ABSTRACT
PIEZO channels are force sensors essential for physiological processes including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, expressed in several tissues including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive channel and contributes to pharyngeal function remains unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recordings to establish that pezo-1 is expressed in the pharynx, including a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity and food quality. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the pezo-1 isoform G (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms.
INTRODUCTION
Mechanosensitive ion channels regulate several physiological processes ranging from osmotic balance in bacteria (Kung, Martinac and Sukharev, 2010), turgor control in plants (Hamilton, Schlegel and Haswell, 2015), touch (Geffeney and Goodman, 2012; Yan et al., 2013; Ikeda et al., 2014; Maksimovic et al., 2014; Ranade et al., 2014; Woo et al., 2014; Chesler et al., 2016), pain (Murthy et al., 2018; Szczot et al., 2018), proprioception (Woo et al., 2015), hearing (Pan et al., 2018), lineage choice (Pathak et al., 2014), and blood pressure regulation in animals (Retailleau et al., 2015; Wang et al., 2016; Rode et al., 2017; Zeng et al., 2018). These channels are ubiquitous, as they transduce mechanical stimuli into electrochemical signals in all kingdoms of life (Kung, Martinac and Sukharev, 2010; Geffeney and Goodman, 2012; Douguet and Honoré, 2019). In 2010, the PIEZO1 and PIEZO2 channels were identified as essential components of distinct, mechanically activated cation channels in mammalian cells (Coste et al., 2010). Since then, many physiological roles have been assigned to these two ion channels (Parpaite and Coste, 2017).
Mammalian PIEZO channels have been associated with several hereditary pathophysiologies (Alper, 2017). Piezo1 gain-of-function (GOF) mutations display slow channel inactivation leading to an increase in cation permeability and subsequent red blood cell dehydration (Zarychanski et al., 2012; Albuisson et al., 2013; Bae et al., 2013; Ma et al., 2018). For instance, the human Piezo1 hereditary mutation R2456H located in the pore domain decreases inactivation and, when substituted by Lys inactivation is completely removed (Bae et al., 2013). Piezo1 global knockouts (KO) are embryonically lethal in mice (Li et al., 2014; Ranade et al., 2014) and cell-specific KOs result in animals with severe defects (Wu, Lewis and Grandl, 2017; Ma et al., 2018). Intriguingly, both Piezo2 KO and GOF mutations are associated with joint contractures, skeletal abnormalities and alterations in muscle tone (Coste et al., 2013; Chesler et al., 2016; Yamaguchi et al., 2019). GOF and loss-of-function (LOF) mutations are useful genetic tools to determine the contribution of PIEZO channels to mechanosensation in various physiological processes and in different animals.
The C. elegans genome encodes an ortholog of the Piezo channel family, namely pezo-1 (wormbase.org v. WS280). Recently, Bai and collaborators showed that pezo-1 is expressed in several tissues including the pharynx (Bai et al., 2020). The worm’s pharynx is a pumping organ that rhythmically couples muscle contraction and relaxation in a swallowing motion to pass food down to the animal’s intestine (Keane and Avery, 2003). This swallowing motion stems from a constant low-frequency pumping maintained by pharyngeal muscles and bursts of high-frequency pumping from a dedicated pharyngeal nervous system (Avery and Horvitz, 1989; Raizen, Lee and Avery, 1995; Trojanowski, Raizen and Fang-Yen, 2016; Lee et al., 2017). In mammals, the swallowing reflex is initiated when pressure receptors in the pharynx walls are stimulated by food or liquids, yet the identity of the receptor(s) that directly evoke this mechanical response remain to be identified (Tsujimura et al., 2019). Interestingly, the Drosophila melanogaster PIEZO ortholog is a mechanosensitive ion channel (Kim et al., 2012) required for feeding while also avoiding food over-consumption (Min et al., 2020; Wang et al., 2020). To date, whether pezo-1 encodes for a mechanosensitive ion channel or regulates worm’s pharyngeal activity has yet to be determined.
