InteractiveFly: GeneBrief

Piezo and Piezo-like: Biological Overview | References

BIOLOGICAL OVERVIEW

Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. This study shows that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a approximately 1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction (Coste, 2012).

Mechanically-activated (MA) currents have been described in various mammalian cells, including inner ear hair cells, somatosensory dorsal root ganglion neurons, vascular smooth muscle cells, and kidney primary epithelia. The majority of these MA currents are cationic with Ca²⁺-permeability, leading to a search for cation channels able to convert mechanical forces into such currents. Few MA channels have been described to date; however, none of the candidates have been shown convincingly to mediate the physiological relevant non-selective cationic MA currents in mammals (Coste, 2012).

Mouse piezo1 (mpiezo1) was recently identified as a protein required for MA currents in a mammalian cell line. Expressing mpiezo1 or related mpiezo2 in a variety of mammalian cell lines induces large MA cationic currents. mpiezo1-induced currents are inhibited by GsMTx4, a toxin widely used to study MA channels. Piezo1 and piezo2 contain over 30 putative transmembrane domains and do not resemble known ion channels or other protein classes. Piezo proteins could be non-conducting subunits of cationic ion channels required for proper expression or for modulating channel properties. Alternatively, piezo proteins may define a novel class of ion channels involved in mechanotransduction (Coste, 2012).

Piezo sequences are present in the genomes of many animal, plant, and other eukaryotic species. Functional analysis of piezos from distant species could demonstrate a conserved role of these proteins in mechanotransduction; furthermore, a comparative analysis of MA currents could elucidate unique pore properties of channels induced by piezos from distinct species. This study focused on the apparently single member of D. melanogaster piezo (dpiezo), as this distant invertebrate species is widely used to study mechanotransduction using genetic approaches. Dpiezo is 24% identical to mammalian piezos, with sequence conservation throughout the length of the proteins. The full length dPiezo cDNA was cloned into pIRES2-EGFP vector. MA currents were recorded from fluorescent HEK293T cells expressing dPiezo-pIRES2-EGFP by applying force to the cell surface while monitoring transmembrane currents at constant voltage using patch-clamp recordings in the whole-cell configuration. Dpiezo, but not mock-transfected cells, showed large MA currents. These currents display a time constant of inactivation τ of 6.2 ± 0.3 ms at -80 mV when fitted with mono-exponential function, which is faster than observed for mpiezo1 (~16 ms) and more comparable to mpiezo2 (~7 ms). Similar to its mammalian counterparts, dpiezo- MA currents are characterized by a linear current-voltage relationship with a reversal potential around 0 mV, consistent with it mediating a non-selective cationic conductance. Dpiezo-induced currents were further characterized in HEK293T cells in response to negative pressure pulses applied through the recording pipette in the cell-attached mode, an alternative mechanosensitivity assay. Overexpression of dpiezo induced stretch-activated currents with a pressure for half-maximal activation (P₅₀) of -31.8 ± 2.8 mm Hg, similar to the P₅₀ calculated for mpiezo1-induced currents (~30 mm Hg). Therefore, mechanosensitivity of the piezo family is conserved in invertebrates. Importantly, the physiological relevance of dpiezo in vivo in an accompanying paper (Coste, 2012).

Fundamental permeation properties of mpiezo1- and dpiezo were compared. Ruthenium red (RR), a polycationic pore blocker of TRP channels, blocks mpiezo1- and mpiezo2-induced MA currents. RR was found to be a voltage-dependent blocker of mpiezo1, with an IC₅₀ value of 5.4 ± 0.9 μM at -80mV : At a concentration of 30 μM, extracellular RR inhibited inward MA currents without affecting outwards currents. Such voltage-dependence is a characteristic of open channel block. A high concentration of RR (50 μM) included in the pipette solution in the whole cell configuration showed no evidence of block, as large MA currents still displayed a linear current-voltage relationship. These results suggest RR blocks the pore of mpiezo1-induced MA channels from the extracellular side. Remarkably, dpiezo-induced MA currents were insensitive to RR concentrations that potently blocked mPiezo1-induced currents. Together, these results demonstrate that overexpression of dpiezo or mpiezo1 gives rise to MA channels with distinct channel properties (Coste, 2012).

Next, the single channel conductance (γ) of MA channels induced by piezo proteins was determined by using negative-pressure stimulations of membrane patches in cell-attached mode. Openings of stretch-activated channels showed a striking difference in amplitude of single channel currents, as determined from the single channel current-voltage relationship for mpiezo1- and dpiezo. Linear regression of these I-V relationships resulted in slope-conductance values in these recording conditions of 29.9 ± 1.9 and 3.3 ± 0.3 pS for mpiezo1- and dpiezo-induced MA currents, respectively. Therefore, dpiezo-dependent channels are 9-fold less conductive than mpiezo1-dependent channels (Coste, 2012).

The pore of the majority of ion channels is formed by the assembly of transmembrane domains from distinct subunits (e.g., voltage-gated K⁺ channels, ligand-gated ion channels) or structurally repetitive domains within a large protein (e.g., voltage gated Na⁺ and Ca²⁺ channels). Since piezos lack repetitive transmembrane motifs presumably they oligomerize to form ion channels. To test this hypothesis, the number of subunits was determined in piezo complexes by expressing GFP-mpiezo1 fusion proteins in Xenopus oocytes, imaging individual spots with total internal reflection microscopy (TIRF), and counting discrete photobleaching steps. N-terminal GFP-mpiezo1 functionality was confirmed by overexpression in HEK293T cells. Several GFP-fusion constructs of ion channels with known stoichiometry were used as controls: voltage-gated Ca²⁺ channel (α1E-GFP; monomer), NMDA receptor (NR1 co-expressed with NR3A-GFP; dimer of dimers), and cyclic nucleotide gated (CNG) channel (XfA4-GFP; tetramer). Complexes of mpiezo1 frequently exhibited at most four photobleaching steps, consistent with the idea that piezos homo-multimerize. Fluorescent mpiezo1 (or CNG) complexes exhibiting bleaching in fewer than four steps can be explained by non-functional GFP or pre-bleached GFP or general bias against noisier multi-step traces during data analysis. Histograms of the number of photobleaching steps observed for mpiezo1 complexes were comparable to histograms obtained from tetrameric CNG channels. These results suggest that in living cells, piezos assemble as homo-multimers (Coste, 2012).

Piezo proteins were further characterized biochemically by heterologously expressing and purifying mpiezo1 C-terminally fused with a glutathione S-transferase (mpiezo1-GST). Functionality of mpiezo1-GST was confirmed by overexpression in HEK293T cells. A protein band at a position near the 260 kDa protein marker on a Coomassie blue-stained denaturing protein gel. Western blot with a GST (S. japonicum form) antibody or a mpiezo1 specific antibody confirmed the presence of mpiezo1-GST in the mpiezo1-GST sample. Using native gel electrophoresis and Coomassie blue staining, a prominent protein band was detected at a position near the 1,236 kDa protein marker only in the mpiezo1-GST sample. Western blot using mpiezo1 antibody confirmed that this major band contains mpiezo1. These data indicate that the purified mpiezo1-GST protein complex has a molecular weight of about 1.2 million Daltons, four times the predicted molecular weight of a single mpiezo1-GST polypeptide (318 kDa). Next, it was asked whether any endogenous proteins are present in this mpiezo1-containing complex. Mass spectrometry of the ~1.2 million Dalton protein complex mainly detected peptides derived from mpiezo1-GST, but not from other endogenous membrane proteins. Although several non-transmembrane proteins were also detected, most of them were also present in the control sample, indicating an absence of specific interacting proteins in the complex. Moreover, mass spectrometry of the whole purified solution samples prior to gel electrophoresis confirmed that no other ion channel protein was detected. This argues that mpiezo1 is not tightly associated with any endogenous pore-forming protein (Coste, 2012).

To further examine whether this piezo complex is indeed a tetramer, the purified mpiezo1-GST protein was treated with the crosslinker paraformaldehyde (PFA) and the samples were subjected to denaturing gel electrophoresis and western blotting. PFA-treated samples contained three major additional higher-order piezo containing bands, with longer PFA treatments increasing the prominence of the higher bands. The distribution of the bands on the 3-8% gradient gel suggests that the four bands correspond to monomer, dimer, trimer and tetramer of mpiezo1-GST. The observation that mpiezo1 is crosslinked by formaldehyde, a crosslinker with a relative short spacer arm (2.3-2.7 Å), suggests that the subunits form a tetramer (Coste, 2012).

