InteractiveFly: GeneBrief

no mechanoreceptor potential C: Biological Overview | References

BIOLOGICAL OVERVIEW

The generation of coordinated body movements relies on sensory feedback from mechanosensitive proprioceptors. The proper function of NompC, a putative mechanosensitive TRP channel, is not only required for fly locomotion, but also crucial for larval crawling. Calcium imaging revealed that NompC is required for the activation of two subtypes of sensory neurons during peristaltic muscle contractions. Having isolated a full-length nompC cDNA with a protein coding sequence larger than previously predicted, nompC function was demonstrated by rescuing locomotion defects in nompC mutants. Antibodies against the novel C-terminus recognize NompC in chordotonal ciliary tips. Moreover, it was shown that the ankyrin repeats in NompC are required for proper localization and function of NompC in vivo and are required for association of NompC with microtubules. Taken together, these findings suggest that NompC mediates proprioception in locomotion and support its role as a mechanosensitive channel (Cheng, 2010).

Mechanosensation is a sensory modality of importance to both prokaryotes and eukaryotes. Most unicellular organisms are capable of detecting membrane tension and distortion caused by mechanical stimuli. In higher organisms, specialized mechanosensitive cells and organs mediate the detection of touch, nociception, hearing, and proprioception. Despite the importance of these modalities, in many instances, especially in the case of proprioception, the identity of the mechanosensitive cells and the molecules required for mechanosensation in these cells are largely unknown (Cheng, 2010).

Proprioception refers to the sensory input and feedback by which animals keep track of and control the different parts of their body for balance and for locomotion. In humans, selective loss of proprioception results in a 'rag doll' state -- a failure to make any coordinated body movement. Proprioception is likely mediated by mechanosensitive stretch receptors located within the muscles, joints and ligaments. Ion channels and neurons important for proprioception have been identified in genetic studies of organisms with stereotypical patterns of locomotion. In C. elegans, mutations in trp-4, which encodes a transient receptor potential (TRP) channel, and unc-8, which encodes an epithelial sodium channel (ENaC), lead to abnormal body movement, suggesting that TRP-4 and UNC-8 mediate proprioception in C. elegans. These studies also identified neurons that contribute to the regulation of proprioception. Two TRP-4-expressing neurons are located in the body wall with extended axons that span nearly the whole length of the body and may function as proprioceptor neurons (Li, 2006). Several UNC-8-expressing sensory neurons, interneurons, and motor neurons may also contribute to proprioception in C. elegans (Cheng, 2010).

The Drosophila larval peripheral nervous system (PNS) provides a model for systematic analysis of the physiological function of morphologically distinct sensory neurons. The Drosophila PNS is composed of segmentally repeated sensory neurons which are classified as either type I or type II neurons. Type I neurons, which have ciliated monopolar dendrites, are located in external sensory organs and chordotonal organs. The primary function of type I neurons is mechanosensation. Type II neurons, also known as multi-dendritic (MD) neurons, are further divided into tracheal dendrite (td) neurons, bipolar dendrite (bd) neurons, and dendritic arborization (da) neurons. Each subtype of MD neuron has characteristic dendrite arborization and axonal targeting patterns, suggesting that different subtypes of MD neurons may be functionally distinct. Previous studies have shown that silencing all MD neurons results in a cessation of larval locomotion, demonstrating that the function of MD neurons is critical for larval locomotion (Song, 2007). Further, simultaneously silencing two specific subtypes of MD neurons, bd and class I da neurons, disrupts larval crawling ability (Hughes, 2007), suggesting that bd and class I da neurons play an essential role in larval locomotion and could function as proprioceptor neurons. However, the molecules required for proprioception in these neurons have not been identified (Cheng, 2010).

The Drosophila TRP channel TRPN1/NompC is a putative mechanosensitive channel that affects fly locomotion. Loss-of-function mutations of nompC abolish mechanoreceptor potentials in fly bristles and a missense mutation of nompC alters adaptation of mechanoreceptor potentials (Walker, 2000). NompC is also required for hearing in Drosophila (Gopfert, 2006; Kamikouchi, 2009; Sun, 2009). In addition, adult nompC mutant flies are severely uncoordinated (Kernan, 1994; Walker, 2000). To substantiate the physiological role of NompC in locomotion, it is important to identify the neurons that require NompC for locomotion, to characterize the subcellular localization of NompC, and to study how NompC function is regulated in vivo (Cheng, 2010).

This study reports the molecular characterization of NompC that establishes its functional role in Drosophila locomotion. NompC is expressed in the dendrites of bd/class I da neurons and is required for activating bd/class I da neurons during larval peristaltic muscle contractions. Proper function of NompC controls the pace of larval crawling. In addition, the ankyrin repeats in NompC are required not only for microtubule association, but also for the proper localization and function of NompC in vivo, suggesting that the ankyrin repeats directly or indirectly connect NompC to the cytoskeleton to enable mechanosensitivity. Taken together, these findings reveal a critical role of NompC in locomotion and support its function as a mechanosensitive channel (Cheng, 2010).

This study reports the identification and characterization of NompC as a critical regulator of Drosophila locomotion. Proper NompC function in bd and class I da neurons is crucial for larval peristaltic crawling behavior; the locomotion speed is greatly reduced in both null-mutants and mutants carrying a missense mutation. Importantly, NompC is required for activating bd and class I da neuron central projections during muscle contractions. Moreover, NompC protein is localized to chordotonal ciliary tips and along sensory neuron dendrites, the potential sites of mechanosensation. Finally, the ankyrin repeats in NompC are required for its association with microtubules and for the proper localization and function of NompC in vivo (Cheng, 2010).

Previous studies have shown that two types of sensory neurons, namely the bd and class I da neurons, are required for Drosophila larval locomotion. Indeed, calcium imaging revealed that bd and class I da neurons are activated during peristaltic muscle contractions, suggesting that they may function as proprioceptor neurons during locomotion. Structurally, bd and class I da neurons are suitable to function as proprioceptor neurons. The bd neuron is found in many different insects. It is situated between muscles in the dorsal body wall. The two dendrites of a bd neuron span the length of the segment and reach the segmental folds on either end, where the dendritic tips are attached to the epidermal cells (Schrader, 2007). Class I da neurons have a relatively simple dendrite branching pattern. Two dorsal class I da neurons project their fan-shaped dendrites anteriorly and posteriorly, respectively (Grueber, 2002). Similar to other da neurons, class I da neurons form a subepidermal plexus sandwiched between the epidermis and muscles. The dendrite morphology of bd and class I da neurons, their location in the segment, as well as their positions relative to muscles and the epidermis suggest that bd and class I da neurons could sense the stretch in muscles and/or epidermis during larval locomotion movement, thus function as proprioceptors. Consistent with the requirement of the function of bd and class I da neurons in locomotion, their axons project to the same dorsal regions of the neuropil (Grueber, 2007), indicating that signals from bd and class I da neurons might be processed in the same circuitry for the feedback regulation of motor neuron activity (Cheng, 2010).

The TRP family of cation channels plays an important role in the detection of various sensory stimuli. This study found that NompC is required for activating bd and class I da neurons during muscle contractions and controls the pace of larval crawling. NompC is also involved in mechanosensation in bristles (Walker, 2000) and hearing in fly (Gopfert, 2006; Kim, 2003). How does a single channel contribute to multiple mechanosensation modalities in different neurons? While NompC is presumably gated by mechanical forces, the specificity in the sensory modality could arise from different dendrite morphology of the neurons in which NompC is expressed. For example, the ciliary structure in hair cells makes NompC responsive to sound stimuli, whereas the two longitudinal dendrites of bd neurons may allow NompC to be tuned to body stretch or bending. Another possibility is that NompC interacts with different partners to achieve the specificity in different neurons (Cheng, 2010).

A critical question in the study of mechanosensation is to understand how the ion channels are gated mechanically. Ankyrin repeats are a common feature in many TRP channels, yet the function of ankyrin repeats remains unclear (Gaudet, 2008). Previous studies implicate NompC as a mechanosensitive channel because nompC null mutants lack the mechanoreceptor potential, whereas a point mutation of nompC alters adaptation of mechanoreceptor potential (Walker, 2000). One question raised by Walker concerns the ability of the ankyrin repeats of NompC to mediate interactions with the cytoskeleton. This study shows that the ankyrin repeats of NompC are essential for NompC-microtubule interaction in transfected cells and for proper targeting of NompC in vivo to the distal ciliary tips, which are wrapped by NompA-enriched dendritic caps. It is noteworthy that two other TRP channels that mediate hearing in Drosophila, Nan and Iav, are localized to the proximal cilium and are absent from the ciliary tips that are in contact with the dendritic caps (Gong, 2004). Therefore, if Nan and Iav are mechanically gated, the forces that gate the channels would have to be transmitted down the axoneme through the ciliary membrane or via other extracellular material. Localization of NompC at distal ciliary tips and its interaction with microtubules raise the possibility that NompC may be anchored to both the cytoskeleton and to the extracelluar matrix of the dendritic caps at the ciliary tip. With mechanical stimulation, the relative displacement between extracelluar matrix and the cytoskeleton might 'pull' the channel open as suggested by the prevailing model for direct mechanogating (Albert, 2007; Ernstrom, 2002; Sukharev, 2004). Although it remains to be tested whether NompC can be directly activated by mechanical forces, studies using atomic force microscopy have demonstrated the elastic properties of ankyrin repeats in response to pulling on both ends of the domain (Lee, 2006). In larval bd neurons, NompC-L-GFP is distributed throughout the two longitudinal dendrites, which are wrapped by glial processes (Schrader, 2007). It is speculated that the shearing forces between the dendrites and the glial wrapping could provide the relevant stimulation to activate the NompC channel in bd neurons, thus leading to Ca²⁺ signals in their central projections in the central nervous system (Cheng, 2010).

NompC TRP channel is essential for Drosophila sound receptor function

The idea that the NompC TRPN1 channel is the Drosophila transducer for hearing has been challenged by remnant sound-evoked nerve potentials in nompC nulls. This study reports that NompC is essential for the function of Drosophila sound receptors and that the remnant nerve potentials of nompC mutants are contributed by gravity/wind receptor cells. Ablating the sound receptors reduces the amplitude and sensitivity of sound-evoked nerve responses, and the same effects ensued from mutations in nompC. Ablating the sound receptors also suffices to abolish mechanical amplification, which arises from active receptor motility, is linked to transduction, and also requires NompC. Calcium imaging shows that the remnant nerve potentials in nompC mutants are associated with the activity of gravity/wind receptors and that the sound receptors of the mutants fail to respond to sound. Hence, Drosophila sound receptors require NompC for mechanical signal detection and amplification, demonstrating the importance of this transient receptor potential channel for hearing and reviving the idea that the fly's auditory transducer might be NompC (Effertz, 2011).

Ever since NompC (also known as TRPN1) was implicated in Drosophila touch sensation, it has been speculated that this transient receptor potential (TRP) channel could be one of the elusive transduction channels for hearing. Bearing a predicted pore region and an N-terminal ankyrin spring, NompC seems structurally qualified for being a gating spring-operated ion channel as implicated in auditory transduction. Though displaying a rather spotty phylogenetic appearance, NompC is required for the function of certain Drosophila and nematode mechanoreceptors and zebrafish hair cells. NompC is also expressed in hair cells of frogs and in mechanoreceptors of the Drosophila ear, but even though NompC demonstrably can serve as mechanotransduction channel, its importance for auditory transduction and hearing remains uncertain: in frog hair cells, NompC localizes to kinocilia that are dispensable for transduction. And in the Drosophila ear, loss of nompC function reduces the amplitude of sound-evoked afferent nerve responses by only approximately one-half (Effertz, 2011).

A possible explanation for the mild latter effect has emerged with the recent discovery that the antennal hearing organ of Drosophila, Johnston's organ (JO), houses sound and gravity/wind receptors: about half of the fly's approximately 480 JO receptor cells preferentially respond to dynamic antennal vibrations and serve sound detection, whereas the other half preferentially respond to static antennal deflections and mediate the detection of gravity and wind. Driving reporter genes via a nompC-Gal4 promoter fusion construct only labeled the sound receptors, suggesting that the sound-evoked nerve potentials that persist in nompC mutants may be contributed by nompC-independent JO gravity/wind receptor cells. nompC-Gal4, however, reproduces endogenous nompC expression only partially, and an antibody detected NompC protein in virtually all receptors of JO. To explore whether the two JO receptor types nonetheless differ in their nompC dependence, JO function was analyzed in nompC mutants and in flies with ablated sound or gravity/wind receptor cells (Effertz, 2011).

To selectively ablate JO sound or gravity/wind receptors, UAS-ricin toxin A was expressed in these cells using receptor type-specific GAL4 drivers in conjunction with the ey-FLP/FRT system to restrict toxin expression to GAL4-expressing cells in the antenna and eye. To assess JO function, the flies were exposed to pure tones of different intensities and the resulting mechanical input and electrical output of JO were simultaneously monitored. The mechanical input was measured as sound-induced displacement of the antenna's arista, whereas the electrical output was recorded in the form of sound-evoked compound action potentials (CAPs) from the receptor axons in the antennal nerve. The frequency of the tones was adjusted to the mechanical best frequency of the antenna, which was deduced from the power spectrum of the antenna's free fluctuations. The intensity of the tones was measured as the sound particle velocity at the position of the fly (Effertz, 2011).

In accord with previous observations, it was found that remnant sound-evoked nerve potentials persist in nompC nulls: varying the sound particle velocity between approximately 0.001 and 10 mm/s evoked CAPs in nompC² and nompC³ null mutants whose maximum amplitudes were ~6 times smaller than those of the wild-type and controls. Mutant flies carrying the weaker allele nompC⁴ displayed equally reduced CAP amplitudes, but the amplitudes were normal when a UAS-nompC-L rescue construct was expressed in all JO receptors of nompC³ nulls. Reduced CAP amplitudes as observed in nompC mutants also ensued from the targeted ablation of JO sound receptors. When JO gravity/wind receptors were ablated, however, CAP amplitudes remained normal, resembling those of wild-type flies and controls. Hence, sound-evoked potentials in the fly's antennal nerve are not only contributed by JO sound receptors: if these receptors are ablated, residual CAPs persist whose amplitudes resemble those of nompC nulls (Effertz, 2011).

Mutations in nompC, in addition to reducing sound-evoked nerve potentials, impair sensitive hearing. This reduction in auditory sensitivity became apparent when the relative CAP amplitudes were plotted against the corresponding sound-induced antennal displacement. In wild-type and control flies, antennal displacements equal to or greater than ~50 nm were sufficient to elicit CAPs, and the CAP amplitude increased monotonously for displacements between approximately 50 and 600 nm. In nompC mutants, this dynamic range of the CAP response consistently shifted up to antennal displacements between approximately 160 and 2000nm, corresponding to an ~3-fold sensitivity drop. This sensitivity drop, which was rescued by expressing UAS-nompC-L in the JO receptors of nompC³ mutants, was also observed in flies with ablated JO sound receptor cells. When the gravity/wind receptors were ablated, however, auditory sensitivity remained unchanged (Effertz, 2011).

When the relative CAP amplitudes were plotted against the sound particle velocity instead of the antennal displacement, the sensitivity drop observed in nompC mutants and flies with ablated sound receptors was even more pronounced. Accordingly, loss of nompC function and loss of sound receptor function reduce both the sensitivity of JO to antennal displacements and, in addition, the mechanical sensitivity of the antenna to sound (Effertz, 2011).

