372) allele results in constituitive dauer entry at 25˚C, and the allele is a useful tool for controlling dauer entry (Vowels and Thomas 1992; Karp 2018). While performing experiments examining the role of
lin-41 during dauer, we observed that
lin-41(0);
daf-7 worms rarely entered dauer. To better understand this phenotype, we quantified the number of dauer and nondauer larvae in synchronous populations of
daf-7(
e1372) larvae grown at the dauer-inducing temperature of 24˚C.
lin-41(0) mutants are sterile, and therefore the strain must be propagated as a heterozygote (Slack et al. 2000). We took advantage of a closely linked transgene, nIs408[LIN-29::mCherry,
ttx-3::gfp] to balance
lin-41(0) (Harris and Horvitz 2011).
lin-41(0) homozygous larvae were identified by the absence of fluorescence. Controls included nIs408-positive siblings, 2/3 of which should be
lin-41(0)/nIs408 and 1/3 of which should be nIs408/nIs408, as well as a strain that is homozygous for the wild-type allele of
lin-41.
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[
MicroPubl Biol,
2020]
Sensation of environmental cues and decisions made as a result of processing of specific sensory cues underlies a myriad of behavioral responses that control every-day life decisions and ultimately survival in many organisms. Despite the appreciation that organisms can sense, process, and translate sensory cues into a behavioral response, the neural mechanisms and molecules that mediate these behaviors are still unclear. Neurotransmitters, such as glutamate, have been implicated in a variety of sensory-dependent behavioral responses, including olfaction, nociception, mechanosensation, and gustation (Mugnaini et al., 1984, Wendy et al., 2013, Daghfous et al., 2018). Despite understanding the importance of glutamate signaling in sensation and translation of contextual cues on behavior, the molecular mechanisms underlying how glutamatergic transmission influences sensory behavior is not fully understood. The nematode, C. elegans, is able to sense a variety of sensory cues. These types of sensory-dependent behavioral responses are mediated through olfactory, gustatory, mechanosensory and aerotactic circuits of the worm (Lans and Jansen, 2004, Milward et al., 2011, Bretscher et al., 2011, Kodama-Namba et al., 2013, Ghosh et al., 2017). Odor guided behavior toward attractants, such as, food cues requires neurotransmitters, that include, glutamate (Chalasani et al., 2007, Chalasani et al., 2010). More specifically, once on a food source, wild type N2 hermaphrodites will generally be retained on a food source (Shtonda and Avery, 2006, Milward et al., 2011, Harris et al., 2019). The types, quality, pathogenicity, and perception of food can modulate food recognition, food leaving rates, and overall navigational strategies towards food (Zhang et al., 2005, Shtonda and Avery, 2006; Ollofsson et al., 2014). These types of behaviors are based on detection of environmental cues, including oxygen, metabolites, pheromones, and odors. Food leaving behaviors have been shown to be influenced by a number of neuronal signals (Shtonda and Avery, 2006, Bendesky et al., 2011, Ollofsson et al., 2014, Meisel et al., 2014, Hao et al., 2018).
