Dattananda Chelur et al. (2001) International C. elegans Meeting "MEC-6, a protein needed for degenerin channel activity, is expressed at the cell surface"

Dattananda Chelur, Marty Chalfie

[International C. elegans Meeting, 2001]

The response to gentle touch in Caenorhabditis elegans requires the activity of at least twelve touch function genes. Among them, mec-4 and mec-10 are likely to encode subunits of a mechanosensitive channel; they belong to the DEG/ENaC ion-channel superfamily, and dominant mutations in them cause degeneration of the touch cells. Since mec-4 and mec-10 -induced degenerations are completely suppressed by mutations in mec-6, wild type MEC-6 protein is essential for this degenerin channel activity. Also, mutations in mec-6 suppress the degenerations caused by deg-1 , unc-8 and partially suppress the hypercontraced phenotype of unc-105 dominant mutation. Thus, mec-6 is more generally required for degenerin channel function. We have cloned mec-6 gene and had earlier reported that it encodes a predicted protein of 377 amino acids with limited sequence similarity to the mammalian enzyme paraoxonase. mec-6 is widely expressed in many cell types including the touch receptor neurons consistent with its requirement for functioning of different degenerins. MEC-6 is a type II transmembrane protein with a single N-terminal transmembrane domain and a large extracellular C-terminal tail. MEC-6 is glycosylated when synthesized in vitro in presence of microsomes. We have now confirmed the predicted topology and subcellular localization of MEC-6 by expressing it in cultured cells. MEC-6 was tagged with HA epitope at the C-terminal end and was transiently transfected into CHO cells. Immunostaining of the non-permeablized cells with anti-HA antibodies revealed surface expression of MEC-6, which appears as discrete dots that are reminiscent of lipid rafts/caveolae. Punctate pattern of expression was also seen in C. elegans when the rescuing mec-6::gfp or the full-length mec-4::gfp constructs were injected. In case of mec-6::gfp , expression in neuronal processes is weak and hence only a few dots could be discerned, but the punctate expression was very clear in body wall muscle cells. Using the two color variants of GFP, we have shown that MEC-6 and MEC-4 are colocalized; coinjection of ectopically expressing mec-4::cfp fusion construct under the control of myo-3 promoter along with mec-6::yfp resulted in overlapping punctate expression in body wall muscle. Furthermore, coinjection of full-length mec-4::yfp with P mec-4 cfp into wild type worms resulted in uniform expression of CFP and punctate expression of YFP along the entire length of all the touch cell processes. However, when the two constructs were introduced into mec-6 mutants, the punctate expression of YFP was totally abolished whereas the CFP expression from mec-4 promoter was unaffected. Thus, MEC-6 affects the stability and/or localization of MEC-4 protein, but not the transcription of the mec-4 gene. Since u3 allele of mec-6 also partially suppresses the hypercontracted phenotype of dominant mutation in unc-105 (a muscle-specific degenerin), we are testing to see if MEC-6 colocalizes with UNC-105. We have expressed epitope tagged MEC-4 (with FLAG), MEC-6 (with HA) and MEC-10 (with MYC) in CHO cells to study the interactions among these proteins. Immunoprecipitation of MEC-6 pulls down both MEC-4 and MEC-10. Similarly, immunoprecipitation of MEC-10 coprecipitates both MEC-4 and MEC-6 thereby demonstrating that all the three proteins physically interact with one another. In addition, MEC-4 and MEC-10 form amiloride-sensitive Na + channels in Xenopus oocytes only when coexpressed with either MEC-6 or MEC-2, a stomatin-like protein (see the abstract by Goodman et al.). These results taken together suggest that MEC-6 either function as a channel subunit and/or is required for clustering of the channel complexes into discrete microdomains of the membrane.

Dattananda Chelur et al. (2005) International Worm Meeting "Targeted Genetic Ablation of C. elegans cells by Combinatorial Expression of Recombinant Caspase Subunits"

Dattananda Chelur, Marty Chalfie

[International Worm Meeting, 2005]