To assess the mechanical sensitivity of the antenna, its displacement varies with sound intensity was determined. In wild-type and control flies, the antenna's displacement nonlinearly increased with sound particle velocity, displaying a compressive nonlinearity that, arising from mechanical activity of JO receptors, enhanced the mechanical sensitivity ~8-fold when sound was faint. Consistent with previous observations, it was found that this nonlinear mechanical amplification was lost in nompC mutants, rendering their antennae mechanically less sensitive to acoustic stimuli so that louder sounds were required to displace their antennae by a given distance, in addition to the larger antennal displacements that were required to elicit CAPs in their antennal nerves. It was also found that this nonlinear amplification could be rescued by expressing UAS-nompC-L in JO receptors and that it specifically required JO sound receptor cells: ablating only the sound receptors abolished mechanical amplification, and the same effect was caused by mutations in nompC. In nompC mutants, this loss of amplification was associated with alterations of the antenna's tuning and fluctuation power that were quantitatively mimicked in flies with ablated sound receptor cells. If the gravity/wind receptors were ablated, however, mechanical amplification remained normal, with the antenna's compressive nonlinearity, its tuning, and its fluctuation power resembling those of wild-type, nompC-L rescue, and control flies. Hence, nonlinear mechanical amplification in the Drosophila ear requires both the NompC channel and JO sound receptors but is independent of JO gravity/wind receptor cells (Effertz, 2011).

Ablating JO sound receptors phenocopies the auditory defects of nompC mutants, suggesting that NompC is essential for the mechanosensory function of these cells. To test this hypothesis, mechanically evoked calcium signals were monitored in the somata of JO receptors of nompC³ null mutants and controls while simultaneously recording the displacement of the antenna and the ensuing CAPs from the antennal nerve. Calcium signals were measured through the cuticle of the antenna using the genetically encoded ratiometric calcium sensor Cameleon2.1 (Cam2.1). To evoke calcium signals, the antenna was sinusoidally actuated at its mechanical best frequency with electrostatic force (Effertz, 2011).

When Cam2.1 was expressed in either the sound receptors alone or all JO receptors, antennal vibrations evoked robust calcium signals in controls. The calcium signals of the sound receptors were entirely abolished in nompC³ mutants, but when Cam2.1 was expressed in all of their JO receptors, small calcium signals were detected that closely resembled those of the gravity/wind receptors of controls. To assess the relation between JO calcium signals and antennal nerve potentials, their respective amplitudes were plotted against the antennal displacement. The large calcium signals of the sound receptors of controls superimposed with the relative amplitudes of the simultaneously recorded CAPs and the CAPs of flies with ablated gravity/wind receptor cells. The small calcium signals of the gravity/wind receptors were shifted to larger antennal displacements and superimposed with the CAPs of flies with ablated sound receptor cells. Calcium signals obtained from all JO receptors of controls had intermediate amplitudes, identifying them as mixed signals contributed by sound and gravity/wind receptor cells. The residual CAPs of nompC³ mutants did not associate with calcium signals in their sound receptors, yet they superimposed with the small calcium signals obtained from all JO receptors of the mutants and from JO gravity/wind receptors of the controls. Although unsuccessful recombination prevented selectively expressing Cam2.1 in the gravity/wind receptors of the mutants, the above findings show that calcium signals that can be ascribed to these receptors are associated with the residual CAPs in nompC nulls. Additional evidence that the calcium signals in the mutants arise from gravity/wind receptors was obtained when the time course of these signals was inspected: in controls, the onset of the calcium signals of all JO receptors followed two exponentials. The exponential with the larger time constant well fitted the calcium signals of their sound receptors. The exponential with the smaller time constant well fitted the calcium signals of their gravity/wind receptors and also those of nompC³ nulls. Hence, instead of being contributed by JO sound receptors, the residual CAPs of nompC mutants are deemed to reflect the activity of JO gravity/wind receptor cells (Effertz, 2011).

Judged from the intracellular calcium signals, the responses of JO gravity/wind receptors to sinusoidal forcing are independent of NompC. Because these receptors preferentially respond to static forcing, the flies' antennae were statically deflected, and the ensuing calcium signals were measured. In accord with previous observations, JO sound receptors hardly responded to antennal deflections, and the calcium signals obtained from all of the JO receptors of nompC³ mutants were indistinguishable from those of controls. Hence, whereas NompC is essential for the mechanosensory function of JO sound receptors, the mechanosensory function of JO gravity/wind receptors seems independent of NompC. Because NompC is detectable in the dendritic tips of virtually all JO receptors other proteins may compensate for the loss of NompC in JO gravity/wind receptors. Possibly, both JO receptor types also use different NompC isoforms, which could also explain why certain nompC promoter fusion constructs are selectively expressed in JO sound receptor cells. The isoform NompC-L rescues the auditory defects of nompC mutants and accordingly seems crucial for JO sound receptor function. Determining NompC isoform patterns in JO may help understanding why gravity/wind receptors express, but apparently do not need, this TRP (Effertz, 2011).

This study has shown that NompC is essential for the mechanosensory function of Drosophila sound receptors, making this TRP channel a strong candidate for the fly's auditory mechanotransducer. Precedence that NompC can serve as a mechanotransduction channel comes from work on C. elegans, and the importance of NompC for Drosophila auditory transduction is supported by its requirement for nonlinear mechanical amplification: in the Drosophila ear, the source of this amplification has been traced down to mechanotransducers that, judged from the present study, reside in the sound receptors. Loss of amplification in flies with ablated sound receptors and in nompC mutants indicates that these auditory transducers require NompC. Clearly, more work is needed to dissect the specific roles of NompC in auditory transduction, and such dissection now seems most worthwhile given the auditory importance of this TRP (Effertz, 2011).

Drosophila TRPN(=NOMPC) channel localizes to the distal end of mechanosensory cilia

A TRPN channel protein is essential for sensory transduction in insect mechanosensory neurons and in vertebrate hair cells. The Drosophila TRPN homolog, NOMPC, is required to generate mechanoreceptor potentials and currents in tactile bristles. NOMPC is also required, together with a TRPV channel, for transduction by chordotonal neurons of the fly's antennal ear, but the TRPN or TRPV channels have distinct roles in transduction and in regulating active antennal mechanics. The evidence suggests that NOMPC is a primary mechanotransducer channel, but its subcellular location - key for understanding its exact role in transduction - has not yet been established. By immunostaining, this study has located NOMPC at the tips of mechanosensory cilia in both external and chordotonal sensory neurons, as predicted for a mechanotransducer channel. In chordotonal neurons, the TRPN and TRPV channels are respectively segregated into distal and proximal ciliary zones. This zonal separation is demarcated by and requires the ciliary dilation, an intraciliary assembly of intraflagellar transport (IFT) proteins (Lee, 2010). The results provide a strong evidence for NOMPC as a primary transduction channel in Drosophila mechansensory organs. The data also reveals a structural basis for the model of auditory chordotonal transduction in which the TRPN and TRPV channels play sequential roles in generating and amplifying the receptor potential, but have opposing roles in regulating active ciliary motility (Lee, 2010).

The focal and zonal signals labeled by the antiserum represent endogenous NOMPC gene products, as evidenced by their absence from nompC null mutants and reduction in nompC^f00642 mutant, and by the mislocalized label in the nompC⁴ missense mutant. They may represent any or all of the predicted alternate spliceforms, all of which include the peptide sequence used as an antigen. The data support a role for NOMPC as a primary transducer channel, because it is located at sites where mechanical signals impinge on the sensory cilia in both external and chordotonal organs. In both receptor types, this is at a site where the ciliary membrane is contacted by the extracellular matrix of the dendritic cap or sheath. This could mean that matrix elements are direct ligands for the TRPN channel. Indeed, nompA mutations detach the cilium from the matrix and eliminate transduction. This is not seen in nompC null mutants, so the channel cannot be the only connection between the cilium and cap. This is also consistent with the limited extracellular exposure of the NOMPC channel's predicted topology. However, no direct interaction has been demonstrated between any identified mechanosensory channel and an extracellular protein or matrix; it is also possible that the sensory transducers are basically stretch-activated channels and that the specialized extracellular and cytoskeletal structures are required to activate the channel indirectly by locally increasing membrane tension (Lee, 2010).

From a detailed analysis of antennal mechanics, sequential roles for TRPN and TRPV in fly auditory transduction have been proposed (Gopfert, 2006). In this model, TRPN is the primary transduction channel that triggers the mechanical amplification of the antennal vibrations, while TRPV functions as a secondary channel required for the generation of action potentials but also downregulates the antennal vibrations. A puzzle is how two TRP channels, both probably cation-selective, have distinct effects in the same cell. Their spatial separation provides one possible explanation: the ciliary dilation (CD), where the membrane and the axonemal microtubules bulge outward to enclose an electron-dense inclusion, may divide the sensory cilium into two functionally distinct zones in chordotonal neurons. In support of this idea, the two zones also differ in their axoneme structures: axonemal dynein-like arms are found only in the proximal zone. It seems likely that the non-motile distal segment is the place where the initial mechanotransduction current occurs, whereas the potentially motile proximal segment amplifies the sound stimuli by actively vibrating the cilium in response to the initial transduction current. TRPV in proximal segment may act as a secondary channel that generates depolarizing current needed to trigger the action potentials. Opening of TRPV may also negatively regulate the active vibration of the proximal segment (Lee, 2010).

In this study, the antibody staining showed that NOMPC is expressed in almost all sensory units of JO, although it is unclear whether every neuron in each sensory unit expresses NOMPC. This result contrasts with the previous report that argued the subgroup-specific expression of NOMPC on the basis of the expression pattern of GAL4 under the control of nompC promoter (Kamikouchi, 2009). But the promoter-fusion construct does not necessarily represent the whole expression pattern of the endogenous gene. Indeed, the nompC-GAL4 does not express GAL4 in sensory neurons of abdominal bristles in which NOMPC is required for mechanosensory transduction. Thus it is reasonable to conclude that NOMPC is expressed in all sensory units of JO, and participated in sensing not only sound but also gravity and wind. Further functional analyses are required to confirm this (Lee, 2010).

In some vertebrates, TRPN channels also have an essential role in sensory hair cell function. Most hair cells include both a true cilium, the kinocilium, and a bundle of actin-based sterecilia that are the probable site of mechanosensory transduction. In Xenopus, TRPN1 localizes only to kinocilium, particularly to its bulbous tip. This suggests that the essential role for frog TRPN1 is in the kinocilium instead of main transduction channel. However, the precise role of TRPN1 in the frog kinocilium is unclear. In zebrafish, TRPN1 is also required for hair cell function but its precise role and subcellular localization in hair cells is not determined yet. It is of interest to note that some fish kinocilia can beat spontaneously or in response to tip stimulation. It will be interesting to examine whether the ciliary motility in vertebrate hair cell transduction requires TRPN1 (Lee, 2010).

NOMPC, a member of the TRP channel family, localizes to the tubular body and distal cilium of Drosophila campaniform and chordotonal receptor cells

Mechanoreception underlies the senses of touch, hearing and balance. An early event in mechanoreception is the opening of ion channels in response to mechanical force impinging on the cell. This study reports antibody localization of NOMPC, a member of the transient receptor potential (TRP) ion channel family, to the tubular body of campaniform receptors in the halteres and to the distal regions of the cilia of chordotonal neurons in Johnston's organ, the sound-sensing organ of flies. Because NOMPC has been shown to be associated with the mechanotransduction process, these studies suggest that the transduction apparatus in both types of sensory cells is located in regions where a specialized microtubule-based cytoskeleton is in close proximity to an overlying cuticular structure. This localization suggests a transmission route of the mechanical stimulus to the cell. Furthermore, the commonality of NOMPC locations in the two structurally different receptor types suggests a conserved transduction apparatus involving both the intracellular cytoskeleton and the extracellular matrix (Liang, 2011).

These data show that NOMPC localizes to the distal cilium in both campaniform and chordotonal mechanoreceptors. Because of the association of NOMPC with mechanotransduction, these results provide new insight into the transduction mechanism in flies (Liang, 2011).

First, the distal localization of NOMPC suggests that the site of transduction is where the extracellular structures make physical contact with the neuron. In the case of the campaniform receptor, deformation of the cuticle squeezes the distal tip of the tubular body. In the case of the chordotonal receptor, joint rotation stretches the cilium due to its distal attachment to the cap. Localization of the transduction apparatus at the contact sites argues against a mechanism in which the mechanical signal is transmitted intracellularly to a distant site, such as the base of the cilium (Liang, 2011).

Second, the localization suggests that the prominent microtubule-based cytoskeletal plays a crucial role in transduction. The most likely role is that the microtubules act as a rigid structure against which compressive or tensile forces exerted through the extracellular cuticle can squeeze or stretch the channel and lead to gating. Because of the high rigidity of both the microtubules and the cuticle, it is likely that the transduction apparatus contains a compliant element to protect the channel from excessive forces. The 29 ankyrin repeats in the N-terminus of the NOMPC protein may serve as such an element, acting as a 'gating spring' to transmit forces arising from cuticle deformations to the gate of the channel (Howard, 2004; Liang, 2011 and references therein].

Third, the presence of NOMPC, the microtubule-based cytoskeleton and the extracellular matrix in the same local region in both cell types, suggest that both receptor types may share a conserved mechanotransduction apparatus that senses force transmitted through a cuticular capping structure. Such conservation is interesting because there are striking structural and mechanical differences between the two cell types. The '9 + 0' cilium of chordotonal receptors has C₉ rotational symmetry, whereas the elliptical distal tip of the tubular body of campaniform receptors has approximate D₂ dihedral symmetry. Furthermore, the chordotonal receptor is excited by longitudinal tension of the cilium, whereas the campaniform receptor is thought to be excited by transverse compression of the tubular body, as in the case of the bristle receptor. Though longitudinal tension is likely to lead to transverse compression of the ciliary dilation, it is not clear how a similar proximate mechanical stimulus could lead to channel gating in both cell types. These differences highlight an uncertainty about the precise roles of NOMPC in these two receptor types (Liang, 2011).

Fourth, the localization of NOMPC suggests that mechanosensory information travels anterogradely from the distal tip to the proximal base of the mechanosensory dendrite. In the proximal part of the chordotonal cilium, below the dilation, two transient receptor potential V (TRPV) ion channels (Inactive and Nanchung) form a complex that is essential for hearing (Kim, 2003; Gong, 2004]. Though mutants of Inactive and Nanchung have no electrical response to sound, they do have active mechanical responses (Gopfert, 2006) indicating that the mechanotransduction apparatus is likely to be still present in these mutants. By contrast, electrical responses are attenuated and mechanical responses are diminished in NOMPC mutants (Gopfert, 2006) consistent with NOMPC being a component of the mechanotransduction apparatus. Mechanical measurements indicate that the TRPV channels are downstream of the mechanotransducer (Gopfert, 2006). These observations, together with NOMPC localization suggest the following model: the electrical signal is initiated in the distal region of the mechanoreceptors by a transduction complex that includes the NOMPC protein, and TRPV channels shape the electrical signal as it is transmitted down the cilium to the cell body, where it initiates action potentials that convey the sensory information to the central nervous system (Liang, 2011).

