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[
Wiley Interdiscip Rev RNA,
2012]
XRN1 is a 5' 3' processive exoribonuclease that degrades mRNAs after they have been decapped. It is highly conserved in all eukaryotes, including homologs in Drosophila melanogaster (Pacman), Caenorhabditis elegans (XRN1), and Saccharomyces cerevisiae (Xrn1p). As well as being a key enzyme in RNA turnover, XRN1 is involved in nonsense-mediated mRNA decay and degradation of mRNAs after they have been targeted by small interfering RNAs or microRNAs. The crystal structure of XRN1 can explain its processivity and also the selectivity of the enzyme for 5' monophosphorylated RNA. In eukaryotic cells, XRN1 is often found in particles known as processing bodies (P bodies) together with other proteins involved in the 5' 3' degradation pathway, such as DCP2 and the helicase DHH1 (Me31B). Although XRN1 shows little specificity to particular 5' monophosphorylated RNAs in vitro, mutations in XRN1 in vivo have specific phenotypes suggesting that it specifically degrades a subset of RNAs. In Drosophila, mutations in the gene encoding the XRN1 homolog pacman result in defects in wound healing, epithelial closure and stem cell renewal in testes. We propose a model where specific mRNAs are targeted to XRN1 via specific binding of miRNAs and/or RNA-binding proteins to instability elements within the RNA. These guide the RNA to the 5' core degradation apparatus for controlled degradation.
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[
Worm Breeder's Gazette,
1999]
Mechanotransduction plays a central role in fundamental physiologic processes such as detection of touch and sound, regulation of cell volume, and control of motility. Mechanosensitive channels have been studied extensively using electrophysiology. Little is known, however, about the molecular structure of mechanosensitive channels or about how mechanical stress is transduced into altered channel gating. We developed techniques to patch-clamp and study ion channels in C. elegans embryonic cells. In cell-attached and inside-out patches, application of gentle suction activated a mechanosensitive current. Suction caused an immediate increase in current amplitude of 6.4 2.8 fold (n = 20) at +100 mV in inside-out patches. The current rapidly inactivated when suction was discontinued and could be repeatedly reactivated by additional suction. When membrane voltage was ramped from +100 to -100 mV at 100 mV/second, the current showed moderate outward rectification (outward:inward current = 2.0 0.05 at 100 mV). Current amplitude was largely unaffected when bath Na+ was replaced with NMDG+ (n = 10). However, replacement of bath Cl- with either gluconate or glutamate reduced inward and outward currents by 46 10% and 39 7%, respectively (n = 9). Replacement of 120 mM bath Cl- with a mixture of 60 mM Cl- and 60 mM SCN- increased the inward current at -100 mV by 3.0 0.4 fold and shifted Erev by 16 2 mV (n=7). We conclude that membrane stretch activates a novel mechanosensitive anion current in inside-out patches from C. elegans embryonic cells.
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[
West Coast Worm Meeting,
1996]
Genetic analyses of behavior have identified gene products important for neuronal function in C. elegans, including putative sensory and neurotransmitter receptors. As a first step in forging a link between these proteins and cellular physiology, we have recorded from neurons in the heads of semi-intact, L1 worms. The technique was modified from that reported previously [WBG 13(5): 32] and regularly yields tight-seal, whole-cell recordings. In current clamp, neurons responded to current steps with graded potentials whose time course depended on the injected current (n=24). Action potentials could not be elicited. Some cells (n=3) exhibited spontaneous, sustained shifts in membrane potential from approximately -60 to -20 mV. At -60 mV, smaller amplitude, transient depolarizations were frequently observed. Voltage pulses to greater than -10 mV elicited an outward current with transient and sustained components. The rate of decay of the transient component varied between cells. The range (tau = 10-100 ms) is similar to that reported for the superfamily of cloned, voltage-gated potassium currents. Thus, the variation in decay rate could reflect heterogeneity in potassium channel expression among different classes of neurons. Hyperpolarizing pulses to less than -80 mV elicited a voltage-dependent inward current with no obvious time dependence. Between -80 and -10 mV, most cells had a region of high resistance. We are currently characterizing the main outward current and investigating how it might contribute to temporal processing in individual C. elegans neurons.