The basic function of many neurons in C. elegans has been elegantly demonstrated by laser ablation. Such laser ablations, however, are not suitable for genetic analysis as they can be performed only on small number of animals. Establishing C. elegans lines that are genetically ablated for specific cell(s) will be a powerful tool in identifying the function of redundant neurons, creating sensitized backgrounds for genetic screens or for identifying the role of specific neurons in the sensory behaviors that require population assays. Although many C. elegans genes have been characterized, cell-specific promoters are rare. Targeting of individual cells can be greatly enhanced by using appropriate combination of promoters that show overlapping expression in a specific cell or group of cells. We have developed a combinatorial method where coexpression, but not individual expression, of two subunits of a caspase enzyme kills cells. Caspases are a family of serine protease enzymes that are essential for programmed cell death. They are synthesized as inactive zymogens and are activated by proteolytic cleavage in the flexible region connecting small and large subunits of the procaspase polypeptide. We cloned separately both subunits of two different executioner caspases (CED-3 from C. elegans and Caspase-3 from humans) under the control of the touch cell-specific promoter Pmec-18. Expression of the individual subunits did not cause cell death. Integrated lines, however, containing interacting versions of both subunits showed apoptotic death of nearly 100% of the touch cells. This apoptotic death does not require the activities of the endogenous ced-3 or ced-4, which confirms that we have generated a constitutively active caspase enzyme. We are also able to induce cell death in cultured human HeLa cell line using recombinant Caspase-3 subunits. We will show some example of combination of promoter used for targeting death of specific cells in C. elegans. This approach can be used as a general method for inducible and/or targeted killing of specific cells from C. elegans to mammals.

Calixto, Andrea et al. (2009) International Worm Meeting "SID-1 expression in neurons enhances neuronal RNAi in C. elegans."

Irini, Topalidou, Martin, Chalfie, Calixto, Andrea, Dattananda, Chelur

[International Worm Meeting, 2009]

SID-1 is a multispan transmembrane protein required for systemic RNAi in C. elegans. The sid-1 gene is expressed in all but neuronal tissue, where systemic RNAi works poorly. We expressed SID-1 in all neurons using the pan-neural promoter from the unc-119 gene (Punc-119sid-1) and found that this protein increased the response of neurons to dsRNA delivered by feeding. This effect was further increased by mutations in the lin-15b and lin-35 genes. dsRNA that did not induce a phenotype in wild-type animals phenocopied known neuronal mutants in Punc-119sid-1 animals. Phenotypes were also discovered for several genes for which no previous information was known. A striking secondary effect produced by sid-1 expression in the neurons was a decrease in the response in non-neuronal cells to RNAi. This effect can be used to uncover neuronal defects for genes that, when eliminated in other tissues, produce lethality. For example, in wild-type animals RNAi for pat-4, which encodes an integrin-linked kinase, results in embryonic and larval lethality. In contrast Punc-119sid-1 animals are selectively touch insensitive, suggesting a previously unidentified role for pat-4 in mechanosensation .

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 .

Robert O'Hagan et al. (2004) West Coast Worm Meeting "DEG/ENaCs Form a Sensory Mechanotransduction Channel Complex in C. elegans Touch Receptor Neurons"

Robert O'Hagan, Martin Chalfie, Miriam B Goodman

[West Coast Worm Meeting, 2004]

Transformation of mechanical energy into ionic currents is essential for touch, hearing, and nociception, as well as for control of bodily homeostasis. Members of two ion channel superfamilies (DEG/ENaCs and TRPs) are believed to be sensory mechanotransduction channels; evidence for this, however, remains indirect. In C. elegans , behavioral responses to touch require a set of six touch receptor neurons and at least twelve genes. We have recently shown that four of these genes form an ion channel complex in heterologous cells (1,2). To determine if such channels are directly activated by external force, we recorded from C. elegans touch neurons in vivo . We found that external force evokes rapidly activating mechanoreceptor currents (MRCs) carried mostly by Na + and blocked by amiloride—characteristics consistent with direct mechanical gating of a DEG/ENaC channel. Like mammalian Pacinian corpuscles, C. elegans touch neurons depolarize with both positive and negative changes in external force but not sustained force. Null mutations in the DEG/ENaC gene mec-4 and genes encoding the accessory ion channel subunits MEC-2 and MEC-6 eliminate MRCs. Elimination of touch neuron-specific, large-diameter microtubules by a null mutation in the mec-7 beta-tubulin gene reduces, but does not abolish, MRCs. Our findings provide the first direct evidence linking application of external force to activation of a molecularly-defined metazoan sensory transduction channel. 1. Chelur, D. S., et al. (2002). Nature 420(6916): 669-73. 2. Goodman, M. B., et al. (2002). Nature 415(6875): 1039-42. Supported by an HHMI predoctoral fellowship to RO; an NIGMS grant to MC; and Baxter Foundation, Sloan Foundation, and Terman Faculty Scholarships to MBG.