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[
Dev Biol,
2015]
Cell polarity is a fundamental characteristic of epithelial cells. Classical cell biological studies have suggested that establishment and orientation of polarized epithelia depend on outside-in cues that derive from interactions with either neighboring cells or the substratum (Akhtar and Streuli, 2013; Chen and Zhang, 2013; Chung and Andrew, 2008; McNeill et al., 1990; Nejsum and Nelson, 2007; Nelson et al., 2013; Ojakian and Schwimmer, 1994; Wang et al., 1990; Yu et al., 2005). This paradigm has been challenged by examples of epithelia generated in the absence of molecules that mediate cell-cell or cell-matrix interactions, notably E-cadherin and integrins (Baas et al., 2004; Choi et al., 2013; Costa et al., 1998; Harris and Peifer, 2004; Raich et al., 1999; Roote and Zusman, 1995; Vestweber et al., 1985; Williams and Waterston, 1994; Wu et al., 2009). Here we explore an alternative hypothesis, that cadherins and integrins function redundantly to substitute for one another during epithelium formation (Martinez-Rico et al., 2010; Ojakian et al., 2001; Rudkouskaya et al., 2014; Weber et al., 2011). We use C. elegans, which possesses a single E-cadherin (Costa et al., 1998; Hardin et al., 2013; Tepass, 1999) and a single -integrin (Gettner et al., 1995; Lee et al., 2001), and analyze the arcade cells, which generate an epithelium late in embryogenesis (Portereiko and Mango, 2001; Portereiko et al., 2004), after most maternal factors are depleted. Loss of E-cadherin(HMR-1) in combination with -integrin(PAT-3) had no impact on the onset or formation of the arcade cell epithelium, nor the epidermis or digestive tract. Moreover, B-integrin(PAT-3) was not enriched at the basal surface of the arcades, and the candidate PAT-3 binding partner -laminin(LAM-1) was not detected until after arcade cell polarity was established and exhibited no obvious polarity defect when mutated. Instead, the polarity protein
par-6 (Chen and Zhang, 2013; Watts et al., 1996) was required to polarize the arcade cells, and
par-6 mutants exhibited mislocalized or absent apical and junctional proteins. We conclude that the arcade cell epithelium polarizes by a PAR-6-mediated pathway that is independent of E-cadherin, -integrin and -laminin.
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[
Neurotoxicology,
2012]
Manganese (Mn) is a well established neurotoxin associated with specific damage to the basal ganglia in humans. The phenotype associated with Mn neurotoxicity was first described in two workers with occupational exposure to Mn oxide (Couper, 1837). Although the description did not use modern clinical terminology, a parkinsonian illness characterized by slowness of movement (bradykinesia), masked facies, and gait impairment (postural instability) appears to have predominated. Nearly 100 years later an outbreak of an atypical parkinsonian illness in a Chilean Mn mine provided a phenotypic description of a fulminant neurologic disorder with parkinsonism, dystonia, and neuropsychiatric symptoms (Rodier, 1955). Exposures associated with this syndrome were massive and an order of magnitude greater than modern exposures (Rodier, 1955; Hobson et al., 2011). The clinical syndrome associated with Mn neurotoxicity has been called manganism. Modern exposures to Mn occur primarily through occupations in the steel industry and welding. These exposures are often chronic and varied, occurring over decades in the healthy workforce. Although the severe neurologic disorder described by Rodier and Couper are no longer seen, several reports have suggested a possible increased risk of neurotoxicity in these workers (Racette et al., 2005b; Bowler et al., 2007; Harris et al., 2011). Based upon limited prior imaging and pathologic investigations into the pathophysiology of neurotoxicity in Mn exposed workers (Huang et al., 2003), many investigators have concluded that the syndrome spares the dopamine system distinguishing manganism from Parkinson disease (PD), the most common cause of parkinsonism in the general population, and a disease with characteristic degenerative changes in the dopaminergic system (Jankovic, 2005). The purpose of this symposium was to highlight recent advances in the understanding of the pathophysiology of Mn associated neurotoxicity from Caenorhabditis elegans to humans. Dr. Aschner's presentation discussed mechanisms of dopaminergic neuronal toxicity in C. elegans and demonstrates a compelling potential role of Mn in dopaminergic degeneration. Dr. Guilarte's experimental, non-human primate model of Mn neurotoxicity suggests that Mn decreases dopamine release in the brain without loss of neuronal integrity markers, including dopamine. Dr. Racette's presentation demonstrates a unique pattern of dopaminergic dysfunction in active welders with chronic exposure to Mn containing welding fumes. Finally, Dr. Dydak presented novel magnetic resonance (MR) spectroscopy data in Mn exposed smelter workers and demonstrated abnormalities in the thalamus and frontal cortex for those workers. This symposium provided some converging evidence of the potential neurotoxic impact of Mn on the dopaminergic system and challenged existing paradigms on the pathophysiology of Mn in the central nervous system.