Miriam B Goodman et al. (2001) International C. elegans Meeting "Touch Cell Proteins Needed for the Formation of Amiloride-sensitive Ion Channels"

Martin Chalfie, Dattananda Chelur, Robert O'Hagan, Glen G Ernstrom, Miriam B Goodman

[International C. elegans Meeting, 2001]

Touch sensation along the body of C. elegans depends on six specialized sensory neurons called touch cells (ALM, AVM, PVM, PLM). Genetic screens for touch insensitive mutants identified twelve mec ( mec hanosensory abnormal) genes needed for the activity, but not the development, of the touch cells. Most of these genes have been cloned. Except for mec -5, all of the cloned genes are expressed in the touch cells. The protein encoded by mec -4 is touch-cell specific and belongs to the DEG/ENaC superfamily of proteins that form amiloride-sensitive ion channels. The mec -10 gene encodes another DEG/ENaC channel subunit expressed in touch cells and four other mechanosensory neurons. These channel subunits are hypothesized to be the core of a mechanotransduction complex. A long-standing question is whether the putative ion channel subunits, MEC-4 and MEC-10, form channels similar to other DEG/ENaC channels. To address this question we measured whole cell currents in Xenopus oocytes injected with cRNAs encoding the channel subunits. We found that heterologous expression of MEC-4 and MEC-10 bearing activating mutations that cause degeneration in vivo fails to produce any current. However, robust amiloride-sensitive currents are observed when either MEC-2 (a protein related to human stomatin) or MEC-6 (a protein related to human paraoxonase) is added. MEC-4, but not MEC-10, is required to produce amiloride-sensitive currents. These currents were Na + -selective, inwardly-rectified, effectively time-independent, and exquisitely sensitive to amiloride. The largest currents were observed when both MEC-2 and MEC-6 were present together with the channel subunits; MEC-2 and MEC-10 altered affinity for amiloride. The expression pattern data, genetic interaction data, and now these oocyte expression data suggest that all four of these proteins (and possibly others) are needed for the formation of a mechanotransduction complex in vivo .

Miriam B Goodman et al. (2003) International Worm Meeting "Mapping the pore of the amiloride-sensitive channel (ASC) needed for touch sensation in C. elegans"

Miriam B Goodman, Zhiwen Liao

[International Worm Meeting, 2003]

The ion channel formed by MEC-4 and MEC-10 is proposed to mediate sensory mechanotransduction in C. elegans touch cells. Initial efforts to reconstitute this channel in heterologous cells have shown that MEC-4 and MEC-10 form an amiloride-sensitive, Na+-selective ion channel complex together with MEC-2 stomatin and MEC-6 paraoxonase (1, 2). We are using double mutant cycle analysis to map the interaction surface between the pore-forming subunits, MEC-4 and MEC-10. Our general approach is to co-express wild-type and mutant forms of MEC-4 and MEC-10 and identify interacting amino acid residues using the antagonist, amiloride, as a probe. Preliminary findings have already identified one such interaction site: the d position. The importance of the d position was first highlighted by gain-of-function mutations in genes encoding ASC proteins in C. elegans (3, 4) that cause neuronal degeneration in vivo. In both MEC-4 and MEC-10, the wild-type residue is an alanine. Thus far, we have analyzed mutations that cause neuronal degeneration in vivo (T, V, and D) and mutations that we predict will be similar to wild-type (S, C). Our initial results are consistent with the idea that d position forms part the interaction surface between MEC-4 and MEC-10. We speculate that residues at this position contribute to a gate that regulates access to the amiloride binding site in the ion channel pore. 1. M. B. Goodman et al., Nature 415, 1039-42. (2002); 2. D. S. Chelur et al., Nature 420, 669-673 (2002); 3. M. Driscoll, M. Chalfie, Nature 349, 588-593 (1991); 4. M. Chalfie, E. Wolinsky, Nature 345, 410-416 (1990).

Krieg, Michael et al. (2013) International Worm Meeting "A Pre-Stressed UNC-70 b-Spectrin Network Governs the Sense of Touch."