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).

Austin L Brown et al. (2004) West Coast Worm Meeting "Accessories to Touch: The Roles of MEC-2 and MEC-6 Studied with Single Channel Recordings."

Miriam B Goodman, Austin L Brown

[West Coast Worm Meeting, 2004]

The sense of touch is still the most enigmatic of the ways organisms learn about their environment. C. elegans is an outstanding organism for the study of sensory mechanotransduction since the response to gentle touch is behaviorally simple and mediated by a set of 6 touch receptor neurons. Genetic analyses have led to the formulation of a molecular model for touch transduction. Central to this model is an ion channel formed by at least four proteins assembled in an unknown stoichiometry: two pore-forming subunits, MEC-4 and MEC-10, and two accessory proteins, MEC-2 and MEC-6. When co-expressed in Xenopus oocytes, MEC-2 and MEC-6 dramatically and synergistically increase current generated by MEC-4/10 ion channels 1,2 . This is consistent with the observation that mutations in any one of the genes encoding these four proteins ruin touch sensation. Moreover, mechanoreceptor currents, recorded in vivo, require intact copies of all four genes (see poster this meeting). Whole-cell recordings in oocytes are unable, however, to distinguish between possible mechanisms for the action of MEC-2 and MEC-6. Each protein must either enhance the single channel conductance, open probability, or some combination of both parameters. Single channel recordings, by contrast, are ideal to differentiate between mechanisms. So far, we have eliminated increases in single channel conductance as an explanation for the effects of MEC-2 and MEC 6 on macroscopic current. We are currently investigating how each accessory protein modulates open probability. 1. Chelur, D. S., et al. (2002). Nature 420(6916): 669-73. 2. Goodman, M. B., et al. (2002). Nature 415(6875): 1039-42. Supported by an AHA Grant-in-Aid, an Alfred P. Sloan and Terman Fellowships to MBG.

Shanehsaz, Bahareh et al. (2011) International Worm Meeting "RNAi screen to identify new genes and pathways regulating PKD-2 ciliary localization and function."

Shanehsaz, Bahareh, Wang, Juan, Barr, Maureen, Patrick, Cory

[International Worm Meeting, 2011]

Cilia are of profound medical importance in human health, yet how sensory cilia develop and signal remains poorly understood. We focus on a subset of male-specific ciliated sensory neurons, which express the C. elegans polycystins (PCs) LOV-1/PC-1 and PKD-2/PC-2 and are required for male mating behavior. In humans, mutations in PKD1 and PKD2, encoding PC-1 and PC-2 respectively, cause autosomal dominant polycystic kidney disease (ADPKD). The evolutionarily conserved polycystin genetic pathway, ciliary localization, and sensory function make the nematode an attractive model to study ciliary formation, morphogenesis, specialization, and signaling in the context of human genetic diseases of cilia. To identify molecules required for polycystin localization and/or function, we are performing a neuron-specific RNAi screen using a strain expressing the SID-1 dsRNA transporter under the control of the pkd-2 promoter, which is amenable to high-throughput and enables knockdown of otherwise essential genes (1). We performed a pilot RNAi screen of 70 candidate genes, identified from the literature based on interaction with PC-1 or PC-2. We observed a variety of PKD-2::GFP ciliary localization (Cil) defects, including abnormal accumulation of PKD-2 along the cilium proper, at the ciliary base, along the dendrite, and in the cell body. We are currently examining RNAi effects on male mating behavior. Our long-term goal is to complete a genome-wide RNAi screen once we work out the logistics of screening and validating candidates. This approach will provide a comprehensive picture of the molecules that influence polycystin channel assembly and localization, and will provide important insight to ciliary receptor trafficking in general. (1) Calixto A, Chelur D, Topalidou I, Chen X, Chalfie M. Enhanced neuronal RNAi in C. elegans using SID-1. Nat Methods. 2010 Jul;7(7):554-9.

Austin L Brown et al. (2007) International Worm Meeting "The Accessory Proteins MEC-2 and MEC-6 Promote an Active State of the MEC-4 Channel Complex."