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[
Mid-west Worm Meeting,
2002]
Calcium-activated potassium channels are present in the C. elegans nervous system, including many neurons of the nerve ring. Properties of these channels have been reported in a heterologous expression system; however, it was of interest to determine whether channels of similar properties could be recorded from C. elegans neurons. We recorded single channels in excised patches from the chemosensory neuron AWA, which were labeled with GFP. Neurons were exposed by cutting the worm with a scalpel blade just behind the terminal bulb. Excised patches were formed in conventional manner. Channels corresponding to BK potassium channels were present in AWA neurons. In patches containing the channels, channels were absent or only activated at high positive potentials (+100 mV). Addition of 100 mM calcium markedly shifted the activation of the channels toward more negative potentials, with channel openings visible at 0 mV and more positive potentials. Mean conductance of these channels was about 65 pS, with a reversal potential of -50 mV.
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[
European Worm Meeting,
2002]
Continuing contractions and extensive intercellular coupling prevent reliable whole-cell current recording from the enzymatically cleaned muscle cells of the C.elegans pharynx. However, confirming data previously obtained with intracellular recording with sharp microelectrodes (Pemberton et al., 2002), it has been shown that sodium omission from the Dent's physiological saline blocks inward currents in response to depolarisations from the holding potential of 120 mV to 40 mV both in wild type worms and egl 19 (
n582). In outside-out patches tested with Cs-internal solution and Dent's saline and held at 80 mV channels with very low opening probability were observed. Amplitude of the unitary currents in these patches was reduced in 0Na+ Dent's saline but not in 0Ca2+ solution. Currents were further reduced and then abolished in 0Na+ 0Ca2+ solution. Exposure of the patches to the Dent's saline with 10-times reduced concentration of Na+ caused a shift in reversal potential smaller that expected in accordance with the Nernst equation. Channels can be blocked by gadolinium ions and their mean opening time increased by veratridine. In inside out patches tested in symmetrical 150 mM Na+ solution containing no Ca2+ and K+ ions opening probability of the recorded channels at 80 mV was 3 times higher than in outside-out patches but was reduced to the level observed in outside-out patches when 3 mM Ca2+ were added to the solution contacting the external surface of the membrane patches. Taken together these data suggest the presence of some sort of sodium permeable channels in pharyngeal muscle cells of the adult worms.
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[
Worm Breeder's Gazette,
1978]
It has proven difficult to study the currents that generate the electrical activity in the somatic muscle cells of Ascaris due to the inability to control the voltage across the excitable membrane. Therefore, we have directed our attention to the pharyngeal muscle, where it is possible to directly measure the voltage and pass large currents across the excitable membrane. We have developed a system which allows us to do current-clamp and voltage-clamp experiments on an isolated segment of the pharyngeal membrane. We find that this membrane has no pacemaker activity. In the absence of nervous input the membrane potential is flat at a level near -40 mV. Two types of spontaneous postsynaptic potentials are frequently seen; one type has a reversal potential near -40 mV and the other has a reversal potential near -10 mV. When the membrane is at the resting level, this second type PSP triggers a positive-going action potential, which reaches a level between +30 and +50 mV. The membrane potential then falls to a plateau near O mV, where it remains until a negative-going PSP triggers a negative-going action potential that reaches about -50 mV (the potassium reversal potential). The membrane potential remains at the plateau level for periods ranging from 100 msec to several minutes. The positive-going action potential is produced by an inward current that appears to be carried by both Na+ and Ca++. This current is prolonged, showing little inactivation by 200 msec after a positive voltage step. Clamping the membrane to positive potentials elicits essentially no delayed-rectification K current, the current that normally repolarizes active membranes. However, stepping the membrane potential back to the resting level after a large positive pulse elicits a strong, transient outward K+ current; this is the current that produces the negative-going action potential. We have done a detailed analysis of this current and have shown that it is a voltage- inverted analogue of the Hodgkin-Huxley Na+ current. It is activated by negative steps in potential. It shows inactivation, being completely inactivated at the resting potential. Conditioning pulses to levels more positive than -10 mV are necessary to remove the inactivation from the channel. This is a new K+ conductance that has not been found in any other animal. This demonstration of unique mechanisms in nematode physiology should serve as a caution in trying to interpret the function of the nematode nervous system. The pharyngeal nervous system must not only generate the signal that triggers the pharynx to contract (open the lumen), but also the signal to relax (close the lumen). Since the relaxation of the pharynx is the 'power stroke' of this muscle, it is not surprising that the membrane has developed a special K+ current to produce a fast, separately-triggerable repolarization.