Krieg, Michael, Goodman, Miriam B, Dunn, Alexander R

[International Worm Meeting, 2013]

Many somatosensory neurons have evolved specialized molecular sensors that convert mechanical stress into behavioral responses. The genetics, development and physiology of the touch receptor neurons (TRNs) in Caenorhabditis elegans nematodes are especially well characterized and this animal has the particular advantage that the TRNs can be studied both in living animals and dissociated in culture. Like other somatosensory neurons, the TRNs use ion channels to convert mechanical stress into electrical signals and ultimately appropriate behaviors. Whereas the protein partners that form these mechanosensitive channels have been known for some time, the nature of the molecular machine important for efficient force transmission from skin to touch receptor neurite is essentially unknown. Here we show that sensation of mechanical forces depends on a continuous, pre-strained spectrin cytoskeleton inside neurons. We observed that mutations in the tetramerization domain of C. elegans b-spectrin (UNC-70), an actin-membrane crosslinker, lead to defective neuron morphologies under compressive stresses in moving animals. We performed AFM force spectroscopy experiments on isolated neurons, laser axotomy and FRET imaging to measure force across single cells and molecules. Our data indicate that spectrin is held under constitutive tension in living animals, which contributes to an elevated pre-stress in TRNs. Based on these results and data obtained from optogenetic and mechanical stimulation on b-spectrin mutants, we suggest that b-spectrin-dependent pre-tension is required for efficient responses to external mechanical stimuli.

Eastwood, Amy L. et al. (2011) International Worm Meeting "The functional importance of the MEC-4 transmembrane domain in force activation of the channel."

Goodman, Miriam B., Eastwood, Amy L.

[International Worm Meeting, 2011]

The C. elegans mechanoelectrical transduction channel MEC-4 is the most well-established force-gated ion channel to date. To gain insight into how it translates mechanical energy into ion flux, we incorporated single-copies of mutant MEC-4 genes into worms lacking the wild-type channel. We focused on two highly-conserved sequences in the channel: (1) the GxxxG motif in the pore-lining helix; and (2) the LxxxfG sequence in the extracellular domain, which may act as a gating hinge by coupling the extracellular domain to the transmembrane domain. To study the role of the GxxxG motif, each glycine was mutated to the slightly larger alanine. Behavioral assays characterizing the responsiveness of mutant worms to gentle touch indicated that even these subtle mutations were enough to disrupt the touch sensitivity of the worm. To study the role of the potential gating hinge, the aromatic tyrosine in the LxxxfG sequence, the asparagine at the top of the first transmembrane helix, and the glutamate at the top of the second transmembrane helix were ablated by mutating each to the much smaller alanine. In contrast to the GxxxG mutations, behavioral assays of these MEC-4 mutants suggested that these residues were not critical for MEC-4 channel function since all retained wild-type sensitivity. Together, these results indicate that force coupling through the transmembrane domain is crucial for the activation of the MEC-4 channel and the resulting aversive behavior to touch. We are working towards understanding how these mutations affect channel function using in vivo electrophysiology.

Lasse, Samuel et al. (2015) International Worm Meeting "Genetic Polymorphisms and the Relationship Between Ambient Temperature and Behavioral Performance in Caenorhabditis elegans."

Goodman, Miriam B., Lasse, Samuel, Andersen, Erik C., Hoelscher, Victoria, McPherson, Kevin P.

[International Worm Meeting, 2015]

Temperature and muscle function are highly correlated. For instance, marathon runners' winning times decrease significantly in a range of 10 to 15degC [1]. To learn more about how genes might influence the temperature dependence of behavioral performance, we turned to C. elegans egg laying as a model. This behavior has several features that make it ideal for the studies currently in progress. First, egg laying involves a simple motor circuit and well-defined set of muscles. Second, the output of this motor program is straightforward to measure. Third, we find that Caenorhabditis elegans, egg-laying rates increase gradually with heating until a maximum is achieved and then decline sharply, similar to observations reported for other behaviors in other ectotherms. Additionally, egg-laying rates vary between C. elegans strains isolated from different environments. For example, the maximal egg-laying rate of the Bristol (N2) hermaphrodites is about three-fold higher and occurs at a lower temperature than that of Hawaiian (CB4856) hermaphrodites (see Lasse, Koulechova & Goodman, IWM 2015).We are investigating the possibility that genetic polymorphisms account for differences in egg-laying rates by scoring a panel of recombinant inbred lines between N2 and CB4856. In brief, we measured egg-laying rates by placing three young adult (two-day old) hermaphrodites per well in a 96-well plate held at defined temperatures and recording rate as eggs/worm/hour during a two-hour assay time. This preliminary QTL analysis points to loci on chromosome IV that may account for variations in egg-laying rates based on measurements at a fixed temperature (25degC). In parallel, we investigated the possibility that ANOH-1 and ANOH-2, C. elegans orthologs of calcium-activated and temperature-sensitive chloride channels anoctamin-1 and anoctamin-2 found in humans [2], might contribute to the temperature sensitivity of egg-laying in N2 hermaphrodites. For these experiments, the egg-laying rate in anoh-1 and anoh-2 mutants was compared with that of wild-type (N2) hermaphrodites at temperatures between 5 C and 35degC. The resulting rate-temperature curves were indistinguishable, suggesting that neither anoh-1 nor anoh-2 plays a significant role in regulating the temperature-dependence of egg laying. References: [1] N Maughan, Scand J Med Sci Sports. 20 (2010), p. 95-102; [2] Y Wang et al., Am J Physiol Integr Comp Physiol. 11 (2013), p. R1376-R1389. .