It is possible that mpiezo1 oligomers associate with other proteins; however such an association might not withstand the GST purification step. To probe this, PFA crosslinking experiments were performed on living cells prior to the purification procedure. On a native gel, the mpiezo1-GST complex purified from PFA-treated cells also migrated to the position near the 1236 kDa protein marker, similar to the sample from untreated cells. On a denaturing gel, on-cell PFA treatment resulted in four distinct Piezo1-specific bands, similar to results of PFA treatment on the purified complex. This suggests that mpiezo1 is not tightly associated with other proteins large enough to discernibly alter its size on denaturing gels, and confirms the results from mass spectrometry. However, cross-linking studies with paraformaldehyde could miss weak interactors with mpiezo1. Regardless, together with the results obtained from single molecule photobleaching analysis in living cells, the biochemical data suggest that mpiezo1 forms a homomultimeric ion channel, most likely as a homotetramer (Coste, 2012).

Finally, to assess if piezo proteins are sufficient to recapitulate the channel properties recorded from piezo-overexpressing cells, purified mpiezo1 proteins were reconstituted into lipid bilayers in two distinct configurations: droplet interface lipid bilayers (DIBs) assembled from two monolayers and proteoliposomes. In the first configuration, mpiezo1 was reconstituted into asymmetric bilayers that mimic the cellular environment: The extracellular facing lipid monolayer is predominantly neutral whereas the intracellular facing leaflet is negatively charged. In contrast, the lipid composition of the bilayer in the second configuration is uniform (Coste, 2012).

In the DIBs setting, representative segments from a 6 minute recording obtained at –100 mV show brief, discrete channel openings blocked by addition of 50 μM RR to the neutral facing compartment. In contrast, no effect was observed when RR was introduced into the negative facing compartment. Efficient block of channel activity was detected even at 5 μM RR. The asymmetric accessibility of RR block of reconstituted channels agrees with the data obtained from mpiezo1-overexpressing HEK293T cells, thereby establishing the fidelity of the assays and validating mpiezo1 protein as an authentic ion channel. The piezo currents exhibit ohmic behavior; records displayed at higher resolution clearly demonstrate the occurrence of unitary events with γ values obtained from conductance histograms of 118 ± 15 pS and 80 ± 6 pS in symmetric 0.5 M KCl from the negative and positive branches of I-V plots, respectively (Coste, 2012).

A similar pattern of activity was obtained from mpiezo1 reconstituted in asolectin liposomes. A selection of recordings shows the presence of two channels in the membrane which reside predominantly in the open state, as discerned in a higher time resolution display. These recordings were obtained in the presence of 50 μM RR inside the recording pipette, to ensure functional selection of a single population of mpiezo1 channels facing the RR-free compartment. mpiezo1 in asolectin proteoliposomes under these conditions (symmetric 0.2 M KCl) exhibits a γ = 110 ± 10 pS at V = –100 mV and 80 ± 5 pS at V = 100 mV. Finally, reconstitution of control samples purified from nontransfected cells as well as heat-denatured purified mpiezo1-GST into either bilayer systems under otherwise identical conditions failed to reproduce this pattern of channel activity (Coste, 2012).

The ability of the reconstituted mpiezo1 to conduct sodium was then tested. Initially, single channel currents were recorded from asymmetric bilayers in symmetric 0.2 M KCl; γ = 58 ± 5 pS. Subsequent addition of 0.2M NaCl in presence of 0.2M KCl increased the unitary conductance of reconstituted channels to 95 ± 5 pS while retaining sensitivity to RR block. These results confirm that these channels conduct both sodium and potassium as would be expected from a cationic non-selective channel. This assertion was further substantiated by recording mpiezo1 currents from proteoliposomes under bi-ionic conditions (0.2 M KCl/0.2M NaCl). A summary of the current–voltage relation for the mpiezo1 channel, extracted from 204,088 events obtained in three experiments, shows that the single channel current is ohmic between –100 and 200 mV with a slope conductance of 102 ± 2 pS. The current reversed direction at 0.0± 0.3 mV demonstrating that the channel does not select between K⁺ and Na⁺, and importantly, displays open channel block by RR (Coste, 2012).

The difference in γ between overexpressed mpiezo1 in cells and reconstituted mpiezo1 in lipid bilayers may be attributed to many variables, including the distinct lipid environments which are known to strongly influence conductance measurements. Moreover the ionic conditions used in the two systems are different, as divalent cations present in HEK293T cell-attached experiments also affect the conductance values. Indeed, when divalent cations are excluded from the recording pipette, γ of mpiezo1-induced currents in HEK293T cells is 58.0 pS ± 1.5 pS (150 mM NaCl solution), compared to 29.9 ± 1.5 pS in the presence of divalent ions. The near equivalence of γ values together with the similar pattern of channel activity demonstrates that reconstitution of mpiezo1 into two distinct bilayer systems produces channels with identical functional properties (Coste, 2012).

Future reconstitution and recording of dpiezo in lipid bilayers will show whether the difference in conductance between mpiezo1 and dpiezo arises from intrinsic properties. The membrane milieu and lipid composition are known to modulate the activity of the embedded channel proteins in a drastic and deterministic manner. It is not entirely surprising that the conditions to emulate the cellular environment in the reconstituted system in terms of the mechanical state of the membrane or its lipid composition have thus far been inadequate to retrieve the activation features of MA ion channels. Furthermore, the complexity of protein clusters and dynamic cytoskeletal interacting partners at the cell membrane introduce regulatory constraints on channel activity. Further investigation may clarify whether piezo ion channel subunits are intrinsically mechanosensitive or use unknown interacting partners to sense membrane tension (Coste, 2012).

This study has provide compelling evidence to support the hypothesis that piezo proteins are indeed ion channels. First, overexpression of dpiezo or mpiezo1 in a human cell line gives rise to MA channels with distinct biophysical and pore-related properties. Second, isolated piezo complexes do not contain detectable amounts of other channel-like proteins. Finally, purified mpiezo1 protein reconstituted into proteoliposomes and planar lipid bilayers in the absence of any other cellular components gives rise to RR-sensitive cationic ion channel activity. The mouse piezo1 complex is estimated to weigh ~1.2 million Daltons with 120-160 transmembrane segments, being the largest plasma membrane ion channel complex identified to date (Coste, 2012).

Piezo-like gene regulates locomotion in Drosophila larvae

To maintain proper locomotive patterns, animals constantly monitor body posture with their proprioceptive receptors. In Drosophila, the chordotonal organs (Cho) are especially important in the regulation of locomotion pattern. However, how Cho neurons that are normally activated with sound (vibration) transduce static displacement caused by body position change remains unclear. This study reports that piezo-like (pzl), a homolog for mammalian piezo1 and 2, is essential for Cho's function in locomotion. The mutant allele of pzl showed severe defects in crawling pattern and body gesture control, which were rescued by expressing Pzl specifically in Cho neurons. The ability of Cho neurons to respond to micrometer-scale body wall displacement requires pzl. Intriguingly, human or mouse Piezo1 can rescue pzl-mutant phenotypes, suggesting a conserved role of the Piezo-family proteins in locomotion (Hu, 2019).

'Proprioception' refers to the sensory input and feedback by which animals keep track of and control different parts of their bodies for balance and correct locomotive patterns. Selective loss of function of proprioceptors results in movement defects in human. Proprioception is thought to be mediated with mechanosensitive proprioceptors. In insects, some chordotonal organs (Cho) serve proprioceptive roles. Perturbation of Cho neurons in Drosophila results in defective locomotion and posture control (Hu, 2019).

Despite Cho's roles in locomotion, the mechanism underlying their mechanosensation to static displacement remains largely unknown. Mechanosensation that mediates the detection of touch, nociception, hearing, and proprioception is an important sensory modality. In many circumstances, especially proprioception, the identity of the mechanosensitive neurons or the channels is largely unknown. In Drosophila, the mechanosensitive channel NompC and other putative channels are crucial for larval crawling. Humans with dominant mutations of Piezo2 suffer from different forms of distal arthrogryposis. The other member of the Piezo family, Piezo1, however, has broader roles. Structures of the mouse Piezo1 protein were recently solved, revealing a trimeric propeller-like structure. Unlike most animals that have two piezo genes, only one ortholog was reported in Drosophila (Dmpiezo). This study reports a gene named piezo-like (pzl; CG45783), a homolog of piezo gene families, and explores its roles in locomotion regulation in Drosophila (Hu, 2019).

In the fruit fly, Cho neurons of the Johnston organ in the antenna are the major sensors for airborne sound, gravity, and wind. Moreover, larval Cho was reported to sense low temperature. Previous work and the current study suggest that Cho neurons are required for Drosophila locomotion. These studies raise the possibility that Cho is capable of integrating multiple sensory cues to facilitate the animals' survival in a complex environment with cross-modal information (Hu, 2019).

It appears that different roles of Cho neurons rely on distinct mechanotransduction channels. This study observed only a mild defect in the pzl-mutant larvae to low-frequency vibration but not to the stimuli to which larval Cho neurons are optimally tuned. Considering that low-frequency vibration may cause stronger displacement at the same sound level, the defect of pzl mutant may result from lower sensitivity to static displacement. Alternatively, it is possible that pzl contributes to sound sensing at certain frequencies. Nevertheless, it appears that pzl plays a more important role in sensing static displacement (Hu, 2019).