Distinct roles of TRP channels in auditory transduction and amplification in Drosophila

Auditory receptor cells rely on mechanically gated channels to transform sound stimuli into neural activity. Several TRP channels have been implicated in Drosophila auditory transduction, but mechanistic studies have been hampered by the inability to record subthreshold signals from receptor neurons. This study develop a non-invasive method for measuring these signals by recording from a central neuron that is electrically coupled to a genetically defined population of auditory receptor cells. It was found that the TRPN family member NompC, which is necessary for the active amplification of sound-evoked motion by the auditory organ, is not required for transduction in auditory receptor cells. Instead, NompC sensitizes the transduction complex to movement and precisely regulates the static forces on the complex. In contrast, the TRPV channels Nanchung and Inactive are required for responses to sound, suggesting they are components of the transduction complex. Thus, transduction and active amplification are genetically separable processes in Drosophila hearing (Lehnert, 2013).

This study has shown that relatively low-intensity sounds (i.e., lower-intensity than previously used to study courtship behavior) can elicit a behavioral response in Drosophila. This provides a motivation for investigating Drosophila auditory transduction near absolute threshold and in particular the mechanisms that specify the sensitivity of the transduction complex. This in turn requires developing a sensitive method for measuring transduction currents from type AB Johnston's organ neurons (JONs), the receptor neurons that are most sensitive to sound (Kamikouchi, 2009; Yorozu, 2009). Anatomical and genetic data demonstrate that giant fiber neuron (GFN) currents are a selective measure of spiking and generator currents in type AB JONs (Lehnert, 2013).

Although this approach involves recording JON activity indirectly via the GFN, the currents recorded are nevertheless relatively fast. Indeed, they have latencies and rise times that are similar to (and even faster than) currents that are recorded directly from the cell bodies of mechanosensitive neurons. Thus, although the signals that were recorded are likely smoothed by cable filtering, the degree of filtering is not necessarily larger than in the case where signals are recorded directly from mechanosensitive neurons. Generator currents were observed in the GFN in response to the smallest step stimulus used, and this stimulus is essentially identical to the threshold stimulus for evoking calcium responses in JONs (Effertz, 2011). The threshold for evoking GFN currents was also essentially the same as the threshold for evoking an antennal nerve field potential response. Finally, these thresholds are just below the threshold for Drosophila auditory behavior. Taken together, these comparisons argue that the approach taken is sensitive enough to report generator currents evoked by near-threshold auditory stimuli (Lehnert, 2013).

The results confirm and extend what is known about the fundamental properties of transduction in Drosophila JONs. First, the measurements show that the transduction complex in type AB JONs is gated by antennal rotations as small as 5 x 10^-4 radians. This rotation corresponds to a 74 nm displacement of the distal end of the 'lever' (the arista) which projects from the most distal segment of the antenna. This measurement of the transduction threshold is consistent with that obtained by a previous study (Effertz, 2011). It is emphasized that the displacement that actually gates the transduction complex is certainly much smaller than this (on the order of a few nm), but because this displacement occurs within the interior of the antenna itself, it cannot be measured directly (Lehnert, 2013).

Second, this study shows that the type AB JONs that provide input to the GFN are depolarized by both lateral and medial rotations. The data suggest that bidirectionality is probably a property of individual JONs of this type, and not just the population as a whole. Indeed, the geometrical arrangement of type A (and perhaps B) JONs within the auditory organ suggests that individual JONs of this type should be stretched by both medial and lateral movements, and thus should respond twice per sound cycle (Lehnert, 2013).

Finally, evidence was found that some transduction channels are open at rest, even in the absence of sound. This conclusion relies on the observation that JONs spike spontaneously, and that the rate of spontaneous activity is substantially reduced by loss of either Nanchung or Inactive. This conclusion is consistent with previous studies which used other techniques to make inferences about JON activity (Lehnert, 2013).

Loss of either Nanchung or Inactive abolishes generator currents. These findings are consistent with previous reports that loss of either Nanchung or Inactive completely eliminates antennal field potential responses to sound. However, antennal field potentials are thought to reflect the spiking activity of JONs rather than subthreshold activity. Thus, it was not clear from this result whether Nanchung and Inactive were required for transduction or merely spike generation (Lehnert, 2013).

Previously, it has been proposed that the role of Nanchung and Inactive is to amplify the transduction signal. However, the latency and speed of the generator currents that were recorded implies that the transduction complex is directly gated by force, rather than gated indirectly by a second messenger. Given this, the Nanchung/Inactive complex is unlikely to merely amplify the transduction signal, because amplification would need to occur within microseconds (which rules out a role for diffusible second messengers), and amplification would need to be >100-fold in magnitude. This level of amplification seems unlikely, given the weak voltage dependence of the channels formed by Nanchung and Inactive. Finally, because the Nanchung/Inactive complex does not colocalize with NompC in the JON dendrite, no amplification mechanism could rely on direct protein-protein interactions between these components (Lehnert, 2013).

Given these considerations, it seems more likely that Nanchung and Inactive form part of the transduction complex itself. Consistent with this conclusion, both Nanchung and Inactive confer calcium responses to hypo-osmotic stimuli in heterologous cells. However, more work will be needed to test the idea that Nanchung and Inactive could function as force-gated ion channels. An alternative possibility is that Nanchung and Inactive are required for the trafficking or function of an unknown channel. Previous work has shown that the loss of Nanchung or Inactive results in abnormally large sound-driven antennal movements, as well as spontaneous oscillatory movement in the absence of sound (Gopfert, 2006). The results show that this phenotype goes hand-in-hand with loss of all measurable transduction in JONs. Together, these findings imply that transduction in JONs inhibits the active amplification of antennal movements, possibly because the transduction complex represents a mechanical load on the amplifier element. The presence of active movements in the absence of transduction is also incompatible with the idea that the active amplification of antennal movement is a direct consequence of transduction channel gating (Lehnert, 2013).

The results demonstrate that NompC is not required for mechanotransduction in the type AB JONs that provide input to the GFN. Moreover, the maximal level of transduction current is essentially normal in the absence of NompC, and the rise time of the current is normal at this maximal level. This result argues that NompC does not specify the intrinsic properties of the transduction channel, such as conductance or ionic selectivity. This result also implies that NompC is not required for the proper trafficking or localization of the transduction complex. These conclusions differ from that of a previous study. That study reported that sound-evoked calcium signals are lost in nompC mutant type AB JONs, and concluded that NompC is absolutely required for transduction in these JONs (Effertz, 2011).

The basis for this discrepancy is not clear, but is likely related to the differences between calcium imaging and electrophysiological recordings. It is possible that the calcium indicator does not report the entirety of the generator current, but rather a small and slow component that does require NompC. Our results imply that the principal role of NompC is not to transduce force into an electrical signal, but rather to modulate the forces on the transduction complex. Specifically, generator currents were found to be more sensitive to movement when NompC is present, which implies that NompC effectively amplifies mechanical input to the transduction channel, given a fixed amount of antennal movement. Thus, NompC is likely to generate force, or to be permissive for a process that generates force, within the interior of the antenna (Lehnert, 2013).

Previous studies have shown that loss of NompC abolishes active amplification of sound-evoked antennal movement, and also reduces spontaneous oscillatory antennal movement. Thus, loss of NompC appears to eliminate or occlude a process that exerts force on the antenna. This is broadly consistent with the conclusion that NompC is involved in a process which generates force within the interior of Johnston's organ. Recent studies have proposed that NompC is part of the transduction channel, or channel gating spring, or is otherwise required for the function of either of these components (Effertz, 2012; Gopfert, 2006); however, the observation that transduction persists in the absence of NompC is not consistent with these ideas. Rather, it is proposed that NompC is permissive for the function of a mechanical amplifier operating between the antennal sound receiver and the transducer. In other words, it is proposed that the force generated within Johnston's organ is exerted on the transduction apparatus as well as the distal antennal segment. In addition to amplifying mechanical input to the transduction complex, NompC appears to be required for balancing the medial and lateral resting forces on the transduction complex. In the presence of NompC, JONs are equally sensitive to medial and lateral movements, suggesting that medial and lateral resting forces on the transduction complex are balanced. By contrast, in the absence of NompC, JONs are less sensitive to medial movements than to lateral movements. Simulations show that this phenotype can result from asymmetrical medial and lateral resting forces on the transduction complex. Thus, a single NompC-dependent process may be responsible for balancing resting forces, as well as actively amplifying stimulus- evoked forces. Adaptation appears to be a separate process, because it does not require NompC (Lehnert, 2013).

In sum, it is proposed that NompC functions in a manner analogous to the role of prestin in the mammalian cochlea (Dallos, 2008). Prestin is expressed by outer hair cells in the cochlea, and is essential for the ability of outer hair cells to mechanically amplify sound-evoked movements of the basilar membrane. In this manner, prestin increases the sensitivity of the transduction apparatus of the inner hair cells to sound stimuli. However, like NompC, prestin is not absolutely required for transduction, and is not colocalized with the transduction apparatus (Lehnert, 2013).

On the basis of its subcellular location, NompC is well-positioned to act as a modulator of mechanical forces. Whereas Nanchung/ Inactive are localized to the proximal dendrite, NompC is localized to the distal dendrite, closer to the point where the dendrite inserts into the connective structures that link it to the moving segment of the antenna (Cheng, 2010; Gong, 2004; Lee, 2010; Liang, 2011). A bundle of microtubules runs longitudinally through the dendrite, and this could provide a substrate for adjustments of tension that propagate from the distal to the proximal dendrite. It is proposed that transduction occurs in the proximal dendritic segment (where Nanchung and Inactive are localized), and this would place NompC in series between the moving segment of the antenna and the transduction complex (Lehnert, 2013).

How might NompC be involved in modulating mechanical force? One possibility is that NompC itself generates force that adjusts the longitudinal tension within a JON. NompC contains an unusually large number of ankyrin repeats. Ankyrin repeats can act as elastic elements, and can. If, for instance, calcium entry into JONs were to modulate the energetics of the unfolded state on a cycle-by-cycle basis, then the refolding force could augment transduction. An alternative possibility is that NompC does not itself generate force, but it is permissive for a process that generates force. For example, calcium influx through NompC might change the state of motor proteins that adjust longitudinal tension within a JON (Lehnert, 2013).

Assuming that NompC forms part of a channel, this channel appears to carry relatively little current, or is otherwise ineffective at exciting the JON. No detectable generator current was found in the absence of either Nanchung or Inactive, meaning that any current must be below the limit imposed by noise in the recordings. That limit is about 100-fold smaller than the generator currents that were measured. Moreover, a previous study reported that sound-evoked calcium signals in JONs are essentially eliminated when Nanchung is absent (Kamikouchi, 2009). Together, these findings argue that any ionic flux through NompC is far less than the flux through the transduction complex itself. This conclusion relies on the idea that NompC can still function when Nanchung is absent. In support of this, it was shown that NompC localizes properly in the absence of Nanchung. Moreover, active amplification of antennal movements is intact when Nanchung is absent (Gopfert, 2006). Because the active amplification of sound-evoked movements requires NompC, this implies that NompC can function without Nanchung. Interestingly, a slow current was observed that persists for hundreds of milliseconds after sound offset, and which absolutely requires both Nanchung and NompC (Lehnert, 2013).

Future studies will be required to fully elucidate the mechanism of NompC's action. What makes this mechanism intriguing is the implication there may be two functionally distinct types of TRP channels involved in Drosophila hearing. One of these (the transduction channel) evidently carries most or all of the current, and requires Nanchung and Inactive. The other -- which requires NompC -- carries comparatively little current, and controls the active generation of force within the auditory organ (Lehnert, 2013).

A NOMPC-dependent membrane-microtubule connector is a candidate for the gating spring in fly mechanoreceptors

Mechanoreceptors contain compliant elements, termed 'gating springs,' that transfer macroscopic stimuli impinging on the cells into microscopic stimuli that open the mechanosensitive channels. Evidence for gating springs comes from mechanical experiments; they have not been identified molecularly or ultrastructurally. This study shows that the filamentous structures that connect the plasma membrane to the microtubules are compliant structural elements in the mechanoreceptive organelle of fly campaniform receptors. These filaments colocalize with the ankyrin-repeat domain of the transient receptor potential (TRP) channel NOMPC. In addition, they resemble the purified ankyrin-repeat domain in size and shape. Most importantly, these filaments are nearly absent in nompC mutants and can be rescued by the nompC gene. Finally, mechanical modeling suggests that the filaments provide most of the compliance in the distal tip of the cell, thought to be the site of mechanotransduction. These results provide strong evidence that the ankyrin-repeat domains of NOMPC structurally contribute to the membrane-microtubule connecting filaments. These filaments, as the most compliant element in the distal tip, are therefore good candidates for the gating springs (Liang, 2013).

Mechanosensory interactions drive collective behaviour in Drosophila

Collective behaviour enhances environmental sensing and decision-making in groups of animals. Experimental and theoretical investigations of schooling fish, flocking birds and human crowds have demonstrated that simple interactions between individuals can explain emergent group dynamics. These findings indicate the existence of neural circuits that support distributed behaviours, but the molecular and cellular identities of relevant sensory pathways are unknown. This study shows that Drosophila melanogaster exhibits collective responses to an aversive odour: individual flies weakly avoid the stimulus, but groups show enhanced escape reactions. Using high-resolution behavioural tracking, computational simulations, genetic perturbations, neural silencing and optogenetic activation it was demonstrated that this collective odour avoidance arises from cascades of appendage touch interactions between pairs of flies. Inter-fly touch sensing and collective behaviour require the activity of distal leg mechanosensory sensilla neurons and the mechanosensory channel NOMPC. Remarkably, through these inter-fly encounters, wild-type flies can elicit avoidance behaviour in mutant animals that cannot sense the odour—a basic form of communication. These data highlight the unexpected importance of social context in the sensory responses of a solitary species and open the door to a neural-circuit-level understanding of collective behaviour in animal groups (Ramdya, 2014).

Drosophila melanogaster is classified as a solitary species but flies aggregate at high densities (~1 fly per cm²) to feed, providing opportunities for collective interactions. Although groups affect circadian rhythms and dispersal in Drosophila, how social context influences individual sensory behaviours is unknown. To study this question, an automated behavioural assay was developed to track responses of freely-walking flies to laminar flow of air or an aversive odorant, 5% carbon dioxide (CO₂). Odour was presented to one half of a planar arena for 2 min. Avoidance behaviour was quantified as the percentage of time a fly spent in the air zone during the second minute of a trial. Unexpectedly, isolated flies spent very little time avoiding this odour, despite the aversion to CO₂ observed in other assays. However, increasing the number of flies was associated with substantial increases in odour avoidance. This effect peaked at 1.13 flies per cm², a density typical for fly aggregates and was only apparent for flies in the odour zone. Time-course analysis revealed that, within only a few seconds after odour onset, a larger proportion of flies in high-density groups had left the odour zone compared to isolated individuals. Additionally, the motion of flies after odour onset was coherent at higher densities, with flies moving in the same direction, out of the odour zone; this effect was not observed for flies in the air zone (Ramdya, 2014).

To determine the basis of these global behavioural differences, the locomotion of individual flies was examined. Single animals are typically sedentary but walk more when exposed to CO₂. In groups, however, it was discovered that 63% of the time, the first walking response of a fly after odour onset coincided with proximity to a neighbouring fly. These Encounters were more frequent with increasing group density. Moreover, walking bouts initiated during an Encounter ('Encounter Responses') were significantly longer than those spontaneously initiated in isolation. These observations indicated that inter-fly interactions might contribute to the enhanced odour avoidance of groups of flies (Ramdya, 2014).