Goodman MB et al. (1998) East Coast Worm Meeting "In situ patch-clamp recording from neurons in C. elegans"

Goodman MB, Chalfie M

[East Coast Worm Meeting, 1998]

We have extended recently described methods for in situ patch-clamp recording from C. elegans neurons (1, 2) to permit recording in adult animals. The main improvement is the use of a fixed-stage, upright microscope mounted on an x-y translation stage. In this way, worms can be visualized from above using a high N.A. water immersion objective (Zeiss 63X/0.9 or 100X/1.0). This arrangement gives superior optics compared to viewing worms on an inverted microscope and makes it possible to expose neurons in adult animals. In addition, methods for exposing neuronal cell bodies in the head were modified to expose neuronal cell bodies in the tail for in situ patch-clamp recording. With this apparatus, we plan to record the response of PLM cells to light touch. 1. Lockery, S. R. & Goodman, M. B. (1998) Tight-seal whole-cell patch clamping of C. elegans neurons. Methods in Enzymology (in press). 2. Goodman, M. B., Hall, D. H., Avery, L. & Lockery, S. R. (1998). Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron (in press).

Petzold, Bryan C. et al. (2011) International Worm Meeting "Influence of Body Mechanics on Force Thresholds for Touch Sensation in C. elegans."

Park, Sung-Jin, Pruitt, Beth L., Goodman, Miriam B., Petzold, Bryan C.

[International Worm Meeting, 2011]

We assessed the interplay between body mechanics and touch sensitivity by modulating muscle tone with Channelrhodopsin-2 and measuring force thresholds with a novel force-clamp metrology. Touch sensation is poorly understood despite the prevalence of disrupted touch and associated pain in pervasive diseases like diabetes. C. elegans is an ideal model for touch with its six touch receptor neurons (TRNs) and behavioral response to gentle touch. Force applied to the body results in stress/strain of nearby TRNs, triggering opening of force-gated ion channels, cellular depolarization, and an avoidance response for sufficiently large forces. Previously, we developed a behavioral force-clamp metrology capable of applying nN-mN forces to moving L4/young adult animals (Park et al, Rev Sci Instr, in press). Using this metrology, we showed that wild-type (N2) animals respond to forces ³ 100 nN, revealing unprecedented mechanical sensitivity. Previously, we showed that the three-layered outer shell (cuticle, hypodermis, and body wall muscle) plays a crucial role in filtering and transmitting applied forces (Park et al, PNAS 104:17376, 2007) and that body wall muscle tone regulates body mechanics (Petzold et al, Biophys J, in press). Now, we are testing the hypothesis that changes in body mechanics modulate touch sensitivity. To do this, we compare force-response curves in un-stimulated and hyper-contracted animals. Preliminary results show that larger forces are needed to evoke avoidance responses in hyper-contracted animals. We used light to manipulate body wall muscle contraction in transgenic animals expressing ChR2 under the control of a body wall muscle-specific promoter. This study provides a way to study the interplay between body mechanics and touch sensitivity in C. elegans and will further our understanding of the role of the outer shell in filtering and transmitting loads to the TRNs.

Nekimken, Adam L. et al. (2015) International Worm Meeting "The force of touch: How gentle is the classical gentle touch assay?"

Nekimken, Adam L., Goodman, Miriam B., Mazzochette, Eileen A., Pruitt, Beth L.

[International Worm Meeting, 2015]

The sense of touch is involved in almost all of our daily activities, yet we do not understand the workings of the proteins that transduce touch. A common method for measuring the touch sensitivity of C. elegans with altered proteins in the touch transduction pathway is to lightly touch them with a fine hair and observe the probability that a behavioral response occurs [1]. While eyebrow touch assays have yielded important results, including mapping the neural circuitry for touch [2], they are limited by its lack of quantitative measurement of the forces being applied. We are measuring the forces applied by volunteers with an eyebrow hair to establish context for studies that use this method. Volunteers touch a force-sensing cantilever with an eyebrow hair, modeling the hair swipe across a nematode. Our results indicate that such a gesture results in minimal horizontal forces relative to the vertical component. The standard deviation of vertical forces that were normalized for each volunteer was 0.475, indicating that 68% of the peak forces applied by a given volunteer are within 47.5% of that volunteer's average force. The average peak downward force across all volunteers was 68 muN. This value is two orders of magnitude higher than the threshold force for touch sensation: 50% probability of reversal with a 0.49 muN stimulus [3]. These results indicate that eyebrow touch assays saturate touch neurons and may not be able to distinguish mutants with less pronounced touch defects.[1] Chalfie M., et al. Assaying mechanosensation (July 31, 2014), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.172.1, http://www.wormbook.org.[2] Chalfie, M. et al., 1985. The neural circuit for touch sensitivity in Caenorhabditis elegans. The Journal of neuroscience : the official journal of the Society for Neuroscience, 5(4), pp. 956-64.[3] Petzold, B.C. et al., 2013. MEMS-based force-clamp analysis of the role of body stiffness in C. elegans touch sensation. Integrative biology : quantitative biosciences from nano to macro, 5, pp.853-64.