In Drosophila, multiple types of proprioceptive neurons were found to participate in the locomotion regulation. Blocking nociceptive class IV da neurons causes the animals to move relatively straight on a plane surface, while silencing class I da neurons and bd neurons resulted in an opposite phenotype-increased number and duration of turning and reduced linear locomotion. Larvae with loss of function of Cho neurons showed more turning and backward movement. It seems that the Cho neurons and class I da/bd neurons converge at least partially onto the same downstream motor pathway (Hu, 2019).

All these proprioceptors may use different mechanotransduction channels to coordinate mechanical cues. Class IV da neurons modulate the extent of linear locomotion via the DEG/ENaC ion channels. In contrast, NompC and Dmtmc function in class I da neurons and bd neurons to regulate stride duration and crawling speed. The present study identified a gene, pzl, and its function in Cho, adding new knowledge to transduction mechanisms in proprioceptive neurons. Notably, RNAi knockdown of pzl appeared to have more head lifting compared with pzl knockout. The mRNA levels of Dmpiezo and nompC were slightly increased in the pzl knockout, suggesting that the mechanotransduction channels may have compensatory roles in regulating animal behaviors (Hu, 2019).

It has been demonstrated that mammalian piezo1 and piezo2 as well as fly DmPiezo are pore-forming channel subunits. Given its conservation with mammalian Piezo, attempts were made to record Pzl'’s channel activity by ectopically expressing pzl in a variety of heterologous systems. However, no channel activity of Pzl was detected in these experimental settings. It is very likely that the Drosophila Pzl cannot achieve a detectable level of plasma membrane proteins, because immunostaining for the protein tags fused to Pzl failed to show any signal (Hu, 2019).

In an in vivo ectopic expression system, however, fluorescence was observed for the Pzl-GFP fusion protein. Still no mechano-gated current was recorded. It is possible that Pzl fails to be trafficked to the plasma membrane at all, because of a lack of necessary molecular partners. Alternatively, additional components may be required for Pzl to form a functional channel. These results revealed an interesting feature of Pzl that distinguishes it from other Piezo proteins: Pzl is more dependent on other partners or naive environments to be fully functional. Besides, although Dmpiezo in flies has been reported to be involved only in nociception, mammalian Piezo proteins, especially Piezo1, are found to be essential in many aspects of mechanotransduction functions. This study showed that pzl has very broad expression in adult flies, suggesting diverse roles of the pzl gene (Hu, 2019).

The role of Drosophila Piezo in mechanical nociception

Transduction of mechanical stimuli by receptor cells is essential for senses such as hearing, touch and pain. Ion channels have a role in neuronal mechanotransduction in invertebrates; however, functional conservation of these ion channels in mammalian mechanotransduction is not observed. For example, No mechanoreceptor potential C (NOMPC), a member of transient receptor potential (TRP) ion channel family, acts as a mechanotransducer in Drosophila melanogaster and Caenorhabditis elegans; however, it has no orthologues in mammals. Degenerin/epithelial sodium channel (DEG/ENaC) family members (see Drosophila Pickpocket) are mechanotransducers in C. elegans and potentially in D. melanogaster; however, a direct role of its mammalian homologues in sensing mechanical force has not been shown. Recently, Piezo1 (also known as Fam38a) and Piezo2 (also known as Fam38b) were identified as components of mechanically activated channels in mammals. The Piezo family are evolutionarily conserved transmembrane proteins. It is unknown whether they function in mechanical sensing in vivo and, if they do, which mechanosensory modalities they mediate. This study examined the physiological role of the single Piezo member in D. melanogaster (Dmpiezo; also known as CG8486). Dmpiezo expression in human cells induces mechanically activated currents, similar to its mammalian counterparts. Behavioural responses to noxious mechanical stimuli were severely reduced in Dmpiezo knockout larvae, whereas responses to another noxious stimulus or touch were not affected. Knocking down Dmpiezo in sensory neurons that mediate nociception and express the DEG/ENaC ion channel pickpocket (ppk) was sufficient to impair responses to noxious mechanical stimuli. Furthermore, expression of Dmpiezo in these same neurons rescued the phenotype of the constitutive Dmpiezo knockout larvae. Accordingly, electrophysiological recordings from ppk-positive neurons revealed a Dmpiezo-dependent, mechanically activated current. Finally, this study found that Dmpiezo and ppk function in parallel pathways in ppk-positive cells, and that mechanical nociception is abolished in the absence of both channels. These data demonstrate the physiological relevance of the Piezo family in mechanotransduction in vivo, supporting a role of Piezo proteins in mechanosensory nociception (Kim, 2012).

These data demonstrate physiological relevance of Piezo family in mechanotransduction in vivo, supporting a role of Piezo proteins in mechanosensory nociception (Kim, 2012).

D. melanogaster is widely used to study mechanotransduction and genetic studies have identified several ion channels that are essential for mechanosensation. However, none of these proteins are shown to be activated by mechanical force when expressed in heterologous systems. Since expression of mouse Piezos in a variety of mammalian cells induces large mechanically activated currents, this study set out to test if the fly counterpart is also sufficient to induce mechanosensitivity. Similar to its mammalian counterparts, the Drosophila piezo gene (CG8486) is predicted to consist of a large number of transmembrane domains. Albeit fly and mammalian piezos do not exhibit extensive sequence conservation (24% identity), expression of Drosophila piezo in cultured human cells induced large mechanically activated cationic currents, suggesting a role of dpiezo in mechanotransduction (Kim, 2012).

To characterize dpiezo expression in flies a fusion between the dpiezo enhancer/promoter region and GAL4 (dPiezoP-GAL4) was used. Four independent dPiezoP-GAL4 transgenic insertions were examined to avoid insertional effects on GAL4 expression. UAS-GFP was used for labeling cells except for arborized neurons that were optimally visualized using the membrane-targeted UAS-CD8::GFP. Fluorescent labeling induced by dpiezo enhancer/promoter region in all types of sensory neurons and several non-neuronal tissues in both adults and larvae. This diverse pattern of dpiezo expression observed in Drosophila is in accord with the expression of Piezo1 and Piezo2 in mice (Kim, 2012).

dpiezo knockout (KO) flies in which all 31 coding exons were deleted were created using genomic FLP-FRT recombination. The knockout flies were viable, fertile and did not show uncoordination or a defect in bristle mechanoreceptor potential. Whether dpiezo KO larvae have mechanical nociception deficits was studied by using a mechanically-induced escape behavior assay. Stimulation with von Frey filaments that ranged from 2 to 60 mN demonstrated that dpiezo KO larvae have a severe response deficit over a wide range. Repeated stimulations of the same larvae resulted in comparable responsiveness in both wild type and dpiezo KO, indicating that the stimuli did not induce considerable damage to the sensory system. A 153 ± 11.0 mN filament elicited responses only to the first of three stimulations in wild type larvae, arguing that this amount of force is damaging. For further experiments, the larvae were stimulated using a 45 mN filament which elicits a substantial response in both wild type and dpiezo mutant larvae. 34 ± 4.4 % of dpiezo KO larvae showed a response to 45 mN filament stimulation, compared to over 80 % of wild type or heterozygote larvae. As a control for the genetic background, larvae were used that carry the dpiezo KO allele on one chromosome and a deficiency in which the entire dpiezo genomic region is deleted on the homologous chromosome. The defect in the trans-heterozygous larvae was similar to the KO homozygote phenotype (51 ± 3.9 %, p = 0.091). In contrast, dpiezo KO larvae were indistinguishable from wild type in an assay for responses to high temperature, a different noxious stimulus that elicits the same escape response. Therefore, dpiezo KO larvae retain a normal ability to elicit the escape behavior in response to noxious stimuli, while dpiezo is specifically required for the mechanical modality of nociception. To evaluate the possible role of dpiezo in other modes of larval mechanical sensing, the sensitivity of dpiezo KO to gentle touch, which is mediated through ciliated neurons, was tested. No defect was observed in the sensitivity of dpiezo KO larvae to innocuous gentle touch (Kim, 2012).

A mechanical nociception phenotype was previously observed in pickpocket (ppk), a DEG/ENaC channel, and painless (pain), a TRPA ion channel. The specificity of dpiezo KO to mechanical nociception resembles the phenotype of ppk since pain is also essential for sensing thermal nociception. Therefore the role of dpiezo was tested in ppk-positive cells using ppk-GAL4, which labels subclasses of multidendritic (MD) neurons. The MD neurons are non-ciliated receptor cells that tile the body wall of the larvae and respond to a variety of external stimuli such as mechanical forces, temperature and light. A green fluorescent protein driven directly by the regulatory regions of the ppk gene (ppk-EGFP) together with DsRed expression in dpiezo-positive cells were used to probe dpiezo and ppk co-expression. Indeed it was observed that all ppk-positive cells also expressed dpiezo. Next a ppk-GAL4 was used to drive the expression of dpiezo RNAi to test whether dpiezo function is specifically required in ppk-expressing cells. The restricted knockdown of dpiezo resulted in a mechanical nociceptive phenotype similar to phenotype observed in dpiezo KO larvae. In a complementary approach, ppk-GAL4 driven expression of dpiezo cDNA was used in an attempt to rescue the mechanical nociception phenotype of dpiezo KO. A fusion between dPiezo and GFP was used to monitor expression levels in ppk cells and dPiezo localization within the neurons. GFP-dPiezo fusion protein induces MA currents in human cell lines, similar to untagged dPiezo, confirming functionality. When expressed in the fly, GFP-dPiezo fluorescence was present throughout cell bodies, axons and dendritic arbors of ppk-positive neurons. Importantly, expression of GFP-dPiezo in ppk-positive neurons alone was sufficient to rescue the mechanical nociception defect of dpiezo KO. These data suggests that dpiezo functions in ppk-positive neurons to mediate mechanical nociception (Kim, 2012).