This possibility was examined initially by computational simulation of the olfactory assay. The dynamics of the simulation were driven by three phenomena observed in behavioural assays. First, flies initiate more spontaneous bouts of walking in odour than in air. Second, flies are more likely to turn and retreat after entering the odour zone from the air zone. Third, close proximity to another fly elicits Encounter Responses in stationary flies. Importantly, these elements could reproduce collective behaviour: higher numbers of simulated flies exhibited greater avoidance. While changing the olfactory parameters preserved stronger responses in groups than isolated individuals, diminishing the Encounter Response probability could abolish and even reverse collective behaviour. These results suggested that Encounter Responses are a crucial component of Drosophila group dynamics. To experimentally test the role of inter-fly interactions in collective behaviour, attempts were made to explain the mechanistic basis of Encounter Responses. Although the olfactory experiments were performed in the dark, the presence of light did not diminish Encounter Response frequency. Volatile chemicals are known modulators of many social behaviours, but putative anosmic flies (lacking known olfactory co-receptors) did not reduce Encounter Responses. By contrast, disruption of the mechanosensory channel NOMPC significantly diminished Encounter Response frequency. These data suggested that mechanosensing is required for Encounter Responses (Ramdya, 2014).

By observing groups of flies at high spatiotemporal resolution, it was found that active flies elicited motion in stationary animals through gentle touch of peripheral appendages (legs and wings). Leg touches took place exclusively on distal segments and resulted in spatially stereotyped walking reactions. These reactions were kinematically indistinguishable from Encounter Responses. This analysis indicates that appendage touch is the stimulus that elicits Encounter Responses. The precise stereotypy of these locomotor responses, similar to cockroach escape reactions, implies their dependence upon somatotopic neural circuits linking touch with movement (Ramdya, 2014).

As fly appendages also house taste receptors, whether mechanical stimulation was sufficient to elicit Encounter Responses was tested by tracking stationary flies following touch of appendages with a metallic disc. A stereotyped relationship was observed between the location of mechanical touch and subsequent walking trajectories, whose associated kinematics were indistinguishable from those of Encounter Responses. Thus, mechanical touch alone can elicit Encounter Responses. Consistently, genetic ablation of flies' oenocytes, to remove cuticular hydrocarbon contact chemosensory signals, had no effect on the ability of these animals to elicit Encounter Responses in wild-type flies. These data imply that Encounter Responses are mediated solely by mechanosensory stimulation (Ramdya, 2014).

Next, mechanosensory neurons required for touch- evoked Encounter Responses were identified by driving tetanus toxin (Tnt) expression with a panel of candidate mechanosensory Gal4 lines. R55B01-Gal4/UAS-Tnt flies exhibited significantly diminished Encounter Responses compared to a gustatory neuron driver line, without reduced ability to produce sustained high-velocity walking bouts. R55B01-Gal4- driven expression of a UAS-CD4:tdGFP reporter was detected in neurons innervating leg and wing neuropils of the thoracic ganglia. Consistently, green fluorescent protein (GFP) labelled neurons in several leg mechanosensory structures: the femoral and tibial chordotonal organs, and distal leg mechanosensory sensilla neurons. Notably, among the screened lines only R55B01- Gal4 drove expression in leg mechanosensory sensilla (Ramdya, 2014).

To ascertain the contribution to Encounter Responses of leg mecha- nosensory sensilla and/or chordotonal structures (which can also sense touch), additional Gal4 driver lines were identified that drove expression in subsets of these neuron classes. By intersecting piezo-Gal4 with cha3-Gal80, a Gal4 suppression line, it was possible to limit leg expression to mechanosensory sensilla neurons (termed 'Mechanosensory Sensilla driver' line). Importantly, silencing neurons with this driver significantly diminished Encounter Response frequency. By contrast, silencing leg chordotonal organs alone had no effect on Encounter Response frequency (Ramdya, 2014).

The sufficiency of leg mechanosensory sensilla neuron activity to elicit Encounter Response-like walking was tested by expressing channelrhodopsin-2 (ChR2) in each class of leg mechanosensory neurons and recording behavioural responses to blue light pulses. Optogenetic stimulation of flies expressing ChR2 in leg mechanosensory sensilla neurons, but not chordotonal organs, resulted in Encounter Response- like walking, consistent with natural elicitation of Encounter Responses by inter-fly touch of distal leg segments (Ramdya, 2014).

Identification of a neuronal basis for Encounter Responses allowed testing of the model's prediction that inter-fly interactions are required for collective odour avoidance. First, leg mechanosensory sensilla neurons were silenced by expressing Tnt with R55B01-Gal4 or the Mechanosensory Sensilla driver. Second, nompC mutants were tested. Each of these perturbations abolished collective odour avoidance, supporting the link between mechanosensation and group behaviour (Ramdya, 2014).

Touch may enhance odour avoidance by increasing awareness of the stimulus. Alternatively, touch may produce an odour-independent Encounter Response reaction that initiates departure from the odour zone. To distinguish between these possibilities, it was asked if odour-insensitive flies displayed increased avoidance in the presence of odour-sensitive animals. Indeed, both in simulations and in real flies, increasing the number of odour-sensitive individuals led to greater avoidance behaviour of odour-insensitive individuals. Thus, in this context, touch-mediated modulation of odour awareness plays little, if any, role in collective avoidance (Ramdya, 2014).

Combining systems-level and neurogenetic approaches, this study has uncovered a hierarchy of mechanisms that drive collective motion in Drosophila. Active flies elicit spatially stereotyped walking responses in stationary flies through appendage touch interactions, requiring the NOMPC mechanosensory channel and distal leg mechanosensory sensilla neurons. Through Encounter Responses, odour reactions of sensitive flies spark cascades of directed locomotion of less sensitive (or even insensitive) individuals, causing a coherent departure from the odour zone. This behavioural positive feedback and group motion are absent among flies in the non-odour zone since they are less likely to initiate walking and, consequently, have a reduced frequency of Encounters. Additionally, flies retreat when encountering the odour while transiting from the air zone. Together these behavioural phenomena cause flies to escape the odour zone and then remain in the air zone, resulting in higher odour avoidance for groups compared to isolated animals. When distal appendage mechanosensory touch detection is impaired, groups of flies cannot produce Encounter Responses, are less likely leave the odour zone, and instead behave like isolated flies. Encounters are likely to have widespread influence on sensory-evoked actions of individuals in groups. For example, movement of flies towards areas of high elevation is also increased in higher density groups (Ramdya, 2014).

Behaviour in animal groups arises from the detection and response to intentional and unintentional signals of conspecifics. While neural circuits controlling pairwise interactions, such as courtship, are increasingly well-understood, little is known about those orchestrating group- level behaviours. The identification of sensory pathways that mediate collective behaviour in Drosophila opens the possibility to understand the neural basis by which an individual's actions may influence—and be influenced by group dynamics (Ramdya, 2014).

The adhesion GPCR Latrophilin/CIRL shapes mechanosensation

G-protein-coupled receptors (GPCRs) are typically regarded as chemosensors that control cellular states in response to soluble extracellular cues. However, the modality of stimuli recognized through adhesion GPCR (aGPCR), the second largest class of the GPCR superfamily, is unresolved. This study study characterizes the Drosophila aGPCR Latrophilin/dCirl, a prototype member of this enigmatic receptor class. dCirl is shown to shapes the perception of tactile, proprioceptive, and auditory stimuli through chordotonal neurons, the principal mechanosensors of Drosophila. dCirl sensitizes these neurons for the detection of mechanical stimulation by amplifying their input-output function. These results indicate that aGPCR may generally process and modulate the perception of mechanical signals, linking these important stimuli to the sensory canon of the GPCR superfamily (Scholz, 2015).

Because of the nature of their activating agents, G-protein-coupled receptors (GPCRs) are established sensors of chemical compounds. The concept that GPCRs may also be fit to detect and transduce physical modalities, i.e., mechanical stimulation, has received minor support thus far. In vitro observations showed that, in addition to classical soluble agonists, mechanical impact such as stretch, osmolarity, and plasma membrane viscosity may alter the metabotropic activity of individual class A GPCR. However, the ratio and relationship between chemical and mechanical sensitivity and the physiological role of the latter remain unclear (Scholz, 2015).

Genetic studies have indicated that adhesion GPCRs (aGPCRs), a large GPCR class with more than 30 mammalian members, are essential components in developmental processes. Human mutations in aGPCR loci are notoriously linked to pathological conditions emanating from dysfunction of these underlying mechanisms, including disorders of the nervous and cardiovascular systems, and neoplasias of all major tissues. However, as the identity of aGPCR stimuli is unclear, it has proven difficult to comprehend how a GPCRs exert physiological control during these processes (Scholz, 2015).

Latrophilins constitute a prototype aGPCR subfamily because of their long evolutionary history. Latrophilins are present in invertebrate and vertebrate animals, and their receptor architecture has remained highly conserved across this large phylogenetic distance. The mammalian Latrophilin 1 homolog was identified through its capacity to bind the black widow spider venom component α-latrotoxin, which induces a surge of vesicular release from synaptic terminals and neuroendocrine cells through formation of membrane pores. Latrophilin 1/ADGRL1 was suggested to partake in presynaptic calcium homeostasis by interacting with a teneurin ligand and in trans-cellular adhesion through interaction with neurexins 1b and 2b. Further, engagement of Latrophilin 3/ADGRL3 with FLRT proteins may contribute to synapse development. The role of Latrophilins in the nervous system thus appears complex (Scholz, 2015).

This study has used a genomic engineering approach to remove and modify the Latrophilin locus dCirl, the only Latrophilin homolog of Drosophila melanogaster. dCirl was shown to be required in chordotonal neurons for adequate sensitivity to gentle touch, sound, and proprioceptive feedback during larval locomotion. This indicates an unexpected role of the aGPCR Latrophilin in the recognition of mechanosensory stimuli and provides a unique in vivo demonstration of a GPCR in mechanoception (Scholz, 2015).

This analysis provides multiple lines of evidence to support that Latrophilin/dCirl, one of only two aGPCRs in the fly, is a critical regulator of mechanosensation through chordotonal neurons in Drosophila larvae. Larval chordotonal organs respond to tactile stimuli arising through gentle touch, mechanical deformation of the larval body wall and musculature during the locomotion cycle, and vibrational cues elicited through sound. First, this study determined that registration of all these mechanical qualities is reduced in the absence of dCirl, based on behavioral assays. Then it was established that behavioral defects can be rescued by re-expression of dCirl in chordotonal neurons, one of several cell types with endogenous dCirl expression. Mechanically stimulated lch5 neurons lacking dCirl were shown to responded with action currents at approximately half the control rate across a broad spectrum of stimulation frequencies, providing direct functional evidence for a role of dCirl in chordotonal dendrites, the site of mechanotransduction and receptor potential generation, or somata, where action potentials are likely initiated. Further, the ability of chordotonal neurons to generate mechanical responses relative to their background spike activity appears to be modulated by dCirl (Scholz, 2015).

Combining dCirl KO with strong hypomorphs of trp homologs, ion channels that are directly responsible for the conversion of mechanical stimulation into electrical signals within chordotonal neurons, implied that dCirl operates upstream of them. Intriguingly, removing dCirl from nompC or nan mutant backgrounds resulted in inverse outcomes, i.e., decreased and increased crawling distances, respectively. This suggests that dCirl enhances nompC activity while curtailing nan function. These experiments demonstrate that dCirl genetically interacts with essential elements of the mechanotransduction machinery in chordotonal cilia. Additional studies showed that the dCirl promoter contains a RFX/Fd3F transcription factor signature that implicates dCirl in the mechanosensitive specialization of sensory cilia. On the basis of these results, it is proposed that dCirl partakes in the process of mechanotransduction or spike initiation and transmission to promote sensory encoding (Scholz, 2015).

The classical model of GPCR activation has become the archetypical example for cellular perception of external signals. It comprises soluble ligands that bind to the extracellular portions of a cognate receptor, whereby receptor conformation is stabilized in a state that stimulates metabotropic effectors. Thus, GPCRs are primarily regarded as chemosensors due to the nature of their activating agents. The concept that GPCRs may also be fit to detect and transduce physical modalities, i.e., mechanical stimulation, has received little support thus far (Scholz, 2015).

aGPCRs display an exceptional property among the GPCR superfamily in that they recognize cellular or matricellular ligands. To date, only one ligand, Type 4 collagen, has proved adequate to induce intracellular signaling, whereas for the vast majority of ligand-aGPCR interactions this proof either failed or is lacking). This implies that sole ligand recognition is generally not sufficient to induce a metabotropic response of aGPCRs. Thus, in addition to ligand engagement, the current results suggest that mechanical load is a co-requirement to trigger the activity of dCIRL, a prototypical aGPCR homolog (Scholz, 2015).

Recent findings place aGPCRs in the context of mechanically governed cellular functions, but how mechanical perception through aGPCR activity impinges on cell responses has not yet been established. In addition, the molecular structure of aGPCR is marked by the presence of a GPCR auto-proteolysis inducing (GAIN) domain, which plays a paramount role in signaling scenarios for aGPCRs. This domain type is also present in PKD-1/Polycystin-1-like proteins, which are required to sense osmotic stress and fluid flow in different cell types and are thus considered bona fide mechanosensors. In addition, studies on EGF-TM7-, BAI-, and GPR56-type aGPCRs further showed that proteolytic processing and loss of NTF may figure prominently in activation of the receptors' metabotropic signaling output and that mechanical forces exerted through receptor-ligand contact are required for receptor internalization (Scholz, 2015 and references therein).

dCirl is not the only aGPCR associated with mechanosensation. Celsr1 is required during planar cell polarity establishment of neurons of the inner ear sensory epithelium. Similarly, the very large G-protein-coupled receptor 1 (VLGR1) exerts an ill-defined developmental role in cochlear inner and outer hair cells, where the receptor connects the ankle regions of neighboring stereocilia. In addition, VLGR1 forms fibrous links between ciliary and apical inner segment membranes in photoreceptors. Both cell types are affected in a type of Usher syndrome, a congenital combination of deafness and progressive retinitis pigmentosa in humans, which is caused by loss of VLGR1 function. Although present evidence derived from studies of constitutively inactive alleles suggests a requirement for Celsr1 and VLGR1 aGPCR for sensory neuron development, their putative physiological roles after completion of tissue differ-entiation have remained unclear and should be of great interest (Scholz, 2015 and references therein).

In the current model on dCirl function, aGPCR activity, adjusted by mechanical challenge, modulates the molecular machinery gating mechanotransduction currents or the subsequent initiation of action potentials and ensures that mechanical signals are encoded distinctly from the background activity of the sen-sory organ. Thereby, dCirl shapes amplitude and kinetics of the sensory neuronal response. Linking adequate physiological receptor stimulation to downstream pathways and cell function is an essential next step to grasp the significance of aGPCR function and the consequences of their malfunction in human conditions. The versatility of the dCirl model now provides an unprecedented opportunity to study the mechanosensory properties of an exemplary aGPCR and to uncover features that might prove of general relevance for the function and regulation of the entire aGPCR class (Scholz, 2015).

