Goodman MB (2013) Curr Biol "Miriam B. Goodman."

Goodman MB

[Curr Biol, 2013]

Miriam B. Goodman (2006) WormBook "Mechanosensation."

Miriam B. Goodman

[WormBook, 2006]

Wild C. elegans and other nematodes live in dirt and eat bacteria, relying on mechanoreceptor neurons (MRNs) to detect collisions with soil particles and other animals as well as forces generated by their own movement. MRNs may also help animals detect bacterial food sources. Hermaphrodites and males have 22 putative MRNs; males have an additional 46 MRNs, most, if not all of which are needed for mating. This chapter reviews key aspects of C. elegans mechanosensation, including MRN anatomy, what is known about their contributions to behavior as well as the neural circuits linking MRNs to movement. Emerging models of the mechanisms used to convert mechanical energy into electrical signals are also discussed. Prospects for future research include expanding our understanding of the molecular basis of mechanotransduction and how activation of MRNs guides and modulates behavior.

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.

Geffeney, Shana L et al. Worm Breeder's Gazette "Worms get a brand new glue"

Geffeney, Shana L, Goodman, Miriam B.

[Worm Breeder's Gazette]

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.