To test if the ppk-positive neurons respond to mechanical stimuli and if dpiezo mediates such responses electrophysiological recordings were performed from isolated cells. Larvae that had GFP labeling in ppk-positive neurons were dissociated using enzymatic digestion and mechanical trituration. Plated fluorescent neurons were then tested using patch clamp recordings in the cell-attached configuration and they were stimulated using a negative pressure through the recording pipette. Stimulating wild type neurons resulted in a current that was rapidly activated and had a half-maximal activation (P₅₀) of 27.6 ± 7.6 mmHg. These currents were not observed in the dpiezo KO mutant neurons. Therefore, ppk-positive neurons which mediate the avoidance response to noxious stimuli display dpiezo-dependent mechanically activated currents (Kim, 2012).

Silencing of ppk cells resulted in a complete abolishment of noxious mechanosensation, in accord with a severe defect observed previously. In contrast, only a moderate deficit is observed upon eliminating or knocking down ppk in the same cells, suggesting that there are multiple pathways for mechanical sensing. Mechanical nociception in larvae that are deficient in dpiezo and either pain or ppk was tested to gain insight into cellular pathways that involve mechanotransduction in these cells. Once again, a 45 mN filament was used, enabling monitoring of both dpiezo-dependent and -independent mechanisms. The dpiezo::pain double mutant had a defect that was comparable to each one of the mutants separately, suggesting that dpiezo and pain might function in the same pathway. Larvae that are heterozygous for both dpiezo and pain showed a response deficit while each one of them separately was normal, further demonstrating their role in a common signaling mechanism. Remarkably, combining both dpiezo and ppk knockdowns resulted in a nearly complete abolishment of responses to noxious mechanical stimuli. Importantly, responses to noxious temperatures and touch were normal in larvae with both dpiezo and ppk knocked-down. These data suggest that dpiezo and ppk function in two parallel pathways in ppk-positive sensory neurons, and that together they constitute the response to noxious mechanical stimuli. There could be many reasons why the mechanically activated currents that were observe are entirely dependent on dPiezo. This can either be because PPK responds to a different modality of mechanical stimulus or due to the specific experimental settings (e.g., level of applied forces, solutions, applied voltage). Future experiments should resolve this issue (Kim, 2012).

Using the Drosophila model system, piezo was demonstrated to be essential for sensing noxious mechanical stimulus in vivo. This is the first demonstration that a Piezo family member is essential for mechanotransduction in the whole animal. Indeed, dpiezo is the first eukaryotic excitatory channel component shown to be activated by mechanical force in a heterologous expression system and required for sensory mechanotransduction in vivo. Piezo2 is expressed in mouse DRG neurons that are involved in sensing nociception, and is required for rapidly-adapting mechanically activated currents in such isolated neurons. This study raises the possibility that mammalian Piezo2 is also required for mechanical pain transduction in vivo. Furthermore, Drosophila genetics can now be utilized to map cellular pathways involved in piezo-dependent mechanotransduction in sensory neurons and beyond (Kim, 2012).

Piezo is essential for amiloride-sensitive stretch-activated mechanotransduction in larval Drosophila dorsal bipolar dendritic sensory neurons

Stretch-activated afferent neurons, such as those of mammalian muscle spindles, are essential for proprioception and motor co-ordination, but the underlying mechanisms of mechanotransduction are poorly understood. The dorsal bipolar dendritic (dbd) sensory neurons are putative stretch receptors in the Drosophila larval body wall. An in vivo protocol was developed to obtain receptor potential recordings from intact dbd neurons in response to stretch. Receptor potential changes in dbd neurons in response to stretch showed a complex, dynamic profile with similar characteristics to those previously observed for mammalian muscle spindles. These profiles were reproduced by a general in silico model of stretch-activated neurons. This in silico model predicts an essential role for a mechanosensory cation channel (MSC) in all aspects of receptor potential generation. Using pharmacological and genetic techniques, the mechanosensory channel, Piezo, was identified in this functional role in dbd neurons, with TRPA1 playing a subsidiary role. It was also shown that rat muscle spindles exhibit a ruthenium red-sensitive current, but no expression evidence was found to suggest that this corresponds to Piezo activity. In summary, this study shows that the dbd neuron is a stretch receptor and demonstrates that this neuron is a tractable model for investigating mechanisms of mechanotransduction (Suslak, 2015).

This study establishes the Drosophila dbd neuron as a useful, accessible and tractable in vivo model for studying the phenomenon of mechanotransduction in stretch receptor neurons. It also shows the utility of an in silico model for identifying components of a mechanosensitive system. Whilst earlier studies have utilised electrophysiology of Drosophila neurons of other sensory modalities, no study utilising this approach in Drosophila has tested in vivo responses to physiologically relevant mechanical stimuli. In combination with the predictive capacity of mathematical modelling, this promises to be a very powerful tool for dissecting the process of mechanotransduction and identifying transducer proteins that are activated by mechanical stimuli in the physiological range. In this study, the contribution of a Piezo protein to a innocuous stretch-activated cellular response in fully differentiated neurons has been directly demonstrated (Suslak, 2015).

Members of three channel families are currently strongly implicated in mechanotransduction: DEG/ENaCs, TRPs, and more recently Piezo proteins. Of these, Piezo protein functions are the least well characterised. Piezo proteins can gate mechanically sensitive currents when expressed in cultured cells, but their in vivo functions are less well known. Recent studies showed that Piezo2b is expressed in zebrafish somatosensory Rohon-Beard cells and is required for behavioural response to light touch, while Piezo2 in mouse is required for touch sensation. In contrast, Drosophila DmPiezo is required in sensory neurons for behavioural responses to noxious touch but not innocuous touch. To these studies, the current findings now demonstrate a role for DmPiezo in a innocuous stretch-mediated receptor response, with direct evidence for an in vivo electrophysiological requirement for DmPiezo. (Suslak, 2015).

The role of Ca²⁺ in the receptor response remains to be explored further. N-methyl-D-glucamine (NMDG) substitution results in a ~20% residual current, suggesting a contribution of Ca²⁺ to the receptor potential. The data show that DmPiezo plays the major role in producing the receptor potential, but it is a non-selective cation channel and likely conducts both Na⁺ and Ca⁺ in dbd neurons. However, as Ca²⁺ is the main permeant ion for TRPA1 channels, the residual current may reflect TRPA1's contribution. Suggestive of this is the observation that the reduction in E_p upon TRPA1 knock-down is quantitatively similar to the current remaining when Na⁺ is removed from the extracellular saline by NMDG substitution. Thus, there seems to be a ~20% contribution of Ca²⁺ to the receptor potential. Conversely, it may be that TRPA1's involvement is indirect, as it can both modulate and co-precipitate with Piezo (Peyronnet, 2013), although in the latter study the modulatory interaction was negative. The essentially complete block of mechanosensory response in the most effective of the two DmPiezo RNAi strains argues more in favour of an interactive regulation between the two channels rather than an independent contribution of TRPA1 to the receptor potential (Suslak, 2015).

A putative sensory transduction role for TRPA1 in dbd neurons had been previously identified, but this was specifically in a thermoreceptive capacity. Further examination of this potentially bimodal sensory role of TRPA1 in the dbd receptor, and how it may interact with DmPiezo may provide useful insights into primary sensory transduction pathways. For example, Ca²⁺ influx through mammalian TRPA1 has a strong role in activating TRPV1 channels in nociceptive neurons. Modelling in this study has indicated that the immediate downstream component of a stretch-transducing MSC may be a voltage-gated channel conducting either Na⁺ or Ca²⁺. It is possible that TRPA1 may fulfill this role, but it has not been reported to be voltage sensitive, this may indicate the involvement of yet another channel (Suslak, 2015).

Amiloride produced a profound inhibition of stretch-evoked responses at only 30μM. While it is possible that this inhibition is secondary to blockade of TRPA1, this seems unlikely as TRPA1 knockdown has only a modest effect. Thus, Piezo channels seem to be directly sensitive to amiloride and, if so, this is the first such report (Suslak, 2015).