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[
J Physiol,
2002]
The properties of K+ channels in body wall muscle cells acutely dissected from the nematode Caenorhabditis elegans were investigated at the macroscopic and unitary level using an in situ patch clamp technique. In the whole-cell configuration, depolarizations to potentials positive to -40 mV gave rise to outward currents resulting from the activation of two kinetically distinct voltage-dependent K+ currents: a fast activating and inactivating 4-aminopyridine-sensitive component and a slowly activating and maintained tetraethylammonium-sensitive component. In cell-attached patches, voltage-dependent K+ channels, with unitary conductances of 34 and 80 pS in the presence of 5 and 140 mm external K+, respectively, activated at membrane potentials positive to -40 mV. Excision revealed that these channels corresponded to Ca2+-activated K+ channels exhibiting an unusual sensitivity to internal Cl- and whose activity progressively decreased in inside-out conditions. After complete run-down of these channels, one third of inside-out patches displayed activity of another Ca2+-activated K+ channel of smaller unitary conductance (6 pS at 0 mV in the presence of 5 mm external K+). In providing a detailed description of native K+ currents in body wall muscle cells of C. elegans, this work lays the basis for further comparisons with mutants to assess the function of K+ channels in this model organism that is highly amenable to molecular and classical genetics.
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[
Brain Res Mol Brain Res,
1992]
Membrane currents were recorded from Xenopus laevis oocytes injected with C. elegans poly(A)+ RNA. In such oocytes glutamate activated an inward membrane current that desensitized in the continued presence of glutamate. Glutamate-receptor agonists quisqualate, kainate, and N-methyl-D-aspartate were inactive. The reversal potential of the glutamate-sensitive current was -22 mV, and exhibited a strong dependence on external chloride with a 48 mV change for a 10-fold change in chloride. The chloride channel blockers flufenamate and picrotoxin inhibited the glutamate-sensitive current. Ibotenate, a structural analog of glutamate, also activated a picrotoxin-sensitive chloride current. Ibotenate was inactive when current was partially desensitized with glutamate, and the responses to low concentrations of glutamate and ibotenate were additive. The anthelmintic/insecticide compound avermectin directly activated the glutamate-sensitive current. In addition, avermectin increased the response to submaximal concentrations of glutamate, shifted the glutamate concentration-response curve to lower concentrations, and slowed the desensitization of glutamate-sensitive current. We propose that the glutamate-sensitive chloride current and the avermectin-sensitive chloride current are mediated via the same channel.
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[
Proc Natl Acad Sci U S A,
2019]
Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all-<i>trans</i> retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode <i>Caenorhabditis elegans</i> ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% F/F increase per 100 mV], and in neurons, integrating circuit activity. ATR fluorescence is very dim, yet, using the retinal analog dimethylaminoretinal, it was boosted 250-fold. eFRET sensors provided sensitivities of 45 to 78% F/F per 100 mV, induced by BWM action potentials, and in pharyngeal muscle, measured in simultaneous optical and sharp electrode recordings, MacQ-mCitrine showed approximately 20% F/F per 100 mV. All sensors reported differences in muscle depolarization induced by a voltage-gated Ca<sup>2+</sup>-channel mutant. Optogenetically evoked de- or hyperpolarization of motor neurons increased or eliminated action potential activity and caused a rise or drop in BWM sensor fluorescence. Finally, we analyzed voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, noninvasive electrophysiology in live, intact <i>C. elegans</i>.
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[
Parasitology,
1994]
Intracellular recordings have been made from neurones in the head ganglia of Ascaris. The neurones had low resting membrane potentials of -21 +9 mV (n = 78) and a relatively high input resistance (e.g. 25 M-OMEGA for a 100 mu-m cell). In all cases the intracellular location of the recording electrode was verified by injection of the fluorescent marker, 5,6-carboxyfluorescein (CBXF). To ascertain whether or not the low membrane potential was due to impalement damage, the same neurone was recorded from using two microelectrodes. The membrane potential following the first impalement by a 20 M-OMEGA 3 M KCl electrode was -38 mV and following the second impalement by a 80 M-OMEGA CBXF (for subsequent intracellular labelling) electrode was decreased to - 34 mV. Input resistance of these cells was estimated using both single and two electrode intracellular recording techniques and in both cases yielded a relatively high value for the size of cell (e.g. 25 M-OMEGA for a 100 mu-m cell). Neurones labelled by intracellular injection of the fluorescent market 5,6-carboxyfluorescein were morphologically simple and lacked extensive arborizations. The dorsal ganglion is a discrete structure consisting of only 3 neurones. Here we compare the morphological properties of these neurones to those described in the dorsal ganglion of Caenorhabditis elegans. The whole mount preparation of Ascaris ganglia thus provides a useful model to study the functional properties of neurones in nematode central nervous system and presents the possibility to assess central sites of action for anthelmintics.