Casar, Jason et al. (2021) International Worm Meeting "Force sensitive upconverting nanoparticles as a direct, noninvasive assay for force generation by muscles"

Casar, Jason, Siefe, Chris, Dionne, Jennifer A., Lay, Alice, McLellan, Claire, Goodman, Miriam B.

[International Worm Meeting, 2021]

The digestive system relies heavily on muscles to breakdown, transport and expel material in order to provide the body the requisite nutrients to live. Additionally, activity of these muscles are key biomarkers of health status. Understanding the fundamental function of the digestive system and its dysfunction in aging and disease is hindered by a lack of suitable in vivo force sensors. Quantifying the forces generated in the alimentary lumen in vivo is currently not feasible through established mechanosensing platforms such as atomic force microscopy, traction force microscopy and Forster Resonant Energy Transfer (FRET)-based molecular strain sensors. To overcome that limitation, we are developing a microscale, biocompatible mechanosensing platform based on upconverting nanoparticles (UCNPs) that can rapidly and noninvasively readout micronewton scale forces within the intestinal and pharyngeal lumen of C. elegans. We synthesize lanthanide-doped ceramic nanoparticles (SrLuF:Yb,Er) embedded in polystyrene microspheres for efficient delivery. When excited in the near-infrared, these mechanosensing UCNPs have an emission spectrum that depends on pressure [PMID: 29927609; PMID: 31592655] and applied force. The ratiometric emission (red/green) increases by 25% with applied forces of 2 micronewtons as measured using dual confocal microscopy and atomic force microscopy. These particles are bright enough to image even the most rapid luminal pressure cycle in the worm (pharyngeal pumping, 5 Hz). We demonstrate the efficacy of using UCNPs to map luminal forces associated with feeding and digestion in wild-type adults. These measurements provide a time-resolved and direct indication of coordinated muscle function. We apply mechanosensing UCNPs concurrently with electrophysiological measurement of pharyngeal activity (ScreenChip), enabling assessment of the relationship between electrical activity and muscle function. We describe how these tools may be further developed into a high throughput platform for muscle function during aging and in C. elegans disease models, as well as how our UCNPs can be adapted to perform analogous measurements in other animals.

Krieg, Michael et al. (2015) International Worm Meeting "Shaping neurons by twist-tension coupling in chiral cytoskeleton networks."

Krieg, Michael, Shen, Kang, Cueva, Juan G., Spilker, Kerry, Goodman, Miriam B., Dunn, Alexander R.

[International Worm Meeting, 2015]

All animals and plants, even protozoa, have evolved specialized molecular sensors that convert mechanical stress into behavioral responses. The touch receptor neurons (TRNs) in Caenorhabditis elegans respond to gentle body touch and are especially well characterized on a physiological and ultrastructural level, a knowledge which is unavailable in other animals. Moreover, C. elegans is a unique model organism to study the mechanics of neurons due to their simple shapes, the known wiring diagram and a rich repertoire of simple behaviors, thus permitting a systems perspective on cell function.As in other animals, neuron morphology is critical for function in C. elegans. Some neurons are highly branched and curved, while others are extremely straight. We have previously shown that a functional, pre-stressed spectrin network is critical for mechanosensation and neuron stability under body-evoked forces (Krieg, Nat Cell Bio, 2014). How the constituent molecules of these different neurons establish a functional organization and how nanometer sized molecules can determine cell shape in the millimeter scale is still not understood. To establish this paradigm, we first compared different neurons and classified their shapes. We then used electron and STED microscopy to investigate how the organization of microtubule bundles and spectrin network determines neuron morphology. We found that TRNs with defective organization in both cytoskeletal elements undergo deformations highly similar to a twisted rod under compression. These experimental results, together with mechanical modeling of the neuron, suggest that spectrin tension and microtubule bundle mechanics are crucial for stabilizing chiral cytoskeletal networks and produce a specialized cell shape that we propose is critical for mechanosensation.