It is interesting to note that the quantitative contribution made by Ca²⁺ to the stretch-activated receptor potential in both systems, the dbd neurons and mammalian muscle spindles, is similar at ~20%. While there have been no reports of specifically TRPA1 in spindles, the expression of TRPC1 and TRPV3 uncovered by this study in muscle spindle afferent terminals could equally be the basis of such a Ca²⁺ current. They may also be the source of the Ca²⁺ influx in spindle terminals responsible for the Ca²⁺-mediated activation of synaptic-like vesicle recycling in these endings (Suslak, 2015).

The similarity of the overall profile of the stretch-evoked receptor potential in dbd neurons and mammalian muscle spindles is striking. The in silico model provides an electrophysiological mechanism to describe these stretch receptor potential behaviours in terms of the Na⁺, K⁺ and Ca²⁺ currents thought to be involved, based on previous studies in invertebrate and mammalian systems. However, it now appears that the specifics of the molecular components responsible for these currents differ between these two systems. While mammalian Piezo2 is expressed in some DRG neurons, including light touch receptors, this study has so far found no evidence for Piezo expression in muscle spindle terminals. Instead, immunocytochemistry, expression and pharmacological evidence suggests that ENaCs play the key role of carrying the Na⁺ current in spindles. The overall complex profile, therefore, seems of great importance whilst the details of the channels responsible for carrying the major, Na⁺- and Ca²⁺-dependent components of the receptor potential may vary (Suslak, 2015).

Parallel mechanosensory pathways direct oviposition decision-making in Drosophila

Female Drosophila choose their sites for oviposition with deliberation. Female flies employ sensitive chemosensory systems to evaluate chemical cues for egg-laying substrates, but how they determine the physical quality of an oviposition patch remains largely unexplored. This study reports that flies evaluate the stiffness of the substrate surface using sensory structures on their appendages. The TRPV family channel Nanchung is required for the detection of all stiffness ranges tested, whereas two other proteins, Inactive and DmPiezo, interact with Nanchung to sense certain spectral ranges of substrate stiffness differences. Furthermore, Tmc is critical for sensing subtle differences in substrate stiffness. The Tmc channel is expressed in distinct patterns on the labellum and legs and the mechanosensory inputs coordinate to direct the final decision making for egg laying. This study thus reveals the machinery for deliberate egg-laying decision making in fruit flies to ensure optimal survival for their offspring (Zhang, 2020).

This study revealed an unexpected complexity of stiffness assessment when female flies select their egg-laying site. Multiple peripheral appendages and mechanosensory channels are employed to determine the stiffness difference between adjacent egg-laying substrates, and the parallel information from different mechanosensory pathways is integrated to make the final decision for softer substrate. At the moderate stiffness range (0.25%-0.5%), a group of nan+ mechanosensory neurons in the leg tarsal bristles are activated. Similarly, a lower stiffness difference (0.25%-0.4%) activates a group of nan+/Dmpiezo+ tarsal bristle mechanosensory neurons. The detection of subtle stiffness differences is small, as 0.05% agarose relies on sd-L and md-L neurons. Activation of each pathway imparts an inhibitory tone on egg laying and thus guides the flies to softer substrate. Although it remains to be tested whether nan+/Dmpiezo+ tarsal mechanosensory neurons can be activated by moderate stiffness or sd-L/md-L neurons can be activated by moderate and mild stiffness, behavioral data argue that there is functional redundancy among the sensory pathways (Zhang, 2020).

Together with previous findings that flies choose egg-laying sites based on internal and external cues, this study demonstrates that the decision-making process for egg-laying sites in female Drosophila is a highly deliberative process that employs multiple sensory modalities and multiple sensory structures within each modality. This deliberateness is essential because choosing the best egg-laying site is the most critical parental behavior among female flies to maximize their offspring's survival. Female flies in the wild certainly face a more difficult task in making such decisions for a far more complicated environment than is available in a lab experiment. Further investigation will be needed to understand how flies make decisions when evaluating complex or conflicting cues from multiple sensory pathways (Zhang, 2020).

This study has revealed the exquisite ability of female flies to discriminate a texture difference as small as 0.05% in agarose. To do this, flies employ both external sensory structures and proprioceptive sensors to assess the stiffness of the surface. Upon touching the substrate with the legs, tarsal bristles are the first structures to be deformed, leading to the activation of mechanosensory neurons underneath the bristles. In the later probing step, as the proboscis pushes against the substrate, the cuticle of the distal labellum starts to be compressed against the substrate. With innervation to most of the labellum bristles, the Tmc+ md-L neurons are well positioned to detect this information. Proboscis extension will also cause a change of the angle between the labellum and haustrum, and consequently activates the proprioceptive Iav+ sd-L neurons. Loss of either md-L or sd-L neurons on the labellum results in a complete disability to identify a subtle stiffness difference, suggesting that the two structures cooperate functionally to detect weak mechanical stimuli. It remains to be explored how these two sensors coordinate to represent stiffness values in the brain to make the final, accurate selection of softer substrate (Zhang, 2020).

Under the experimental conditions used in this study, the labellum and legs are the predominant appendages that detect substrate stiffness during egg laying. Nevertheless, the role of the ovipositor structure that executes the oviposition maneuver cannot be overlooked. This notion is supported by a previous study, but the exact neurons or genes remain elusive due to the structural complexity of the ovipositor. Moreover, a female fly pushes her lower abdomen against the substrate in order to insert the eggs into the substrate, and this abdominal bending action may require proprioceptive feedback to represent her body position and strength, although this notion requires further experimental evidence. Although it is possible to build a cumulative picture of mechanosensory regulation of decision making, a comprehensive understanding cannot be achieved before the roles of ovipositor and abdominal proprioception are elucidated (Zhang, 2020).

So far, a bona fide center in the fly brain for the integration of mechanosensory inputs has not been established. Unlike visual or olfactory pathways, each of which are encoded and represented by discrete brain regions, mechanosensory inputs appear sparsely distributed throughout the brain and neural transduction from the peripheral to the central nervous system (CNS) seems to be largely parallel. In the egg-laying neuronal circuit, the labellum mechanosensory neurons for detecting subtle stiffness differences project extensive arborizations over the SEZ, a brain region critical for gustatory perception. By contrast, leg bristle neurons that sense greater stiffness send their axons to the ventral nerve cord (VNC) and the projections are then relayed to the higher brain regions including the SEZ, ventrolateral protocerebrum (VLP), superior lateral protocerebrum (SLP), and others. This segregation complicates the identification of brain circuitry that integrates parallel mechanosensory inputs from different appendages to direct egg-laying decision making. Previous studies have raised working models for this interaction, most of which are supported by the fact that mechanosensory and gustatory pathways antagonize or facilitate each other in the local SEZ circuits. Based on the results that leg mechanosensory neurons project to multiple brain regions, however, it would seem more likely that integration may also occur at higher brain areas outside the SEZ (Zhang, 2020).

Furthermore, mechanosensory and gustatory information unambiguously influence one another during decision making for egg laying or feeding. Wu (2019) found that Tmc neurons were required for the loss of softness preference when sugar was provided. This study more symmetrically deciphered the mechanosensory pathways involved in the stiffness detection. Both studies agree that the tarsus and labellum are essential for the flies to choose egg-laying substrates of the optimal stiffness. Wu focused on the discrimination between 0.5% and 1.5% agarose whereas this study focuses on substrates from 0.25% to 0.5% agarose. A major difference in the two experimental setups for these two studies is that the stiffness difference ranges in this study were smaller (0.25%-0.5%), which allowed uncovering of additional mechanosensory mechanisms underlying egg-laying site choice. Nevertheless, the two studies are mutually complementary in deciphering how female flies recognize and integrate substrate texture and chemical cues into final decision making for egg-deposition sites (Zhang, 2020).

A significant question in the field asks how multiple mechanotransduction channels function in overlapping or parallel pathways to coordinate behavioral responses, as more than one channel type is typically expressed in the same type of mechanosensory neurons. This study found that the mechanosensory channels Nanchung and DmPiezo are required for the discrimination of a mild stiffness difference. However, how the combination of these two channels drives the function of the same neurons remains elusive. Two possibilities are suggested: first, multiple mechanosensitive channels co-express and function in the same neurons in a parallel manner. For example, DmPiezo and PPK function in larval class VI da neurons to mediate mechanical nociceptive response. Another case comes from larval class I da neurons, in which both NompC and Tmc are required for proprioceptive feedback to control larval locomotion. In this scenario, Nanchung and DmPiezo channels may function in parallel signaling pathways required for normal preference to 0.25% over 0.4%. When either pathway is disrupted, females would show a decreased ability to distinguish stiffness differences. Second, the two channels may function in series in the same pathway, with one acting as a sensor and the other as an amplifier. For instance, in fly Cho organ neurons, three TRP channels, Nanchung, NompC, and Inactive, are all required for sound transduction. Nanchung is expressed in most mechanosensory neurons for hearing and proprioception. It is plausible that Nanchung maintains basal neuronal activity and DmPiezo functions as a specific receptor for mechanical force. The current data support this view, as a nanGal4 mutant lost nearly all spike firing whereas DmpiezoKO still maintained a reduced firing activity. Behaviorally, the nanGal4 mutant showed much more severe defects in selecting softer substrate than DmpiezoKO in the mild range. The data also implicate other mechanosensors such as NompC as working in concert with Nanchung in bristle mechanosensory neurons (Zhang, 2020).