Austin L Brown, Miriam B Goodman

[International Worm Meeting, 2007]

The stomatin-related protein MEC-2 and the paraoxonase-related protein MEC-6 are required for mechanical activation of native force detection channels in C. elegans touch receptor neurons [1]. Prior work indicates that such channels contain at least two pore-forming degenerin subunits, MEC-4 and MEC-10. Both MEC-4 and MEC-10 belong to the DEG/ENaC superfamily of amiloride-sensitive Na+ channel subunits. Wild-type copies of all four proteins are needed for touch sensation in vivo. Co-expressing MEC-2 and MEC-6 with constitutively-active isoforms of MEC-4 and MEC-10 in Xenopus oocytes leads to a dramatic and synergistic increase in amiloride-sensitive whole-cell Na+ current. This effect is not explained by a MEC-2- or MEC-6-dependent increase in the amount of MEC-4 or MEC-10 protein in the plasma membrane [2, 3], suggesting that MEC-2 and MEC-6 alter the activity of individual channels in the plasma membrane. To test this, we analyzed single, amiloride-sensitive Na+ channels in membrane patches drawn from oocytes expressing MEC-4 and MEC-10 in the presence or absence of MEC-2, MEC-6 or both MEC-2 and MEC-6. We found that while MEC-2 coexpression leads to a modest, but significant increase in single channel conductance, <font face=symbol>g</font>, the effect is too small to explain the MEC-2-dependent increase in whole-cell current. Otherwise, <font face=symbol>g</font> and steady-state open probability (P_o) are not significantly altered by the presence or absence of MEC-2 and MEC-6. We conclude that, in the absence of MEC-2 and MEC-6, the vast majority of channels occupy a non-conducting state despite correct expression at the plasma membrane. 1.O''Hagan, R., M. Chalfie, and M.B. Goodman, Nat Neurosci, 2005. 8(1):43 2.Chelur, D.S., et al., Nature, 2002. 420(6916):669 3.Goodman, M.B., et al., Nature, 2002. 415(6875):1039.

Kalogeropoulou, E. et al. (2017) International Worm Meeting "Caenorhabditis elegans exhibits an increase in the probability of pirouettes and head swing amplitude in the absence of the SAA class of neurons."

Hope, I.A., Cohen, N., Kalogeropoulou, E.

[International Worm Meeting, 2017]

Many of the behaviours of C. elegans are mediated by the worm's ability to orient itself towards chemical gradients or other sensory cues. Two mechanisms of orientation have been observed in the worm: pirouettes and steering. Past studies have identified a number of neuron classes that contribute to navigation by linking neuronal laser ablations and observations of mutants to changes in body postures and locomotion statistics1,2. Recently, optogenetic manipulation has also shed light on the neuronal control of the worm's chemotactic behaviour3. SMB motor neurons have been postulated to be part of the navigation circuit. We have generated a chromosomally-integrated transgenic line (UL4230) where the SMBs are genetically ablated in early larvae. The line demonstrates a loopy phenotype similar to that previously observed following laser-ablation of SMBs1. We have also targeted another class of neuron, located close to and highly connected via synapses to the SMBs but not previously explored: the SAA interneurons. We initially used expression of GFP to confirm that the selected promoters drove expression in the location originally described. A combination of lad-2 and unc-42 promoters was used to genetically ablate the SAAs by targeted expression of the worm's caspase, as encoded by two distinct parts of the ced-3 gene, such that the intact enzyme is produced only in these neurons specifically4. Absence of GFP expression confirmed the specific and targeted ablation of the SAAs. The generated strains, with SMBs or SAAs ablated (UL4230 and UL4207), demonstrated a phenotype different to that of N2 in both spontaneous and evoked locomotion. Locomotion was evoked using a radial gradient of NH4Cl. Initial results suggest that both SMBs and SAAs suppress the probability of pirouettes and decrease the amplitude of sinusoidal undulations. Additional experiments are underway to explore the contributions of SAA and SMB, separately and together, to undulations and steering, and to link those to the pirouette initiation pathway. 1. Gray J. M., et al., (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A, 102(9). 2. Iino Y., and Yoshida K. (2009). Parallel use of two behavioral mechanisms for chemotaxis in Caenorhabditis elegans. Journal of Neuroscience, 29(17). 3. Kocabas A., et al., (2012). Controlling interneuron activity in Caenorhabditis elegans to evoke chemotactic behaviour. Nature, 490(7419). 4. Chelur D. S., and Chalfie M. (2007). Targeted cell killing by reconstituted caspases. Proc. Natl. Acad. Sci., 104(7).