Ankyrin repeats convey force to gate the NOMPC mechanotransduction channel

How touch is sensed is fundamental for many physiological processes. However, the underlying mechanism and molecular identity for touch sensation are largely unknown. This study reports on defective gentle-touch behavioral responses in brv1 loss-of-function Drosophila larvae. RNAi and Ca(2+) imaging confirmed the involvement of Brv1 in sensing touch and demonstrated that Brv1 mediates the mechanotransduction of class III dendritic arborization neurons. Electrophysiological recordings further revealed that the expression of Brv1 protein in HEK293T cells gives rise to stretch-activated cation channels. Purified Brv1 protein reconstituted into liposomes were found to sense stretch stimuli. In addition, co-expression studies suggested that Brv1 amplifies the response of mechanosensitive ion channel NOMPC (no mechanoreceptor potential C) to touch stimuli. Altogether, these findings demonstrate a molecular entity that mediates the gentle-touch response in Drosophila larvae, providing insights into the molecular mechanisms of touch sensation (Zhang, 2018).

Touch sensation is the ability to sense and respond to mechanical stimuli, and it is essential for fundamental behaviors, including harm avoidance, environmental exploration, and social exchange. The mechanotransduction of touch relies on mechanosensitive ion channels that transform mechanical stimuli into electrical signals in sensory neurons. Genetic dissection of touch transduction in Drosophila larvae has led to the identification of several candidates that may sense mechanical forces. Painless, a TRPA channel subunit expressed in a subset of larval type II multiple dendritic neurons, is involved in mechanical nociception. The DEG/ENaC channel Pickpocket and Drosophila PIEZO (dPIEZO) are essential for noxious mechanosensation in class IV dendritic arborization (da) neurons. The Drosophila TRPN channel NOMPC (no mechanoreceptor potential C) is highly expressed in class III da neurons and is required for gentle-touch sensation (Yan, 2013). Among these channels, dPIEZO and NOMPC have been shown to form mechanically activated channels with distinct biophysical and pore-related properties. Although these findings have advanced knowledge of the physics and physiology of touch sensation, new molecular entities and underlying mechanisms in sensory neurons remain to be identified and characterized to unravel the complexity of touch (Zhang, 2018).

Brv1 is a member of the TRPP (TRP polycystin) subfamily of TRP ion channels and is known for its role in cold sensation in the fly antenna (Gallio, 2011). Brv1 shares sequence homology with the mammalian TRPP2 channels (also known as PKD2). TRPP2 is a Ca2+-permeable cation channel and functions as part of a polycystin receptor-channel complex that senses fluid flow and induces a Ca2+ signal response. These findings suggest a role of TRPP2 in mechanosensation; however, whether Brv1 participates in mechanical responses has remained unclear (Zhang, 2018).

This study reports the role of Drosophila Brv1 in touch sensation. Behavioral assay and Ca2+ responses indicated that Brv1 is required for class III da sensory neurons to sense gentle touch. It was further demonstrated that Brv1 confers stretch-activated currents when heterologously expressed in a mammalian expression system and reconstituted into proteoliposomes. In addition, a co-expression study suggested that Brv1 amplifies the response of NOMPC channels in touch sensation. These findings demonstrate that Brv1 mediates the gentle-touch response in Drosophila larvae (Zhang, 2018).

The foundational observation here is that Brv1, a member of the TRPP subfamily, is required for Drosophila larvae to sense gentle touch. This study electrophysiologically characterized Brv1 as a pore-forming subunit of a cation channel that is activated in response to stretch, suggesting the direct role of Brv1 as a mechanosensitive channel in the touch response. Brv1 is characterized as distinct from other mechanosensitive ion channels, such as NOMPC. NOMPC functioned as a Drosophila touch sensor in response to multiple mechanical forces, including stretching and poking, while Brv1 was insensitive to poking. Touch is a complex sense comprising diverse modalities. A diverse array of specialized mechanoreceptors is involved in detecting a range of mechanical stimuli, including light brush, stretch, vibration, deflection of hair, and pressure. For example, slowly adapting type 2 (SA2) afferents, which innervate Ruffini cylinders, are primarily sensitive to skin stretch (Birznieks, 2009). The kinds of forces experienced during object manipulation activate SA2 stretch-sensitive afferents, which contribute to touch sensation such as grip control. So it may not be a surprise that Brv1 acts as a stretch-sensitive channel to mediate touch sensation. The distinctions in the detection of variable forces may offer the first hints of differences in the activation mechanisms between Brv1 and NOMPC. NOMPC has been suggested to adopt a tether gating mechanism by which the N-terminal ankyrin repeats form a tether linking the channel and the microtubules to convey force (Zhang, 2015). The finding that Brv1 was mechanically gated in the liposome suggests that mechanical force is transmitted directly to the Brv1 channel through lateral membrane tension. However, detailed structural information would be needed to elucidate the gating mechanism of Brv1 in touch sensation (Zhang, 2018).

NOMPC is also recognized as required for gentle-touch sensation in class III neurons (Yan, 2013), which raises the questions of whether Brv1 and NOMPC work together to mediate touch sensation and how they are required for responding to gentle touch. This study found that co-expression of Brv1 and NOMPC in HEK293T cells led to increased amplitude and delayed adaptation of the poke current compared to NOMPC expression alone. Extracellular recordings from class III neurons in abdominal segments of larval fillets has revealed that touching the body wall induces a burst of action potentials, which adapt with a time constant of ∼150 ms, but heterologous expression of NOMPC in S2 cells reveals that the poking currents adapt quickly, within 2 ms. Brv1 might act as an amplifier of NOMPC in response to poking, thus enhancing the excitability of class III da neurons in the touch response. A similar mechanism, by which TRPP2 inhibits the stretch-activated ion channels in vascular smooth muscle cells to regulate pressure sensing, has been reported (Sharif-Naeini, 2009). These data suggest double roles of Brv1 in touch sensation: the direct role as a mechanosensitive channel and perhaps the indirect role as a modulator for the NOMPC channel. This may partially explain why Brv1 or NOMPC LOF mutants cause severe defects in the behavioral response; however, the comprehensive role of Brv1 in touch sensation and the underlying molecular mechanisms need to be resolved in the future (Zhang, 2018).

A neural circuit integrates pharyngeal sensation to control feeding

Swallowing is an essential step of eating and drinking. However, how the quality of a food bolus is sensed by pharyngeal neurons is largely unknown. This study finds that mechanical receptors along the Drosophila pharynx are required for control of meal size, especially for food of high viscosity. The mechanical force exerted by the bolus passing across the pharynx is detected by neurons expressing the mechanotransduction channel NOMPC (no mechanoreceptor potential C) and is relayed, together with gustatory information, to IN1 neurons in the subesophageal zone (SEZ) of the brain. IN1 (ingestion neurons) neurons act directly upstream of a group of peptidergic neurons that encode satiety. Prolonged activation of IN1 neurons suppresses feeding. IN1 neurons receive inhibition from DSOG1 (descending subesophageal neurons) neurons, a group of GABAergic neurons that non-selectively suppress feeding. These results reveal the function of pharyngeal mechanoreceptors and their downstream neural circuits in the control of food ingestion (Yang, 2021).

Overconsumption is harmful for animals. Although the drive to ingest can be overwhelming for a hungry animal in the initial stage of a meal, inhibition becomes more dominant with the processes of food intake. This study found that food flowing across the pharynx accumulates the satiety state in the brain, demonstrating that multiple strategies are used by the nervous systems to avoid overeating. These pharyngeal sensory neurons are sensitive to sugar and mechanical force, serving as a flow meter that monitors food quality and amount so that the brain knows how much food is ingested even before the food reaches the intestine. This circuit may coordinate with other satiety signals, such as those conveyed by mechanical feedback from the intestine, to control feeding (Yang, 2021).

Gustatory and mechanosensory neurons are well separated on the fly labellum before their axons reach the SEZ, where they interact with each other to regulate the perception of food quality. In contrast, the sensory neurons in the pharynx seem to adapt a different coding mechanism. Some of the pharyngeal neurons are polymodal because they respond to chemical and mechanical stimuli, with PM neurons being an example. A 'generalist' versus 'specialist strategy has been found in other sensory organs too. Being able to evaluate multiple properties of a bolus in the pharynx allow the animals to effectively control the feeding amount. There are sensory neurons in the pharynx that may be tuned to gustatory or mechanosensory cues. For example, the R41E11-GAL4 and nompC-QF labeled approximately 10 pairs of neurons in LSOs along the pharynx, similar to the number observed for mechanosensory neurons. Most of those neurons are likely 'generalist' and are tuned to mechanical stimuli only. It would be valuable to determine the full repertoire of these sensory neurons to understand how the swallowing maneuver is initiated, sustained, and terminated (Yang, 2021).

It has been proposed that IN1 neurons may function as a key node of the feeding control circuits to govern rapid feeding decisions. Previous studies have revealed that IN1 neurons are directly downstream of pharyngeal GRNs and that activation of IN1 neurons to sugar stimulation is correlated with a fly's motivation to feed. Because activation of IN1 neurons triggers proboscis extension to food, they are likely upstream of the motor circuit that controls feeding. IN1 neurons thus appear to function as a hub that integrates sensory information to initiate food ingestion. This study found that IN1 neurons' activity is under control of the fly's feeding states. IN1 neurons are directly downstream of DSOG1 neurons that non-selectively suppress ingestion. In fed flies, DSOG1 neurons impart inhibition on IN1 neurons, resulting in a transient and moderate response to a sugar sip that triggers a robust and sustained calcium response in fasted flies (Yang, 2021).

It has been proposed that DSOG1 neurons impart constant inhibition on the neuronal circuits that initiate food ingestion. However, the upstream circuit of DSOG1 neurons has not been identified. A cohort of neuropeptide receptor genes has been screened, but none of them seemed to function on DSOG1 neurons in feeding control. This study found that interrupting signaling of the neuropeptide MIP phenocopied overfeeding in flies with silenced DSOG1 neurons. It is tantalizing to hypothesize that MIP neurons are upstream of the DSOG1 circuit, either directly or indirectly. Because the receptors of MIP have not yet been identified, further experiments are need to differentiate between the two possibilities (Yang, 2021).

Besides PM neurons, there are many NOMPC-expressing mechanosensory neurons along the fly pharynx. Because of the lack of specific driver lines and the technique to record a single neuron's activity during feeding, their roles in feeding regulation are interesting open questions and await in-depth investigation. Moreover, the receptors of MIP peptide have not been identified, especially the ones involved in feeding regulation, making it difficult to establish a connection between MIP neurons and DSOG1 neurons (Yang, 2021).

Drosophila auditory organ genes and genetic hearing defects

The Drosophila auditory organ shares equivalent transduction mechanisms with vertebrate hair cells, and both are specified by atonal family genes. Using a whole-organ knockout strategy based on atonal, this study has identified 274 Drosophila auditory organ genes. Only four of these genes had previously been associated with fly hearing, yet one in five of the genes that were identified has a human cognate that is implicated in hearing disorders. Mutant analysis of 42 genes shows that more than half of them contribute to auditory organ function, with phenotypes including hearing loss, auditory hypersusceptibility, and ringing ears. This study not only discovered ion channels and motors important for hearing, but also showed that auditory stimulus processing involves chemoreceptor proteins as well as phototransducer components. These findings demonstrate mechanosensory roles for ionotropic receptors and visual rhodopsins and indicate that different sensory modalities utilize common signaling cascades (Senthilan, 2012).

Mechano-, photo-, and chemo-receptors are developmentally specified by bHLH transcription factors across taxa. Using the Drosophila Johnston's organ (JO) as an example, this study has shown that null alleles of these transcription factors provide a background against which the genetic repertoire of the respective receptors can be defined. Ion channels and motors for fly hearing are identified that, judging from mutant phenotypes, contribute to auditory signal transduction. Some of the newly defined genes for hearing are also found in vertebrate cochleae, extending the genetic parallels between the ears of vertebrates and flies. In the fly, photo- and chemoreceptor proteins are expressed in the auditory organ and contribute to sound detection, adding new levels of complexity to auditory signal processing and shedding light on the evolution of ato-dependent receptor organs and sensory signaling cascades (Senthilan, 2012).

Force-gated ion channels and adaptation motors are key constituents of auditory transduction modules, and their interplay provides mechanical amplification in the fly's ear. The best candidate for the fly's auditory transducer is the NOMPC TRPN1 channel, whose Caenorhabditis elegans ortholog is a bona fide mechanotransduction channel, which itself is essential for mechanical amplification. Because JO neurons are ciliated and because their cilia seem to bear dynein arms, these cells were surmised to use axonemal dyneins as adaptation motors. The current analysis shows that auditory phenotypes as reported for nompC nulls also result from mutations in TRPC (TRP, TRPL) and TRPA (WTRW) channels, including the loss of mechanical amplification and sensitive nerve responses. The current analysis also shows that several axonemal dyneins are expressed in -- and essential for the function of -- JO neurons and that mutations in, e.g., the axonemal dynein gene CG9313 lead to auditory defects as observed in nompC nulls. Collectively, the results thus support axonemal dyneins as the presumptive adaptation motors in JO neurons and identify TRP channels (TRP, TRPL, WTRW) that, judging from their requirements for transducer-based amplification, contribute to transduction in the ear of the fly (Senthilan, 2012).

Although JO neurons and hair cells are endowed with different sensory organelles and presumably use different channels and motors for auditory transduction and amplification, this analysis confirms and extends the genetic parallels between the ears of vertebrates and flies: 89 of the 274 JO genes have vertebrate homologs, and several of these homologs occur in vertebrate ears: of the 27 auditory relevant JO genes, for example, calmodulin is found in hair cells where it regulates transducer adaptation. This adaptation actuates active hair bundle motions, which promote (or contribute to) cochlear amplification in vertebrate ears: low Ca2+ concentrations enhance amplification and lead to self-sustained hair bundle oscillations, consistent with the hyperamplification and ringing caused by mutations in Drosophila Cam. Hyperamplification also ensues from mutations in bw, which encodes an ATP-binding cassette (ABC) transporter. ABCs also occur in the mouse cochlea, but whether they contribute to cochlear function is unclear (Senthilan, 2012).