Wang, J., Jiang, J., Yang, X., Zhou, G., Wang, L., Xiao, B. (2022). Tethering Piezo channels to the actin cytoskeleton for mechanogating via the cadherin-beta-catenin mechanotransduction complex. Cell Rep, 38(6):110342 PubMed ID: 35139384

Tethering Piezo channels to the actin cytoskeleton for mechanogating via the cadherin-beta-catenin mechanotransduction complex

The mechanically activated Piezo channel plays a versatile role in conferring mechanosensitivity to various cell types. However, how it incorporates its intrinsic mechanosensitivity and cellular components to effectively sense long-range mechanical perturbation across a cell remains elusive. This study shows that Piezo channels are biochemically and functionally tethered to the actin cytoskeleton via the cadherin-beta-catenin mechanotransduction complex, whose perturbation significantly impairs Piezo-mediated responses. Mechanistically, the adhesive extracellular domain of E-cadherin interacts with the cap domain of Piezo1, which controls the transmembrane gate, while its cytosolic tail might interact with the cytosolic domains of Piezo1, which are in close proximity to its intracellular gates, allowing a direct focus of adhesion-cytoskeleton-transmitted force for gating. Specific disruption of the intermolecular interactions prevents cytoskeleton-dependent gating of Piezo1. Thus, a force-from-filament model is proposed to complement the previously suggested force-from-lipids model for mechanogating of Piezo channels, enabling them to serve as versatile and tunable mechanotransducers. This study shows biochemical and functional findings to demonstrate that Piezo channels are physically linked to the actin cytoskeleton via the cadherin-β-catenin-vinculin mechanotransduction complex and the direct interaction of E-cadherin to key mechanogating domains of Piezo1, prompting a proposed force-from-filament or tether model for the Piezo channel. In line with this proposed gating model, previous studies have shown that pharmacological disruption of the actin cytoskeleton impairs Piezo1-mediated currents evoked by cell indention or elastomeric pillar deflection at cell-substrate contact points (Wang, 2022).

Several lines of evidence suggest that the regulatory effect of E-cadherin on Piezo1 might not depend on E-cadherin's homotypic interactions between neighboring cells. First, the interaction and functional regulation of Piezo is preserved in the E-cadherin-W2 mutant, which is defective in homotypic dimerization. Second, Piezo1-E-cadherin interactions and functional regulation do not depend on high concentration of Ca2+, which is required for E-cadherin's homotypic dimerization. Third, functional potentiation of Piezo1-mediated currents by E-cadherin could be detected in individual cells without contacting cells and in cell-attached membranes. Together, these data demonstrate that E-cadherin-mediated adhesions junctions might not be necessarily needed for the proposed tethered gating of Piezo channels. Instead, F-actin-derived force transduction might modulate the mechanosensitivity of Piezo channels. In line with this, endogenous traction forces have been reported to locally activate Piezo1 (Wang, 2022).

On the basis of the identification of the transmembrane gate located in the inner helix (IH) and the cytosolic lateral plug gates that physically block the three lateral ion-conducting portals, it is proposed that Piezo channels might use a dual-gating mechanism, in which the transmembrane gate is dominantly controlled by the top extracellular cap, while the lateral plug gates are controlled by the peripheral blade-beam apparatus through a plug-and-latch mechanism. The cap domain is embedded in the center of the nano-bowl shaped by the curved Piezo1-membrane system and sits right on the top of the transmembrane pore of Piezo1 (see A proposed mechanogating model for Piezo channel integrating both the cadherin-mediated tether model and the intrinsic force-from-lipids model). Structural analysis has revealed that motion of the cap domain is strictly coupled to the transmembrane gate residing in the pore-lining inner helix. Furthermore, either deleting or crosslinking the cap completely abolished mechanical activation of Piezo1. These studies have led to a proposal that the cap domain functions as a critical mechanogating domain to predominantly control the transmembrane gate. Following the transmembrane pore, Piezo channels use three lateral portals equipped with three spliceable lateral plug gates as their intracellular ion-conducting routes. Interestingly, the three lateral plug gates physically block the lateral portals and are strategically latched onto the central axis for coordinated gating. Remarkably, this study has identified that the extracellular ectodomain of E-cadherin directly interacts with the extracellular cap domain of Piezo1, while its cytosolic tail with the TM region might interact with the cytosolic linker and CTD of Piezo1, which are in close proximity to the lateral plug gates. Thus, a physical interaction of E-cadherin with Piezo1 might allow a direct focus of cytoskeleton-transmitted force on the extracellular cap domain and the intracellular linker and CTD to gate the transmembrane gate and cytosolic lateral plug gates, respectively, providing a structural basis for a tether model for mechanogating of both gates. Furthermore, the Cap domain plays a critical role in determining the inactivation kinetics of Piezo channels. The direct interaction of the extracellular domain of E-cadherin with the Cap domain might provide a structural basis underlying the effect of E-cadherin in slowing the inactivation kinetics. Thus, these findings are consistent with the existence of tether-mediated gating of Piezo channel (Wang, 2022).

In addition to the tether model proposed in the present study, the Piezo channel might use its signature bowl-shaped structural feature and intrinsic mechanosensitivity to adopt the force-from-lipids model for mechanogating. Structural analysis reveals large conformational changes of the highly curved blades, which might be converted to the cytosolic lateral plug gates by a lever-like motion of the featured beam structure. The highly curved blade has been shown to undergo reversible flattening at biologically relevant pressures. Furthermore, this study has identified a serial of key mechanotransduction sites along the blade-beam-lateral plug gate, which might constitute an intramolecular mechanotransduction pathway for converting long-range conformational changes from the distal blades to the cytosolic lateral plug gates. Collectively, these studies suggest that the blade-beam apparatus might serve as the molecular basis for a force-from-lipids model to gate the cytosolic lateral plug gates (Wang, 2022).

Given the existence of the structurally and functionally distinct molecular bases for the proposed force-from-filament and force-from-lipids models, it is proposed that the Piezo channel might incorporate these two distinct yet non-exclusive gating models to serve as a versatile mechanotransducer. Using the dual mechanogating models, Piezo channels are not only able to constantly monitor changes in local membrane curvature and tension but also overcome the obstacle of limited membrane tension propagation within an intact cellular membrane to effectively detect long-range mechanical perturbation across a cell or between cells. Indeed, Piezo1 can mediate both localized and whole-cell mechanical responses regardless of whether the mechanical stimuli are either exogenously applied or endogenously originated (Wang, 2022).

The dual mechanogating model might also allow Piezo channels to flexibly tune to variable cellular context and to distinct forms of mechanical stimuli. For instance, the two gating models might act synergistically to sensitize the mechanosensitivity of Piezo channels. In line with this, the mechanosensitivity of Piezo1 was measured higher in intact cell membranes (T50 ~ 1.4 mN/m) than in artificial lipid bilayers (T50 ~ 3.4 mN/m) and membrane blebs that are presumed to lack actin cytoskeleton (T50 ~ 4.5 mN/m). Importantly, the convergence of the tether model and force-from-lipids model at the cytosolic lateral plug gates might allow a synergistic gating. The two models might also be preferentially used in a cellular context-dependent manner. For instance, actin-directed force rather than membrane tension might act dominantly to activate Piezo1 at the cell-substrate interface. In contrast, localized perturbation of membrane tension might mainly activate Piezo1 via the force-from-lipids model (Wang, 2022).

The identification of a direct interaction between the Piezo channel-based and the cadherin-based mechanotransduction systems might open a novel avenue in understanding of cellular mechanotransduction in general. Although this study has focused on E-cadherin to illustrate the proof of principle for the proposed tethered gating model of the Piezo channel, cadherins constitute a large superfamily of adhesion receptors, which are widely expressed in epithelium, endothelium, and the nervous system. Indeed, this study has found that N-cadherin, P-cadherin, and VE-cadherin all biochemically and functionally interact with Piezo1. Furthermore, the demonstration of a physical interaction between Piezo channels and cadherin raises the possibility that Piezo channels might also influence cadherin-mediated mechanotransduction process through at least two foreseeing mechanisms: Piezo channel-mediated Ca2+ signaling (e.g., the observed bell-shaped dose dependence of Piezo1-E-cadherin interaction on Ca2+ and a reciprocal force transmission from Piezo channels to the E-cadherin-mediated mechanotransduction complex. For instance, membrane tension-induced conformational changes of the bowl-shaped Piezo-membrane system might disturb the embedded Cap domain and consequently the ectodomain of E-cadherin, which is involved in both trans and cis dimerization of E-cadherin and the formation of adhesion junctions. Future studies will investigate how the two prominent mechanotransduction systems are integrated to regulate cellular mechanotransduction processes (Wang, 2022).

