Robert O'Hagan

The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals

Transformation of mechanical energy into ionic currents is essential for touch, hearing and nociception. Although DEG/ENaC proteins are believed to form sensory mechanotransduction channels, the evidence for this role remains indirect. By recording from C. elegans touch receptor 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, these neurons depolarized with both positive and negative changes in external force but not with sustained force. Null mutations in the DEG/ENaC gene mec-4 and in the accessory ion channel subunit genes mec-2 and mec-6 eliminated MRCs. In contrast, the genetic elimination of touch neuron–specific microtubules reduced, but did not abolish, MRCs. Our findings link the application of external force to the activation of a molecularly defined metazoan sensory transduction channel.

MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation.

Touch sensitivity in animals relies on nerve endings in the skin that convert mechanical force into electrical signals. In the nematode Caenorhabditis elegans, gentle touch to the body wall is sensed by six mechanosensory neurons that express two amiloride-sensitive Na+ channel proteins (DEG/ENaC). These proteins, MEC-4 and MEC-10, are required for touch sensation and can mutate to cause neuronal degeneration. Here we show that these mutant or ‘d’ forms of MEC-4 and MEC-10 produce a constitutively active, amiloride-sensitive ionic current when co-expressed in Xenopus oocytes, but not on their own. MEC-2, a stomatin-related protein needed for touch sensitivity, increased the activity of mutant channels about 40-fold and allowed currents to be detected with wild-type MEC-4 and MEC-10. Whereas neither the central, stomatin-like domain of MEC-2 nor human stomatin retained the activity of full-length MEC-2, both produced amiloride-sensitive currents with MEC-4d. Our findings indicate that MEC-2 regulates MEC-4/MEC-10 ion channels and raise the possibility that similar ion channels may be formed by stomatin-like proteins and DEG/ENaC proteins that are co-expressed in both vertebrates and invertebrates. Some of these channels may mediate mechanosensory responses.

The Tubulin Deglutamylase CCPP-1 Regulates the Function and Stability of Sensory Cilia in C. elegans

BACKGROUND Posttranslational modifications (PTMs) such as acetylation, detyrosination, and polyglutamylation have long been considered markers of stable microtubules and have recently been proposed to guide molecular motors to specific subcellular destinations. Microtubules can be deglutamylated by the cytosolic carboxypeptidase CCP1. Loss of CCP1 in mice causes cerebellar Purkinje cell degeneration. Cilia, which are conserved organelles that play important diverse roles in animal development and sensation, contain axonemes comprising microtubules that are especially prone to PTMs. RESULTS Here, we report that a CCP1 homolog, CCPP-1, regulates the ciliary localization of the kinesin-3 KLP-6 and the polycystin PKD-2 in male-specific sensory neurons in C. elegans. In male-specific CEM (cephalic sensilla, male) cilia, ccpp-1 also controls the velocity of the kinesin-2 OSM-3/KIF17 without affecting the transport of kinesin-II cargo. In the core ciliated nervous system of both males and hermaphrodites, loss of ccpp-1 causes progressive defects in amphid and phasmid sensory cilia, suggesting that CCPP-1 activity is required for ciliary maintenance but not ciliogenesis. Affected cilia exhibit defective B-tubules. Loss of TTLL-4, a polyglutamylating enzyme of the tubulin tyrosine ligase-like family, suppresses progressive ciliary defects in ccpp-1 mutants. CONCLUSIONS Our studies suggest that CCPP-1 acts as a tubulin deglutamylase that regulates the localization and velocity of kinesin motors and the structural integrity of microtubules in sensory cilia of a multicellular, living animal. We propose that the neuronal degeneration caused by loss of CCP1 in mammals may represent a novel ciliopathy in which cilia are formed but not maintained, depriving the cell of cilia-based signal transduction.

The Multipurpose 15-protofilament Microtubules in C. elegans Have Specific Roles in Mechanosensation

Because microtubules perform many essential functions in neurons, delineating unique roles attributable to these organelles presents a formidable challenge. Microtubules endow neurons with shape and structure and are required for developmental processes including neurite outgrowth, intracellular transport, and synapse formation and plasticity; microtubules in sensory neurons may be required for the above processes in addition to a specific sensory function. In Caenorhabditis elegans, six touch receptor neurons (TRNs) sense gentle touch and uniquely contain 15-protofilament microtubules. Disruption of these microtubules by loss of either the MEC-7 beta-tubulin or MEC-12 alpha-tubulin or by growth in 1 mM colchicine causes touch insensitivity, altered distribution of the touch transduction channel, and a general reduction in protein levels. We show that the effect on touch sensitivity can be separated from the others; microtubule depolymerization in mature TRNs causes touch insensitivity but does not result in protein distribution and production defects. In addition, the mec-12(e1605) mutation selectively causes touch insensitivity without affecting microtubule formation and other cellular processes. Touching e1605 animals produces a reduced mechanoreceptor current that inactivates more rapidly than in wild-type, suggesting a specific role of the microtubules in mechanotransduction.

The DEG/ENaC Protein MEC-10 Regulates the Transduction Channel Complex in Caenorhabditis elegans Touch Receptor Neurons

Gentle touch sensation in Caenorhabditis elegans is mediated by the MEC-4/MEC-10 channel complex, which is expressed exclusively in six touch receptor neurons (TRNs). The complex contains two pore-forming subunits, MEC-4 and MEC-10, as well as the accessory subunits MEC-2, MEC-6, and UNC-24. MEC-4 is essential for channel function, but beyond its role as a pore-forming subunit, the functional contribution of MEC-10 to the channel complex and to touch sensation is unclear. We addressed this question using behavioral assays, in vivo electrophysiological recordings from TRNs, and heterologous expression of mutant MEC-10 isoforms. Animals with a deletion in mec-10 showed only a partial loss of touch sensitivity and a modest decrease in the size of the mechanoreceptor current (MRC). In contrast, five previously identified mec-10 alleles acted as recessive gain-of-function alleles that resulted in complete touch insensitivity. Each of these alleles produced a substantial decrease in MRC size and a shift in the reversal potential in vivo. The latter finding indicates that these mec-10 mutations alter the ionic selectivity of the transduction channel in vivo. All mec-10 mutant animals had properly localized channel complexes, indicating that the loss of MRCs was not attributable to a dramatic mislocalization of transduction channels. Finally, electrophysiological examination of heterologously expressed complexes suggests that mutant MEC-10 proteins may affect channel current via MEC-2.

The tubulin repertoire of C. elegans sensory neurons and its context‐dependent role in process outgrowth

Microtubules contribute to key cellular processes and are composed of αβ-tubulin heterodimers. Neurons in Caenorhabditis elegans express cell type–specific isoforms in addition to a shared repertoire and rely on tubulins for neurite outgrowth. Isoform function varies between in vivo and in vitro contexts.

Regulation of tubulin glutamylation plays cell-specific roles in the function and stability of sensory cilia

Microtubules (MTs) are post-translationally modified, but the functions of post-translational modifications (PTMs) have in many cases remained unknown. Most PTMs, such as polyglutamylation, occur on the protruding C-terminal tail (CTT) of tubulins, are reversible, and have been proposed to play a role in regulation of MT-associated proteins (MAPs), molecular motors, and MT-severing proteins. Several PTM enzymes have been identified, including a carboxypeptidase in mice known as CCP1, which reduces polyglutamylation on the CTT of MTs, and causes cell-specific neurodegeneration when mutated.

corrigendum: MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation

This corrects the article DOI: 4151039a