Also TRPC channels are found in vertebrate cochleae and outer hair cells reportedly express TRPC3. These cells also display a TRPC-like conductance that contributes to Ca2+ homeostasis and is activated via diacylglycerol (DAG). This study found that mutations in the Drosophila DAG kinase gene rdgA cause auditory phenotypes as observed in TRPC channel mutants, and judged from the RIKEN full-length enriched cDNA library a related DAG kinase, DGKZ, is expressed in the mouse inner ear. The same library also includes ZSCAN22, the homolog of gl, mutations in which impair JO function. DYX1C1, the homolog of the newly defined Drosophila deafness gene CG14921, in turn, is present in the Wackym-Soares normalized rat vestibular cDNA library, which also includes Ank2 and the Arr2 homolog Arrb1. DYX1C1 is also expressed in the zebrafish otic vesicle, as is zgc:63660, the zebrafish homolog of the Drosophila deafness gene CG11253. Several of the fly genes for hearing thus seem present in vertebrate cochleae, putting forward new candidates for auditory relevant vertebrate genes (Senthilan, 2012).

ato, apart from specifying chordotonal organs, directs the formation of Drosophila photoreceptors and chemosensory coeloconic sensilla. All these receptors are thought to have evolved from an ato-dependent 'protosensory' organ that presumably consisted of chordotonal sensilla because they are serially arranged along the body and distributed widely among arthropod groups. Photoreceptors detect light with Rhs and coeloconic chemoreceptors detect volatile chemicals with IRs. The moderate auditory defects caused by mutations in ionotropic receptors, along with the expression of Ir94b in JO supporting cells, suggest that these ion channels indirectly modulate JO neuron function, possibly by contributing to ion homeostasis in JO. Rhs, by contrast, are expressed in JO neurons and their disruption gravely impairs neuron function. Equally severe phenotypes result from the disruption of INAD, which holds together the fly's visual transduction complex. Judging from this analysis, many components of this complex are expressed in JO, and Rhs occur in JO cilia and are required for proper mechanotransduction channel gating. Rhs, apart from sensing photons, have recently been put forward as thermosensors, documenting that they serve sensory functions other than detecting light. The involvement of IRs and Rhs in mechanosensory chordotonal organ function now suggests that these proteins already served roles in sensation before chemo- and photoreceptors have diversified. Given the presumed closeness of the 'protosensory' organ and chordotonal organs, it is anticipated that dissecting IR and Rh functions in JO may help defining archetypical roles of these proteins, with the prospect of gaining a molecular understanding of how sensory modalities and signaling cascades evolved (Senthilan, 2012).

The neural basis of Drosophila gravity-sensing and hearing

The neural substrates that the fruitfly Drosophila uses to sense smell, taste and light share marked structural and functional similarities with ours, providing attractive models to dissect sensory stimulus processing. This study focused on two of the remaining and less understood prime sensory modalities: graviception and hearing. The fly has implemented both sensory modalities into a single system, Johnston’s organ, which houses specialized clusters of mechanosensory neurons, each of which monitors specific movements of the antenna. Gravity- and sound-sensitive neurons differ in their response characteristics, and only the latter express the candidate mechanotransducer channel NompC. The two neural subsets also differ in their central projections, feeding into neural pathways that are reminiscent of the vestibular and auditory pathways in the human brain. By establishing the Drosophila counterparts of these sensory systems, these findings provide the basis for a systematic functional and molecular dissection of how different mechanosensory stimuli are detected and processed (Kamikouchi, 2009).

To gain first insights into the molecular mechanisms that account for the functional differences between deflection- and vibration-sensitive JO neurons, which JO neurons express the candidate mechanotransducer channel NompC (no mechanoreceptor potential C, also known as TRPN1) was analyzed. To identify nompC-expressing neurons, GAL4 was expressed under the control of the nompC promoter (nompC-GAL4) (Liu, 2007). In contrast to F-GAL4, which expresses Gal4 under the control of the nanchung promoter and labels almost all JO neurons, only some JO neurons were labelled by nompC-GAL4. Projection analysis revealed that nompC-GAL4 labels JO neurons of a limited number of the five subgroups that target distinct zones of the antennal mechanosensory and motor centre in the brain. Hence, whereas the TRPV channel Nanchung is expressed by almost all JO neurons, the TRPN channel NompC seems specific for sound-sensitive JO neurons (subgroups AB but not CE of JO neurons). This differential expression presumably explains why disrupting NompC reduces, but does not abolish, mechanically evoked responses in the fly’s antennal nerve, supporting NompC as a candidate mechanotransducer for hearing and indicating that gravity transduction is independent of NompC (Kamikouchi, 2009).

As judged from their central projections, gravity- and sound-sensitive JO neurons target distinct primary centres in the antennal mechanosensory and motor centre (AMMC) in the brain and feed into distinct brain circuits. To trace these circuits, 3,939 GAL4 enhancer trap lines were screened for higher-order neurons in the Drosophila brain that arborize in the AMMC. The target zones of subgroups A and B in the AMMC, which form the primary auditory centres, are both characterized by a close association with the inferior part of the ventrolateral protocerebrum (VLP), which is also directly supplied by a subset of subgroup-A and can be regarded as the secondary auditory centre: various interneurons were identified that arborize in both the VLP and the target zones of subgroups AB in the AMMC. These zones are also characterized by extensive commissural connections, with interneurons connecting the contralateral zones by means of commissures above and below the oesophagus. Also the giant fibre neuron (GFN), a large descending neuron that controls jump escape behaviour, arborizes in zone A and in the inferior VLP. The GFNs of both sides are connected by means of the giant commissural interneurons, a feature not observed in the other descending neurons. All higher-order neurons identified arborized only in the target zone of either subgroup A or B, pointing to a parallel organization of the auditory pathway that might explain why silencing only one subgroup of vibration-sensitive neurons suffices to abolish the flies’ sound-evoked behaviour (Kamikouchi, 2009).

Aside from a few JO neurons of subgroups CE that directly cross the midline, no commissural connections were found between the target zones of subgroups CE. No connections between these zones and the ventrolateral protocerebrum (VLP) were identified either. These zones, however, were abundantly contributed to by descending and ascending neurons to and from the thoracic ganglia. Together, the tight commissural connection in the pathways downstream of sound-sensitive JO neurons and abundant descending tracts downstream of gravity-sensitive JO neurons are reminiscent of the connectivities of mammalian auditory and vestibular pathways, the former of which has extensive binaural interactions between the secondary centres of both hemispheres whereas the latter has direct descending pathways from the primary centre to the spinal cord (Kamikouchi, 2009).

Housing almost 480 primary mechanosensory neurons, JO is the largest mechanosensory organ of the fruitfly. This study has shown that the JO organ serves at least two mechanosensory submodalities that are segregated at the level of the primary neurons. JO neurons of subgroups AB respond preferentially to antennal vibrations; they differ in their frequency characteristics, express the NompC channel, and have a role in sound detection. JO neurons of subgroups CE respond preferentially to static deflections, provide information about the forcing direction, do not express the NompC channel, and are required for gravity sensing. As judged from imaging data and antennal nerve recordings, JO neurons of subgroups CE respond to tiny displacements imposed by the Earth’s gravitational field. Subgroups-CE neurons also respond to large antennal displacements as may be imposed by air jets or wind , indicating either that the same subgroups-CE neurons mediate gravity and wind detection or, alternatively, that sensitive, gravity-responsive CE neurons and less-sensitive, wind-responsive CE neurons may coexist (Kamikouchi, 2009).

Since all JO neurons attach to the same antennal receiver, how do their distinct response characteristics come about? The opposing calcium signals evoked by receiver deflections are likely to reflect the opposing connections of JO neurons with the antennal receiver, indicating that these neurons are hyperpolarized by compression and depolarized by stretch. The vibration- and deflection-sensitivities of distinct JO neuron subgroups may reflect differences in the molecular machineries for transduction; JO neurons reportedly harbour adapting channels that transduce dynamic receiver vibrations but fully adapt within milliseconds during static receiver deflection. Because deflecting the receiver statically for several seconds evokes sustained large-amplitude calcium signals in subgroups CE, however, also less- or non-adapting channels seem to exist. Transduction channels with different adaptation characteristics seem to occur in many mechanosensory systems, including the mammalian cochlea and also Drosophila bristle neurons, which reportedly display mechanically evoked adapting, NompC-dependent and also non-adapting, NompC-independent currents (Walker, 2000). In the fly's JO, such functional and molecular specializations of the transduction machineries could explain why some neurons preferentially respond to gravity whereas others preferentially respond to sound. The segregation of gravitational and auditory stimuli in the Drosophila JO may thus take place at the very first stage of neuronal signal processing (Kamikouchi, 2009).

TRPA channels distinguish gravity sensing from hearing in Johnston's organ

Although many animal species sense gravity for spatial orientation, the molecular bases remain uncertain. Therefore, Drosophila, which possess an inherent upward movement against gravity-negative geotaxis, was studied. Negative geotaxis requires Johnston's organ, a mechanosensory structure located in the antenna that also detects near-field sound. Because channels of the transient receptor potential (TRP) superfamily can contribute to mechanosensory signaling, it was asked whether they are important for negative geotaxis. Distinct expression patterns were discovered for 5 TRP genes; the TRPV genes nanchung and inactive were present in most Johnston's organ neurons, the TRPN gene nompC and the TRPA gene painless were localized to 2 subpopulations of neurons, and the TRPA gene pyrexia was expressed in cap cells that may interact with the neurons. Likewise, mutating specific TRP genes produced distinct phenotypes, disrupting negative geotaxis (painless and pyrexia), hearing (nompC), or both (nanchung and inactive). These genetic, physiological and behavioral data indicate that the sensory component of negative geotaxis involves multiple TRP genes. The results also distinguish between different mechanosensory modalities and set the stage for understanding how TRP channels contribute to mechanosensation (Sun, 2009).

These data show that several TRP superfamily channels in Johnston's organ are required for Drosophila to respond to gravity. nan and iav are expressed in chordotonal neurons throughout Johnston's organ, and their mutation disrupted both hearing and geotaxis. Previous studies of hearing suggested that Nan and Iav form a TRPV channel that acts downstream of the primary mechanotransducer and enhances the relay of excitatory signals toward the cell body. It is speculated that they may serve a similar role in gravity sensing (Sun, 2009).

In contrast to nan and iav, pain expression was limited to a subset of chordotonal neurons, and its mutation disrupted negative geotaxis, but not hearing. Might the Pain channel be a mechanosensor? This possibility is intriguing because the Pain channel also mediates mechanical nociception in fly larvae. However, pain also contributes to thermal and chemical nociception in larvae and heat-induced currents when expressed in heterologous cells, and thus a specific role in detecting mechanical stimuli remains speculative (Sun, 2009).

The contribution of pyx to geotaxis was distinct from the other TRP subunits by its expression in cap cells rather than neurons. Cap cells form structural links between chordotonal neurons and the moving joint between second and third antennal segments. Interestingly, in the cap cells of the katydid Caedicia simplex auditory sensilla, acoustic stimuli generate a slow, graded hyperpolarizing membrane potential and a train of fast, biphasic spikes, and the spikes correlated temporally with depolarizing spikes in the chordotonal sensory neurons. Those results raise the possibility that in Drosophila gravity perception cap cells and their Pyx channels might actively respond to motion and participate in mechanosensory signaling. In addition, cap cells in Drosophila larval chordotonal organs contain numerous aligned microtubules, and motility of those microtubules is thought to modulate tension in the chordotonal organ. Thus, another possibility is that Pyx channels trigger contraction or microtubule motility in Johnston's organ cap cells to influence gravity signals (Sun, 2009).

Pitch, roll, and yaw in either direction trigger action potential firing from Johnston's organ. Once the rotation stops, the electrical activity returns to basal levels. Interestingly, recent data indicated that continuous mechanical displacement of the arista to mimic the effect of gravity (or wind) induces a tonic increase in intracellular Ca²⁺ concentration, [Ca²⁺]_i, in some Johnston's organ neurons. Methodological factors likely explain the apparent difference in phasic vs. tonic responses. First, the dynamic level of [Ca²⁺]_i may not precisely predict the timing of action potentials in a neuron, and thus phasic action potential firing is not necessarily inconsistent with a tonic elevation of [Ca²⁺]_i. Second, in this study body rotations mimicked real-life experience of a fly moving in the gravitational field, and the antennal receiver (third segment including the arista) was free to respond to transient angular accelerations accompanying body rotations. Elastic properties of the antenna and muscle control of antennal movement might have played a role in these studies. In studies measuring [Ca²⁺]_i, a probe statically controlled the position of the antennal receiver, and the first and second antennal segments were immobilized to prevent muscle-based antennal movement. In the future, a comparison between [Ca²⁺] signals and action potential firing in the same experimental setting may yield a better understanding of how Johnston's organ codes sensory information of gravity, acceleration, and orientation (Sun, 2009).

This study also has limitations. First, a caveat to the gene expression data are use of promoter-Gal4 transgenic constructs. A putative enhancer/promoter fragment may not contain the complete information to precisely reproduce endogenous gene expression. Moreover, the location of a transgene in the fly genome may affect expression patterns and levels. In the future, it will be desirable to examine the anatomical and subcellular localization of these TRP channels with specific antibodies. Second, the nompC and pain lines were hypomorphs rather than nulls. Third, although the rotating device for electrophysiological recordings has the advantage that it may mimic gravitational changes, it could introduce unappreciated vibration of the antennal receiver. In addition, programmable control of movement would be desireable (Sun, 2009).

These data combined with recent studies indicate that geotaxis, hearing, and wind detection use the same general structures, Johnston's organ scolopidia and its chordotonal neurons and support cells. They also indicate that subsets of these structures are specialized for distinct senses. Based on the observations that the promoter-Gal4 constructs of pain, pyx, and nompC label different cell populations in Johnston's organ and that their mutants display specific defects in either geotaxis or auditory tests, it is speculated that the expression and function of these different TRP channels may be key for Johnston's organ to distinguish between gravity and sound (Sun, 2009).

Specification of auditory sensitivity by Drosophila TRP channels

Ears achieve their exquisite sensitivity by means of mechanical feedback: motile mechanosensory cells through their active motion boost the mechanical input from the ear. Examination of the auditory mechanics in Drosophila mutants shows that the transient receptor potential (TRP) channel NompC is required to promote this feedback, whereas the TRP vanilloid (TRPV) channels Nan and Iav serve to control the feedback gain. The combined function of these channels specifies the sensitivity of the fly auditory organ (Göpfert, 2006).

In mechanosensory cells for hearing, several transient receptor potential (TRP) ion channels coexist. Drosophila auditory neurons, for example, putatively express NompC (also known as TRPN1) - a channel that has not yet been definitely shown to localize to these cells - and demonstrably express the two interdependent vanilloids (TRPVs) Nan and Iav (encoded by CG4536, or iav), which are deemed to form a heteromultimeric Nan-Iav channel. Likewise, in vertebrate auditory hair cells, NompC and/or TRPA1 seem to occur along with TRPV1 and TRPV4. Most of these TRPs have been implicated in mechanosensory transduction, yet their relative positions in the auditory pathway and their respective contributions to the process of hearing are little understood. Mechanosensory transduction in the context of hearing is linked to nonlinear amplification: the mechanosensory cells that mediate hearing actively generate motions to specifically augment the minute, sound-induced vibrations that they transduce. This mechanical feedback was assayed in the ears of Drosophila nompC, nan and iav mutants to gain insights into the diverse auditory roles of TRPs (Göpfert, 2006).