A definitive proof of the proposed tether model might require structural determination and analysis of the complex composed of Piezo1, E-cadherin, β-catenin, vinculin, and actin cytoskeleton. Furthermore, the physiological importance of the Piezo-cadherin-&betql-catenin-F-actin complex requires further investigation (Wang, 2022).

Identification of motor neurons and a mechanosensitive sensory neuron in the defecation circuitry of Drosophila larvae

Defecation allows the body to eliminate waste, an essential step in food processing for animal survival. In contrast to the extensive studies of feeding, its obligate counterpart, defecation, has received much less attention until recently. This study reports the characterizations of the defecation behavior of Drosophila larvae and its neural basis. Drosophila larvae display defecation cycles of stereotypic frequency, involving sequential contraction of hindgut and anal sphincter. The defecation behavior requires two groups of motor neurons that innervate hindgut and anal sphincter, respectively, and can excite gut muscles directly. These two groups of motor neurons fire sequentially with the same periodicity as the defecation behavior, as revealed by in vivo Ca(2+) imaging. Moreover, a single mechanosensitive sensory neuron was identified that innervates the anal slit and senses the opening of the intestine terminus. This anus sensory neuron relies on the TRP channel NOMPC but not on INACTIVE, NANCHUNG, or PIEZO for mechanotransduction (Zhang, 2014).

This study establishes the Drosophila larva as a model system for studying the defecation behavior. Drosophila larvae were found to exhibit periodic defecation cycles, involving sequential contractions of the hindgut and the anal sphincter. Two groups of neurons were found that innervate the hindgut and anal sphincter respectively, and can excite the hindgut and anal sphincter muscle in a sequential manner. In addition, a single sensory neuron was found that could sense the opening of the anal slit and send feedback to the motor neurons. Studies of C. elegans as a model system have investigated the defecation circuit. Studies of the adult fly have identified neurons regulating defecation behaviors subject to dietary and reproductive modulation. In this study of the defecation behavior in Drosophila larvae, not only the motor neurons innervating gut muscles were identified but also a sensory neuron strategically located to sense radial stretch during defecation were and provide feedback to the central nervous system (Zhang, 2014).

Previous studies of the defecation behaviors of the adult fly have revealed that its defecation rate is regulated by both the internal state and environment, rather than showing a robust rhythm. However, at the larval stage, the motor neurons and gut muscles as well as the sensory neuron responding to anus movement, all show very robust rhythmic activities. Given that feeding and defecation are dominant behaviors for third-instar larvae, conceivably robust rhythmic feeding and defecation behaviors may facilitate their nutrition intake and waste expulsion. In contrast, adult flies will likely encounter more complex environments and may need to conduct their defection behaviors in a more controllable manner (Zhang, 2014).

Mechanosensation serves a number of important physiological functions in Drosophila larvae. The radial stretch sensation is a special type of mechanosensation essential for the function of many organs with luminal structures such as the digestive system and the blood vessels. However, how the organs sense radial stretch remains unclear (Zhang, 2014).

This study has identified a sensory neuron that can sense radial stretch with its highly specialized morphology in Drosophila larvae. In addition, the TRP channel NOMPC but not other TRP channels tested, such as IAV that is often associated with NOMPC function, is required for normal ASN mechanotransduction. Interestingly, the ASN could be labeled by both class III da neuronal marker and class IV da neuronal maker, raising the question whether it might have the dual functions to sense different stimuli. The ASN may provide a neuronal model to study the distinct and cooperative roles of different channels in a single neuron in the sensing of different intensity of stimulation (Zhang, 2014).

The two motor neurons and the sensory neuron ASN provide an entry point to elucidate defecation circuitry. The two motor neurons appear to be functionally connected, possibly involving synaptic connections between them, although the possibility cannot be excluded of multiple neurons being engaged in their functional connections. It remains to be determined as to how they are entrained with this rhythmic firing pattern, and whether it involves a central pattern generator upstream of PDF neurons. Interestingly, PDF is a peptide that has important roles in multiple neuropeptide signaling pathways in the fruit fly; it would be interesting to test whether this neuropeptide also plays a role in the regulation of defecation behaviors by PDF neurons in the VNC. It is also of interest to explore possible contributions of indirect effects of PDF over muscle contraction, such as an influence of tracheal branching in the hindgut that may affect muscle contractions. Recently, a study has suggested a novel role of HGN1 neurons in regulating the long-term food intake behaviors of adult flies. In the current study it was found that HGN1 neurons control the rhythmic pattern of larval defecation. These two studies suggest that Drosophila HGN1 neurons at different developmental stages might have multiple functions in regulating feeding and defecation behaviors (Zhang, 2014).

Though separated in evolution millions years ago, the structures of Drosophila gut and human gut share striking similarity. There are circular and longitudinal muscles lining the gut ending with the anal sphincter that controls defecation. It remains an open question as to the extent of similarity of the mechanisms that control the gut movements. Diseases such as Hirschsprung's disease and anorectal malformation with failure to pass meconium are caused by developmental abnormality related to the gut and its innervation. Several genes and specific regions on the chromosomes have been shown or suggested to be associated with Hirschsprung's disease. Mutations in two human genes could lead to the absence of certain nerve cells in the colon. With the powerful genetic tools, further study of the Drosophila larval gut rhythmicity and its neural modulation will help identify evolutionarily conserved features as well as strategies that may have been adopted by different organisms for their fitness (Zhang, 2014).

The role of PPK26 in Drosophila larval mechanical nociception

In Drosophila larvae, the class IV dendritic arborization (da) neurons are polymodal nociceptors. This study shows that ppk26 (CG8546) plays an important role in mechanical nociception in class IV da neurons. Immunohistochemical and functional results demonstrate that ppk26 is specifically expressed in class IV da neurons. Larvae with mutant ppk26 showed severe behavioral defects in a mechanical nociception behavioral test but responded to noxious heat stimuli comparably to wild-type larvae. In addition, functional studies suggest that ppk26 and ppk (also called ppk1 or pickpocket) function in the same pathway, whereas Piezo functions in a parallel pathway. Consistent with these functional results, Ppk and Ppk26 are interdependent on each other for their cell surface localization. This work indicates that Ppk26 and Ppk might form heteromeric DEG/ENaC channels that are essential for mechanotransduction in class IV da neurons (Guo, 2014: PubMed).

Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila.

A major gap in understanding of sensation is how a single sensory neuron can differentially respond to a multitude of different stimuli (polymodality), such as propio- or nocisensation. The prevailing hypothesis is that different stimuli are transduced through ion channels with diverse properties and subunit composition. In a screen for ion channel genes expressed in polymodal nociceptive neurons, this study identified Ppk26, a member of the trimeric degenerin/epithelial sodium channel (DEG/ENaC) family, as being necessary for proper locomotion behavior in Drosophila larvae in a mutually dependent fashion with coexpressed Ppk1 (Pickpocket), another member of the same family. Mutants lacking Ppk1 and Ppk26 were defective in mechanical, but not thermal, nociception behavior. Mutants of Piezo, a channel involved in mechanical nociception in the same neurons, did not show a defect in locomotion, suggesting distinct molecular machinery for mediating locomotor feedback and mechanical nociception (Gorczyca, 2014).

Piezo1 links mechanical forces to red blood cell volume

Red blood cells (RBCs) experience significant mechanical forces while recirculating, but the consequences of these forces are not fully understood. Recent work has shown that gain-of-function mutations in mechanically activated Piezo1 cation channels are associated with the dehydrating RBC disease xerocytosis, implicating a role of mechanotransduction in RBC volume regulation. However, the mechanisms by which these mutations result in RBC dehydration are unknown. This study shows that RBCs exhibit robust calcium entry in response to mechanical stretch and that this entry is dependent on Piezo1 expression. Furthermore, RBCs from blood-cell-specific Piezo1 conditional knockout mice are overhydrated and exhibit increased fragility both in vitro and in vivo. Finally, it was shown that Yoda1, a chemical activator of Piezo1, causes calcium influx and subsequent dehydration of RBCs via downstream activation of the KCa3.1 Gardos channel, directly implicating Piezo1 signaling in RBC volume control. Therefore, mechanically activated Piezo1 plays an essential role in RBC volume homeostasis (Cahalan, 2015).

Gain-of-function mutations in the mechanically activated ion channel PIEZO2 cause a subtype of Distal Arthrogryposis

Mechanotransduction, the pathway by which mechanical forces are translated to biological signals, plays important but poorly characterized roles in physiology. PIEZOs are recently identified, widely expressed, mechanically activated ion channels that are hypothesized to play a role in mechanotransduction in mammals. This study describes two distinct PIEZO2 mutations in patients with a subtype of Distal Arthrogryposis Type 5 characterized by generalized autosomal dominant contractures with limited eye movements, restrictive lung disease, and variable absence of cruciate knee ligaments. Electrophysiological studies reveal that the two PIEZO2 mutations affect biophysical properties related to channel inactivation: both E2727del and I802F mutations cause the PIEZO2-dependent, mechanically activated currents to recover faster from inactivation, while E2727del also causes a slowing of inactivation. Both types of changes in kinetics result in increased channel activity in response to a given mechanical stimulus, suggesting that Distal Arthrogryposis Type 5 can be caused by gain-of-function mutations in PIEZO2. It was further shown that overexpression of mutated PIEZO2 cDNAs does not cause constitutive activity or toxicity to cells, indicating that the observed phenotype is likely due to a mechanotransduction defect. These studies identify a type of channelopathy and link the dysfunction of mechanically activated ion channels to developmental malformations and joint contractures (Coste, 2013).

Piezo1 plays a role in erythrocyte volume homeostasis

Mechanosensitivity is an inherent property of virtually all cell types, allowing them to sense and respond to physical environmental stimuli. Stretch-activated ion channels represent a class of mechanosensitive proteins which allow cells to respond rapidly to changes in membrane tension; however their identity has remained elusive. The piezo genes have recently been identified as a family of stretch-activated mechanosensitive ion channels. This study set out to determine the role of piezo1 during zebrafish development. Morpholino-mediated knockdown of piezo1 impairs erythrocyte survival without affecting hematopoiesis or differentiation. These results demonstrate that piezo1 is involved in erythrocyte volume homeostasis, disruption of which results in swelling/lysis of red blood cells and consequent anemia (Faucherre, 2014).

Mutations in PIEZO2 cause Gordon syndrome, Marden-Walker syndrome, and distal arthrogryposis type 5

Gordon syndrome (GS), or distal arthrogryposis type 3, is a rare, autosomal-dominant disorder characterized by cleft palate and congenital contractures of the hands and feet. Exome sequencing of five GS-affected families identified mutations in piezo-type mechanosensitive ion channel component 2 (PIEZO2) in each family. Sanger sequencing revealed PIEZO2 mutations in five of seven additional families studied (for a total of 10/12 [83%] individuals), and nine families had an identical c.8057G>A (p.Arg2686His) mutation. The phenotype of GS overlaps with distal arthrogryposis type 5 (DA5) and Marden-Walker syndrome (MWS). Using molecular inversion probes for targeted sequencing to screen PIEZO2, mutations were found in 24/29 (82%) DA5-affected families and one of two MWS-affected families. The presence of cleft palate was significantly associated with c.8057G>A. Collectively, although GS, DA5, and MWS have traditionally been considered separate disorders, these findings indicate that they are etiologically related and perhaps represent variable expressivity of the same condition (McMillin 2014).

Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells

Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. This study found that the mechanically gated ion channel Piezo1 is expressed by brain-derived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca(2+), a known modulator of differentiation, in a substrate-stiffness-dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. It is proposed that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells (Pathak, 2014).

Piezo2 is the major transducer of mechanical forces for touch sensation in mice

The sense of touch provides critical information about physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. This study shows that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. Piezo2 was precisely localized to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. These results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, it was found that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation (Ranade, 2014).

Inactivation of mechanically activated Piezo1 ion channels is determined by the C-terminal extracellular domain and the inner pore helix

Piezo proteins form mechanically activated ion channels that are responsible for our sense of light touch, proprioception, and vascular blood flow. Upon activation by mechanical stimuli, Piezo channels rapidly inactivate in a voltage-dependent manner through an unknown mechanism. Inactivation of Piezo channels is physiologically important, as it modulates overall mechanical sensitivity, gives rise to frequency filtering of repetitive mechanical stimuli, and is itself the target of numerous human disease-related channelopathies that are not well understood mechanistically. This study identifies the globular C-terminal extracellular domain as a structure that is sufficient to confer the time course of inactivation and a single positively charged lysine residue at the adjacent inner pore helix as being required for its voltage dependence. The results are consistent with a mechanism for inactivation that is mediated through voltage-dependent conformations of the inner pore helix and allosteric coupling with the C-terminal extracellular domain (Wu, 2017).

Search PubMed for articles about Drosophila Piezo

Cahalan, S. M., Lukacs, V., Ranade, S. S., Chien, S., Bandell, M. and Patapoutian, A. (2015). Piezo1 links mechanical forces to red blood cell volume. Elife 4. PubMed ID: 26001274

Coste, B., Xiao, B., Santos, J. S., Syeda, R., Grandl, J., Spencer, K. S., Kim, S. E., Schmidt, M., Mathur, J., Dubin, A. E., Montal, M. and Patapoutian, A. (2012). Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483: 176-181. PubMed ID: 22343900

Coste, B., et al. (2013). Gain-of-function mutations in the mechanically activated ion channel PIEZO2 cause a subtype of Distal Arthrogryposis. Proc Natl Acad Sci U S A 110(12): 4667-4672. PubMed ID: 23487782

Faucherre, A., Kissa, K., Nargeot, J., Mangoni, M. E. and Jopling, C. (2014). Piezo1 plays a role in erythrocyte volume homeostasis. Haematologica 99(1): 70-75. PubMed ID: 23872304

Gorczyca, D. A., Younger, S., Meltzer, S., Kim, S. E., Cheng, L., Song, W., Lee, H. Y., Jan, L. Y. and Jan, Y. N. (2014). Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila. Cell Rep 9: 1446-1458. PubMed ID: 25456135

Guo, Y., Wang, Y., Wang, Q. and Wang, Z. (2014). The role of PPK26 in Drosophila larval mechanical nociception. Cell Rep 9: 1183-1190. PubMed ID: 25457610

Hu, Y., Wang, Z., Liu, T. and Zhang, W. (2019). Piezo-like gene regulates locomotion in Drosophila larvae. Cell Rep 26(6): 1369-1377. PubMed ID: 30726723

Kim, S. E., Coste, B., Chadha, A., Cook, B. and Patapoutian, A. (2012). The role of Drosophila Piezo in mechanical nociception. Nature 483: 209-212. PubMed ID: 22343891

McMillin, M. J., et al. (2014). Mutations in PIEZO2 cause Gordon syndrome, Marden-Walker syndrome, and distal arthrogryposis type 5. Am J Hum Genet 94(5): 734-744. PubMed ID: 24726473

Pathak, M. M., Nourse, J. L., Tran, T., Hwe, J., Arulmoli, J., Le, D. T., Bernardis, E., Flanagan, L. A. and Tombola, F. (2014). Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells. Proc Natl Acad Sci U S A 111(45): 16148-16153. PubMed ID: 25349416

Peyronnet, R., Martins, J. R., Duprat, F., Demolombe, S., Arhatte, M., Jodar, M., Tauc, M., Duranton, C., Paulais, M., Teulon, J., Honore, E. and Patel, A. (2013). Piezo1-dependent stretch-activated channels are inhibited by Polycystin-2 in renal tubular epithelial cells. EMBO Rep 14: 1143-1148. PubMed ID: 24157948

Ranade, S. S., et al. (2014). Piezo2 is the major transducer of mechanical forces for touch sensation in mice. Nature 516(7529): 121-125. PubMed ID: 25471886

Suslak, T. J., Watson, S., Thompson, K. J., Shenton, F. C., Bewick, G. S., Armstrong, J. D. and Jarman, A. P. (2015). Piezo is essential for amiloride-sensitive stretch-activated mechanotransduction in larval Drosophila dorsal bipolar dendritic sensory neurons. PLoS One 10: e0130969. PubMed ID: 26186008

Wang, J., Jiang, J., Yang, X., Zhou, G., Wang, L., Xiao, B. (2022). Tethering Piezo channels to the actin cytoskeleton for mechanogating via the cadherin-beta-catenin mechanotransduction complex. Cell Rep, 38(6):110342 PubMed ID: 35139384

Wu, J., Young, M., Lewis, A. H., Martfeld, A. N., Kalmeta, B. and Grandl, J. (2017). Inactivation of mechanically activated Piezo1 ion channels is determined by the C-terminal extracellular domain and the inner pore helix. Cell Rep 21(9): 2357-2366. PubMed ID: 29186675

Wu, S. F., Ja, Y. L., Zhang, Y. J. and Yang, C. H. (2019). Sweet neurons inhibit texture discrimination by signaling TMC-expressing mechanosensitive neurons in Drosophila. Elife 8. PubMed ID: 31184585

Zhang, L., Yu, J., Guo, X., Wei, J., Liu, T. and Zhang, W. (2020). Parallel mechanosensory pathways direct oviposition decision-making in Drosophila. Curr Biol. PubMed ID: 32649914

Zhang, W., Yan, Z., Li, B., Jan, L. Y. and Jan, Y. N. (2014). Identification of motor neurons and a mechanosensitive sensory neuron in the defecation circuitry of Drosophila larvae. Elife 3. PubMed ID: 25358089

date revised: 1 June 2024

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