To test for nonlinear amplification, the antennal sound receivers of wild-type flies were exposed to pure tones of different intensities. The frequency of the tones was adjusted to match the individual best frequency of each receiver. The intensity of the tones was monitored as the stimulus particle velocity at the receiver's position, and the resulting mechanical response of the receiver was measured as the Fourier amplitude of its displacement at the frequency of stimulation. This phase-locked response linearly scaled with intensity for stimulus particle velocities less than ~0.1 mm s^-1 and greater than ~10 mm s^-1. For intermediate particle velocities (0.1–10 mm s^-1), however, a nonlinear scaling was found: the response of the receivers increased with only the two-third power of the stimulus intensity, leading to an ~10-fold gain in sensitivity to low intensity sounds. This nonlinear amplification, which was frequency specific like that in vertebrate hearing, was affected by the disruption of TRPs. The null allele nompC³ abolished nonlinear amplification, leading to linear responses of constantly low sensitivity throughout the intensity range. This loss of amplification was observed in homozygous nompC³cn bw mutants and when the nompC³ mutation was uncovered by a deficiency (Df(2L)cl^h2), but not in homozygous cn bw and balanced nompC³cn bw/Cy cn and Df(2L)cl^h2/Cy cn controls. The opposite effect - that is, facilitation of amplification - was found for null alleles in nan and iav: in homozygous nan^36a and iav¹ mutants, the receiver's response nonlinearly increased with approximately the one-third power for intermediate particle velocities, boosting the sensitivity gain to ~85. This excessive amplification, which was also observed in homozygous nan^dy5 and iav³⁶²¹ mutants but not in balanced controls, regressed toward that in the wild type (gain ~14) when a single wild-type transgene of iav was expressed in the iav¹ mutant background. Hence, whereas NompC is required for amplification, Nan-Iav is required to adjust the amplificatory gain. The excess amplification caused by the disruption of Nan-Iav was found to depend on NompC function: in nompC³; nan^36a double mutants, amplification was completely abolished, leading to a linear response as found for the single mutation nompC³. nompC³ is thus epistatic to nan^36a, placing NompC downstream of Nan-Iav in the regulatory pathway that, by controlling the extent of nonlinear amplification, specifies the mechanical sensitivity of the ear (Göpfert, 2006).

A striking signature of amplification in hearing is sound that is spontaneously emitted by some ears. This ringing sound, which in rare cases can be loud enough to be heard by passersby, is deemed to reflect self-sustained oscillations caused by excessive mechanical feedback in the ear. It was found that self-sustained oscillations arise from the disruption of TRPVs. In nan and iav mutants, but not in nompC³ mutants, wild-type flies or controls, the receiver continuously oscillated in the absence of acoustic stimulation, with displacement amplitudes as large as 0.5 microm. The oscillations were sharply tuned, giving rise to a sharp peak in the power spectrum at frequencies between approximately 100 Hz and 250 Hz and increasing the fluctuation power, measured as the total power spectral density in the frequency band between 100 Hz and 1,500 Hz, by a factor of ~155. In nompC³; nan^36a double mutants, the oscillations were abolished and the fluctuation power was nearly as low as in single nompC³ mutants. Hence, both the excess amplification and the self-sustained oscillations arising from the disruption of Nan-Iav require NompC function; by negatively controlling the gain of NompC-dependent amplification, Nan-Iav prevents oscillations in the ear (Göpfert, 2006).

Mutations in nan and iav are the first genetic defects shown to cause excessive mechanical feedback in hearing and ringing in the ear. This ringing, like many chronic forms of tinnitus, is associated with hearing loss: nan and iav mutants reportedly lack sound-induced afferent nerve responses and, accordingly, are deaf. Because of this deafness and the pressure activation of heterologously expressed Nan and Iav, the prevailing view is that these TRPVs serve as the fly's transducer channel for hearing. These findings do not support this view: transducers are essential components of amplificatory feedback loops; loss of transducer function will inevitably disrupt the feedback and abolish amplification, much as is found for mutations in nompC. Hence, both the requirement for feedback amplification and the functional placement of NompC in the auditory pathway support NompC, but not Nan-Iav, as a candidate transducer channel for hearing in the Drosophila ear. As disrupting NompC reduces, but does not abolish, afferent nerve responses, this channel may well participate in transduction, but it is unlikely to be the only transducer component the fly uses to hear. The complete loss of nerve responses in nan and iav mutants, in turn, suggests that signaling by Nan-Iav is bidirectional, controlling mechanical amplification in a NompC-dependent manner and propagating electrical signals to the nerve. Both functions may be Ca²⁺ mediated: like mammalian TRPV4, heterologously expressed Nan and Iav form Ca²⁺-permeable channels and promote Ca²⁺ spikes. As Ca²⁺-regulated amplification and TRP-dependent mechanosensation also occur in vertebrate hearing, the auditory roles of TRPs revealed by this study may not be restricted to flies: spontaneous sound emissions from the ears of vertebrate TRPV knockouts have not yet been reported, but listening for such sounds seems warranted (Göpfert, 2006).

Hypothesis: A helix of ankyrin repeats of the NOMPC-TRP ion channel is the gating spring of mechanoreceptors

Sensory reception by mechanically sensitive cells is mediated by an ion channel coupled to a molecular spring (Gillespie, 2001). This gating spring is thought to transmit an external force (e.g. from a sound wave or a tactile stimulus) to the ion channel. The force alters the probability of the ion channel being open. This produces an electrical signal that is conveyed to the central nervous system. One such channel has recently been identified as a member of the NOMP-C subfamily of TRP channels. However, the molecular identity of the gating spring remains unknown. This paper proposes that the gating spring is a helix (or bundle of helices) formed by the 29 ankyrin domains of this mechanoreceptive subfamily of TRP channels (Howard, 2004).

The NOMP-C proteins in the bristle sensilla of Drosophila and in the inner ear and lateral line of zebrafish contain 29 consecutive ankyrin domains (ANK repeats) at their amino termini. A similar channel has been identified in worms. Crystallographic studies of a fragment containing 12 ANK repeats revealed a highly regular structure that forms approximately 40% of one turn of a helix. Extrapolation of this structure suggests that 29 ANK repeats would form almost exactly one helical turn. This hypothetical structure is called the 'ANK helix'. Depending on the number of NOMP-C subunits, the channel complex may contain up to four ANK helices surrounding the intracellular face of the pore-forming region of the channel. It is suggested that an arrangement of the ANK helices with their axes perpendicular to the plasma membrane has several mechanical and structural properties that would ideally suit a gating spring (Howard, 2004).

First, a full turn of a helix allows transmission of force from one end of the structure to the other without creating a torque. This geometric constraint provides an explanation for the remarkable conservation of exactly 29 ANK repeats in this otherwise highly divergent protein subfamily found in worms, flies and fish (Howard, 2004).

Second, the stiffness of an ANK helix is expected to be similar to that measured for the gating spring. The stiffness k of a helical spring composed of a material with shear modulus G is given by k=Gr⁴/4NR³, where r is the radius of the material, N is the number of turns and R is the radius of the helix. Substituting values of G = 1 GPa (characteristic of a rigid protein, as well as r = 0.75nm, N = 1 and R = 4.5 nm from the structural model gives k = 0.9 mN/m (1 mN/m = 1 pN/nm). Four ANK helices in parallel would be four times as stiff. This is an upper limit to the stiffness, as structural defects would tend to increase the compliance. This stiffness is similar to the stiffness of the elastic element of fly bristle hair receptors (3 mN/m), as well as to that of of the gating spring in vertebrate hair cells (~1 mN/m) (Howard, 2004).

Third, the ANK helix should be highly deformable and should be able to withstand compression or extension by a factor of two, even though the constituent protein cannot be deformed more than a few percent without yielding. Thus, the ~20 nm long ANK helix should act as a linear spring for compressions and extensions of up to 10-20 nm. From a mechanical point of view, the importance of the postulated arrangement is that the ANK helices are expected to contribute perhaps most of the compliance of the mechanosensory gating apparatus. This compliance is necessary to allow the channel to fluctuate rapidly between closed and open states such that small deformations of the cell will lead to a graded change in the opening probability (Howard, 2004).

The model locates the gating spring on the intracellular side, directly adjacent to the structural elements that are thought to constitute the gate of channels in the potassium-channel superfamily. This location accords with the ultrastructure of the arthropod sensillum, which shows no obvious extracellular compliant elements. By contrast, on the intracellular side of the plasma membrane there are 'membrane-integrated cones', which connect the plasma membrane to the microtubule cytoskeleton in the tubular body, the likely site of mechanotransduction. The resting dimension of a cone corresponds to that of a cluster of ANK helices. Because these cones are compressed by excitatory deflections of the bristle and stretched by inhibitory deflections, it is predicted that compression of the ANK helix in bristle receptors will open the TRP channel (Howard, 2004).

The ANK helices may be the gating springs for vertebrate hair cells, although there are several caveats. First, an intracellular location of the gating spring contradicts the prevailing view that extracellular filaments, termed tip links, are the gating springs of hair cells. However, this view has recently been challenged by electron microscopy of the tip link that showed a structure that is probably too rigid and inextensible to serve as the gating spring. On the other hand, the 'insertional plaques', located where the tip links connect to the plasma membranes and the underlying actin cytoskeleton, appear quite compliant and are large enough to accommodate a bundle of ANK helices. Thus, the hypothesis that the ANK helix is the gating spring is not inconsistent with the ultrastructure of the hair cell. A second potential problem is that because the vertebrate channels open in response to tension in the tip link (caused by shear between the stereocilia), the hypothesis predicts that it is extension, rather than compression, of the ANK helix that gates the channel open. How structural differences between the vertebrate and invertebrate channels could reverse the sign of gating is not clear. The third caveat is that the vertebrate gating spring is highly extensible: it has constant stiffness up to ~50 nm and can extend up to 100 nm. While these large extensions could be achieved if the ANK repeats were to unfold like the Immunoglobulin repeats of titin, thereby protecting the channel from damage due to large deformations of the hair bundle, it is not clear that the dynamics and nonlinearity of protein folding and unfolding are compatible with the gating process. A final caveat is that genes encoding TRP channels with 29 ANK repeats have not been found in the human or mouse databases (Howard, 2004).

The promiscuity of the ANK protein-protein interaction domain could allow connection to the microtubule cytoskeleton in invertebrate mechanoreceptors and to the actin cytoskeleton in hair cells. In the case of the hair cell, the channel may connect to the actin filaments via the myosin motors that mediate adaptation. The large number of ANK repeats in a tetramer of ANK helices could provide the scaffold necessary for assembling the large ensemble of myosins hypothesized to form the adaptation apparatus in hair cells, though the binding of the motors will decrease the compliance of the spring (Howard, 2004).

In summary, it is postulated that the ANK helical bundle forms the gating spring, a compliant element that transmits an externally derived force to the molecular gate of a mechanoreceptive ion channel. In this view, the ANK helix is functionally homologous to the ligand-binding domain of calcium-gated potassium channels. It is possible that the ANK repeats found in other TRP channels are not simply anchoring domains, but play an active role by transmitting mechanical force to the channel's gate. Even ankyrin itself, which contains 24 ANK repeats that likely form a nearly complete helix, may function to transmit tension from the cytoskeleton to ion transporters so that ion flux across the plasma membrane can be regulated by the mechanical state of the cell (Howard, 2004).

TRPN gene family founding member no mechanoreceptor potential-C is essential for Drosophila mechanosensation

Mechanosensory transduction underlies a wide range of senses, including proprioception, touch, balance, and hearing. The pivotal element of these senses is a mechanically gated ion channel that transduces sound, pressure, or movement into changes in excitability of specialized sensory cells. Despite the prevalence of mechanosensory systems, little is known about the molecular nature of the transduction channels. To identify such a channel, Drosophila mechanoreceptive mutants were analyzed for defects in mechanosensory physiology. Loss-of-function mutations in the no mechanoreceptor potential C (nompC) gene virtually abolishes mechanosensory signaling. nompC encodes a new ion channel that is essential for mechanosensory transduction. As expected for a transduction channel, D. melanogaster NOMPC and a Caenorhabditis elegans homolog were selectively expressed in mechanosensory organs (Walker, 2000).

Our capacity to hear a whisper across a crowded room, detect our position in space, and coordinate our limbs during a stroll through the park is conferred by the mechanical senses. Mechanosensory transduction is the process that converts mechanical forces into electrical signals. When mechanoreceptors are stimulated, mechanically sensitive cation channels open and produce an inward transduction current that depolarizes the cell. The opening of mechanosensory transduction channels in vertebrate hair cells takes place within a few microseconds after the onset of a stimulus, too quickly for the generation of second messengers. Mechanical stimuli are therefore hypothesized to directly gate these channels. This mode of activation is in sharp contrast to other sensory modalities, such as vision, olfaction, and taste, which use stereotypical G protein-coupled cascades to modulate transduction channels (Walker, 2000).

Most models of mechanosensory signaling propose that transduction channels be anchored on both sides of the membrane, so that relative movements between the extracellular matrix and the cytoskeleton produce the mechanical tension that gates these channels. In the gating-spring model of mechanosensory transduction in vertebrate hair cells, deflection of the mechanically sensitive hair bundle produces shear between adjacent stereocilia that stretches the gating springs. This increase in tension 'pulls' the transduction channels open, depolarizes the cell, and triggers neurotransmitter release. Although biophysical data support this model for transduction in hair cells, the molecular identity of the mechanically gated ion channel remains unknown. This is largely due to the paucity of sensory tissue and the small number of transduction channels in each hair cell (Walker, 2000).

Genetic approaches are ideally suited for identifying rare molecules involved in mechanosensory transduction. The isolation of genetic mutations does not depend on any assumptions about the nature or abundance of the target molecules, other than loss of their function results in a recognizable phenotype. The most extensive genetic dissection of mechanosensory behavior was based on screens for Caenorhabditis elegans touch-insensitive mutants. These studies identified genes involved in the development, survival, function, and regulation of touch receptor neurons. Of particular interest were those that likely function in the mechanoelectrical transduction process. This group included degenerins, collagen, stomatin, and tubulins, a finding consistent with the expectation that mechanosensory signaling involves finely orchestrated interactions between ion channels, extracellular matrix, and cytoskeletal components (Walker, 2000).

Degenerins (MEC-4, MEC-10, DEG-1, UNC-8, and UNC-105) are a family ofC. elegans ion channels related to vertebrate epithelial sodium channels. Because of their critical role in the touch receptor neurons, degenerins have been proposed to function as mechanosensory transduction channels. More recently, a C. elegans transient receptor potential (TRP) family member, OSM-9, was shown to be involved in mechanotransduction because it is expressed in sensory dendrites of a subset of ciliated sensory neurons and is required for osmosensation and nose touch (Colbert, 1997). Although these genetic studies demonstrated the requirement for degenerins and OSM-9 in mechanoreception, there are no electrophysiological data supporting a role for these channels in the actual transduction process (Walker, 2000).

Drosophila is an attractive model to dissect mechanosensation because it is possible to combine genetic manipulations with electrophysiological recordings from mechanoreceptor neurons. The fly's mechanosensory repertoire includes touch, proprioception, and hearing, mediated by the complement of sensory bristles, campaniform sensilla, chordotonal organs, and type II mechanoreceptors. Of these, sensory bristles are particularly amenable to physiological manipulation in the intact animal. Each mechanosensory bristle organ is composed of a hollow hair shaft whose base impinges on the dendritic tip of a bipolar sensory neuron. The shaft thus acts as a tiny lever arm in which deflections of the external bristle compress the neuron's dendritic tip and gate the transduction channels. The mechanosensory dendrite is bathed in an unusual high-K⁺, low-Ca²⁺ fluid, which provides a large positive driving force into the neuron; opening of transduction channels depolarizes the cell and promotes neurotransmitter release (Walker, 2000).

To identify components of the mechanotransduction machinery, Drosophila touch-insensitive and proprioceptive mutants were screened for defects in the physiology of mechanosensory responses. Those mutants that most likely defined transduction molecules were then characterized (Walker, 2000).

To gain electrical access to the sensory neuron, the tip of the hollow sensory bristle was removed, a recording/stimulation pipette was placed over its end, and calibrated mechanical stimuli were delivered while recording transduction currents with a voltage-clamp apparatus. Responses from wild-type Drosophila, were analyzed focusing on electrophysiological features that characterize vertebrate mechanosensory transduction systems: directional sensitivity, steep displacement-response relations, submillisecond latencies between stimulus and response, and sensitivity to displacements of only a few angstroms (Walker, 2000).

Asymmetries in the ultrastructure and transduction machinery of vertebrate mechanosensory organs endow them with directional sensitivity. It was reasoned that similar asymmetries may confer directional selectivity to fly bristles. Mechanoreceptor currents (MRCs) were recorded from macrochaete bristles throughout the thorax, and all displayed strong directional sensitivity. For instance, when an anterior notopleural bristle was deflected toward the surface of the body, it generated a robust response. In contrast, stimuli in all other directions elicited minimal transduction currents. Hereafter, stimuli in the excitatory direction will be referred to as 'positive,' and those in the opposite direction will be referred to as 'negative' (Walker, 2000).

To characterize the range of responses of a macrochaete, sensory bristles were given positive and negative step stimuli that ranged between +35 and -17.5 microm. During positive displacements, a transient increase was recored in the MRC that peaked at ~210 pA and was followed by a gradual, but incomplete, decline to the resting current level. During negative displacements, only a small negative MRC was observed (-6 pA). Because the neuron adapted to this new negative position, the return of the bristle to its resting state is sensed as a positive deflection and results in a concomitant 100-pA transient current. A displacement-response curve derived from 20 thoracic bristles was fitted using a three-state model; the results showed that the mechanoreceptor neuron is most sensitive to stimuli between 0 and 10 microm and saturates at ~35 microm (Walker, 2000).

Recording of fly mechanoreceptor responses under conditions that allow the detection of microsecond-scale events showed latencies of ~200 micros. Because this response time is ~100 times as fast as the fastest known second-messenger cascade, fly mechanosensory transduction is unlikely to rely on second messengers (Walker, 2000).

Vertebrate hair cells detect mechanical stimuli of atomic dimensions. Although it was not possable to deliver displacements this small, small transduction currents were elicited by stimuli of only 100 nm. Because of the lever action of the bristle shaft, however, a 100-nm stimulus at the end of a cut bristle produces a much smaller displacement at the neuronal dendritic tip. On the basis of the geometry of the fly macrochaete bristles, it is estimated that the corresponding displacement at the base of the bristle would be ~50-fold less, or 2 nm. This level of sensitivity would allow the neuron to perceive displacements of only one-half the thickness of its plasma membrane (Walker, 2000).

Adaptation permits mechanoreceptors to continuously adjust their range of responsiveness, thus enabling the cell to detect new displacements in the presence of an existing stimulus. In vertebrate hair cells, the adaptation machinery restores nearly the full dynamic range of response with each maintained displacement. To investigate adaptation in fly mechanoreceptors, the response to a series of test stimuli was measured before and during adapting steps that varied between -14 and +14 microm. Responses obtained before the adapting steps were then used to produce an I(X) curve that was shifted along the displacement axis to fit the data generated during each adapting stimulus. By plotting the size of the shift as a function of the size of the adapting step, how much of the cell's response is retained at each adapting step was measured. The adaptation process preserved ~85% of the dynamic range. Incomplete adaptation may allow the cell to continue to 'perceive' the sustained stimulus yet remain receptive to new stimuli. This level of adaptation closely resembles that seen in vertebrate hair cells; the similarity also extended to the time course (time constant = 18 ms) of the adaptation process. Together, these results suggest that the core transduction components in fly bristles and vertebrate hair cells are functionally related (Walker, 2000).

To identify components of the transduction machinery, 27 different Drosophila mechanosensory transduction mutants were screened for defects in transduction currents. On the basis of uncoordinated phenotypes, these mutants fell into 20 complementation groups. One of these, nompC, was particularly interesting. At a behavioral level, three of the nompC alleles showed severe uncoordination, whereas another (nompC⁴) showed moderate clumsiness. The three severe mutants (nompC¹, nompC², and nompC³) displayed a dramatic loss of MRC, with transduction currents of ~10% that of control animals. In contrast, the nompC⁴ allele exhibited almost normal MRC amplitudes but displayed severely defective adaptation. The time constant of adaptation in nompC⁴ was 50 ms, versus 277 ms for control flies. Because the MRC and the adaptation process are intimately tied to the function and regulation of the mechanically gated ion channel, it is suspected that the nompC gene product was either a component of the adaptation machinery or a transduction channel (Walker, 2000).

Why are nompC⁴ flies behaviorally uncoordinated, given that they have normal response amplitudes? One possibility is that the abnormally fast decay of the MRP would decrease the number of action potentials by limiting the time in which the cell is depolarized. To test this postulate, control and nompC⁴ animals were stimulated with a step stimulus while recording action potentials through the bristle. As hypothesized, the number of action potentials in nompC⁴ was less than half that of control flies. These results explain the behavioral phenotype of nompC⁴ and further support nompC as a critical player in the transduction process (Walker, 2000).

nompC was mapped to position 25D7 on the left arm of the second chromosome. Three overlapping cosmid clones spanning this interval were tested for rescue of the nompC phenotype by P element-mediated germ line transformation. Cosmid C fully rescued the physiological and behavioral defects of nompC mutants. Sequences from cosmid C were used to screen a Drosophila antennal cDNA library, and two 6.1-kb cDNAs were isolated. Sequence analysis of the full 33-kb cosmid and the two cDNA clones showed a single transcriptional unit encoding a predicted polypeptide of 1619 amino acids. This gene is split into 13 exons, spanning ~18 kb of genomic DNA. Using the polymerase chain reaction (PCR), this candidate gene was isolated from nompC¹, nompC², nompC³, and nompC⁴ mutants and their nucleotide sequences was determined. All four alleles have single base changes that result in either nonsense or missense mutations. nompC¹,nompC², and nompC³ each have nucleotide changes that introduce premature termination codons; in contrast, nompC⁴ has an A --> T change at residue 4820 that results in a C --> Y change at amino acid residue 1400 (Walker, 2000).

A search of protein and nucleotide databases revealed that the NOMPC gene encodes a previously unidentified ion channel with an exceptional feature: the 1150 NH₂-terminal amino acid residues consist of 29 ankyrin (ANK) repeats. The remaining 469 residues share low but significant sequence similarity with ion channels of the TRP family. A search of the C. elegans (Ce) database identified a homologous ion channel, Ce-NOMPC, that shares ~40% amino acid identity with NOMPC. The homology extends throughout the entire molecule, including the six transmembrane segments and the presence of 29 ANK repeats. ANK repeats are 33-residue motifs that mediate specific protein-protein interactions with a diverse repertoire of macromolecular targets. Although the function of the ANK repeats in NOMPC is not known, it is notable that ANK repeats are particularly prominent in the assembly of macromolecular complexes between the plasma membrane and the cytoskeletal network (Walker, 2000).

TRPs are a diverse family of cation channels found in both vertebrates and invertebrates and are implicated in calcium signaling, pain transduction, and chemosensory transduction. In all, pairwise comparison between the channel domains of NOMPC and the various TRP family members revealed ~20% identity (~40% similarity), establishing NOMPC as a new distant member of this channel family (Walker, 2000).

To examine the expression pattern of the nompC transcript, RNA in situ hybridizations to tissue sections of late-stage pupae were performed. It was found that NOMPC is selectively expressed in ciliated mechanosensory organs, including microchaetes, macrochaetes, and bristles on the fly's proboscis. Control hybridizations with sense probes produced no specific signals in any of these cells. Given the strong uncoordinated phenotype of nompC mutants, it was reasoned that nompC should also be required in proprioceptive organs, which include the ciliated chordotonal neurons. Indeed, NOMPC is expressed in chordotonal organs of the halteres, as well as in the leg joints and Johnston's organ. The expression profile of nompC in mechanoreceptive bristles and chordotonal organs accords with the physiological (loss of MRC) and behavioral (uncoordination) phenotypes of nompC mutants and supports NOMPC as a mechanosensory transduction channel (Walker, 2000).

It is interesting to ask why Ce-NOMPC was not isolated in the various screens for C. elegans touch-insensitive mutants. As it turns out, body-touch sensitivity in C. elegans is mediated by nonciliated touch cells. To determine the expression profile of the C. elegans nompC gene, 4.5 kb of upstream sequences and the first four ANK repeats of Ce-NOMPC were fused to a green fluorescent protein (GFP) reporter. The construct was injected into worms, and the transformed progeny was inspected for GFP expression. Multiple transformants were examined, and in all cases, fluorescent signals were observed in CEPV, CEPD, and ADE neurons. These mechanosensory neurons have ciliated dendrites and may be the functional equivalent of the fly ciliated mechanosensory neurons. Notably, the C. elegansNOMPC-GFP fusion localized to the sensory dendrites, the proposed site of mechanosensory transduction in these cells (Walker, 2000).

Thus, several lines of evidence support NOMPC's role as a mechanosensory transduction channel. First, at the primary sequence level, NOMPC has similarity to bona fide ion channels. Second, loss-of-function mutations in nompC virtually eliminate mechanoreceptor responses, and a single point mutation in the channel alters the behavior of the transduction currents. Third, nompC is selectively expressed in mechanosensory organs in Drosophila. Furthermore, the C. elegans homolog localizes to the presumed site of mechanoelectrical transduction. Last, it is expected that transduction channels are tethered to the cytoskeleton; the 29 ANK repeats of NOMPC are ideally suited to interact with the cytoskeleton and transduction partners. This number of ANK repeats is the largest found in any protein (Walker, 2000).

Like many other ion channels, NOMPC may form a multimeric channel. If individual subunits are linked to the cytoskeleton or the extracellular matrix, then mechanical gating can be reduced to simply altering tension between the NOMPC subunits. In this model, deflection of the bristle deforms the dendritic tip, which shifts the position of the channel's anchor points in relation to each other. The resulting tension across the molecule would trigger a conformational change that opens the molecular gate of the NOMPC transduction channel. At least two that NOMPC may be integrated into the transduction apparatus are anticipated. In one, NOMPC could be attached on both sides of the plasma membrane: to the cytoskeleton through the extensive ANK repeats and to the extracellular matrix through a different channel subunit or additional binding proteins. Alternatively, NOMPC need not be linked to the extracellular matrix. Instead, the cytoplasmic anchoring of individual subunits or membrane stress may provide sufficient tension to modulate the molecular gate (Walker, 2000).

Although null mutations in nompC virtually eliminated the transduction current, there is a tiny mechanically gated residual response in these mutants, suggesting the presence of an additional mechanically gated channel. In view of NOMPC's similarity to TRP channels, which together with the TRP-like ion channel generate the light-activated conductance in Drosophila photoreceptors, NOMPC might participate in a transduction current with another channel (Walker, 2000).

Are there vertebrate NOMPC channels? The transduction physiology of Drosophila mechanoreceptor bristles mirrors that of vertebrate hair cells, including the presence of a high-K⁺, low-Ca²⁺ receptor endolymph, directional sensitivity, microsecond latencies, sensitivity to displacements of molecular dimensions, and similar adaptation profiles. In addition, the development of vertebrate hair cells and Drosophila mechanoreceptor organs employ homologous cell-signaling molecules, insinuating common downstream targets. It will be of great interest to determine if there are NOMPC homologs in vertebrates and whether they underlie any sensory deafness or disequilibrium disorders (Walker, 2000).

Search PubMed for articles about Drosophila NompC

Albert, J. T., Nadrowski, B. and Gopfert, M. C. (2007). Mechanical signatures of transducer gating in the Drosophila ear. Curr. Biol. 17: 1000-1006. PubMed ID: 17524645

Birznieks, I., Macefield, V. G., Westling, G. and Johansson, R. S. (2009). Slowly adapting mechanoreceptors in the borders of the human fingernail encode fingertip forces. J Neurosci 29(29): 9370-9379. PubMed ID: 19625527

Cheng, L. E., et al. (2010). The role of the TRP channel NompC in Drosophila Larval and adult Locomotion. Neuron 67: 373-380. PubMed ID: 20696376

Colbert, H. A., Smith, T. L., Bargmann, C. I. (1997). OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J. Neurosci. 17: 8259-69. PubMed ID: 9334401

Dallos, P. (2008). Cochlear amplification, outer hair cells and prestin. Curr Opin Neurobiol 18: 370-376. PubMed ID: 18809494

Effertz, T., Wiek, R. and Göpfert, M. C. (2011). NompC TRP channel is essential for Drosophila sound receptor function. Curr. Biol. 21(7): 592-7. PubMed ID: 21458266

Gopfert, M. C., Albert, J. T., Nadrowski, B. and Kamikouchi, A. (2006). Specification of auditory sensitivity by Drosophila TRP channels. Nat Neurosci 9: 999-1000. PubMed ID: 16819519

Ernstrom, G. G. and Chalfie, M. (2002). Genetics of sensory mechanotransduction. Annu. Rev. Genet. 36: 411-453. PubMed ID: 12429699

Gallio, M., Ofstad, T. A., Macpherson, L. J., Wang, J. W. and Zuker, C. S. (2011). The coding of temperature in the Drosophila brain. Cell 144(4): 614-624. PubMed ID: 21335241

Gaudet, R. (2008). A primer on ankyrin repeat function in TRP channels and beyond. Mol. Biosyst. 4: 372-379. PubMed ID: 18414734

Gillespie, P. G. and Walker, R. G. (2001). Molecular basis of mechanosensory transduction, Nature 413: 194-202. PubMed ID: 11557988

Gong, Z., et al. (2004). Two interdependent TRPV channel subunits, Inactive and Nanchung, mediate hearing in Drosophila. J. Neurosci. 24: 9059-9066. PubMed ID: 15483124

Grueber, W. B., Jan, L. Y. and Jan, Y. N. (2002). Tiling of the Drosophila epidermis by multidendritic sensory neurons. Development 129: 2867-2878. PubMed ID: 12050135

Grueber, W. B., et al (2007). Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology. Development 134: 55-64. PubMed ID: 17164414

Howard, J. and Bechstedt, S. (2004). Hypothesis: a helix of ankyrin repeats of the NOMPC-TRP ion channel is the gating spring of mechanoreceptors. Curr Biol 14: R224-R226. PubMed ID: 15043829

Hughes, C. L. and Thomas, J. B. (2007). A sensory feedback circuit coordinates muscle activity in Drosophila. Mol. Cell. Neurosci. 35: 383-396. PubMed ID: 17498969

Kamikouchi, A., Inagaki, H. K., Effertz, T., Hendrich, O., Fiala, A., Gopfert, M. C. and Ito, K. (2009). The neural basis of Drosophila gravity-sensing and hearing. Nature 458: 165-171. PubMed ID: 19279630

Kernan, M., Cowan, D. and Zuker, C. (1994). Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila. Neuron 12: 1195-1206. PubMed ID: 8011334

Kim, J., et al. (2003). A TRPV family ion channel required for hearing in Drosophila. Nature 424: 81-84. PubMed ID: 12819662

Lee, G., et al. (2006). Nanospring behaviour of ankyrin repeats. Nature 440: 246-249. PubMed ID: 16415852

Lee, J., Moon, S., Cha, Y. and Chung, Y.D. (2010). Drosophila TRPN(=NOMPC) channel localizes to the distal end of mechanosensory cilia. PLoS ONE 5(6): e11012. PubMed ID: 20543979

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date revised: 22 April 2022

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