Here, we found a strong and diverse expression of the pezo-1 gene in pharyngeal tissues by imaging a pezo-1::GFP transgenic reporter strain. By leveraging genetic dissection, electrophysiological measurements, and behavior analyses, we also established that PEZO-1 is required for proper low frequency electric activity and pumping behavior. Analyses of pezo-1 KO and GOF mutants demonstrated that decreasing or increasing PEZO-1 function upregulates pharyngeal pumping frequency. Likewise, mutants display distinct pharyngeal activities triggered by the neurotransmitter serotonin or with various buffer osmolarities. By using elongated bacteria as a food source, we demonstrated that pezo-1 KO decreases pharyngeal pumping frequency, whereas the GOF mutant increases it. Finally, electrophysiological recordings of pezo-1 expressing cells from C. elegans embryo cultures and Spodoptera frugiperda (Sf9) cell line demonstrate that pezo-1 encodes a mechanosensitive ion channel. Altogether, our results show that pezo-1 is a mechanosensitive ion channel involved in a novel biological function regulating pharyngeal pumping and food sensation.
MATERIALS AND METHODS
Strains and Maintenance
Worms were propagated as previously described (Brenner, 1974). N2 (var. Bristol) was referred as wild type (WT) throughout the manuscript. The following strains were used: VVR3 unc119(ed3)III;decEx1(pRedFlpHgr)(C10C5.1[20789]::S0001_pR6K_Amp_2xTY1ce_EGFP_FRT_rpsl_neo_FRT_3xFlag)dFRT::unc-119-Nat, COP1553 (KO: 6,616 bp deletion) pezo-1 (knu508) IV, COP1524 (GOF: R2373K) pezo-1 (knu490) IV, LX960 lin-15B&lin-15A(n765) X; vsIs97 [tph-1p::DsRed2 + lin-15(+)], DA572 eat-4(ad572) III, and DA1051 avr-15(ad1051) V. Transgenic strain VVR3 was obtained by microinjecting a fosmid construct (from the TransgeneOme Project) in a unc-119(ed3) strain from InVivo Biosystems. COP1553 and COP1524 were obtained using the CRISPR-Cas9 method (InVivo Biosystems). Transgenic worm VVR3 expressing GFP under the control of Ppezo-1::GFP was crossed with pezo-1 mutants COP1553 and COP1524 to obtain VVR69 and VVR70, respectively. LX960 was kindly provided by Dr. Kevin Collins (University of Miami).
Imaging
Worms were selected individually and dropped in 15 μL of M9 buffer (86 mM NaCl, 42 mM Na2HPO4, 22 mM KH2PO4, 1mM MgSO4) paralyzed on a glass slide containing 2% agarose pads containing 150 mM 2,3-butanedione monoxime (BDM). Bright field and fluorescence imaging were done on a Zeiss 710 Confocal microscope using either a 20X or 40X objective. Images were processed using Fiji ImageJ (Schindelin et al., 2009) to enhance contrast and convert to an appropriate format.
Worms’ synchronization
For all pharyngeal pumping assays, worms were synchronized by picking young adults onto fresh nematode growth media (NGM) plates seeded with E coli strain OP50 and left to lay eggs for two hours at 20°C. Then, the adults were removed, and the plates incubated at 20°C for three days.
Pharyngeal pumping
Serotonin profile
A serotonin aliquot (InVivo Biosystems) was diluted in M9 Buffer prior to experiments and discarded after three hours. 42 synchronized worms were picked and transferred in 200 μL of M9 Buffer supplemented with 2-, 5-, 10- or 20-mM serotonin and incubated at 20°C for 30 minutes before being loaded inside the microfluidic chip (SC40, The ScreenChip™ System, InVivo Biosystems).
Control E. coli assay
OP50 was grown in liquid LB medium under sterile conditions at 37°C and diluted to an optical density of 1.0. Bacterial cultures were stored at 4°C for up to a week.
Spaghetti-like E. coli assay
OP50 colonies were picked from a fresh LB plate and incubated in 2 mL of LB overnight the day before the experiment. The following day, 0.5 mL of the pre-incubation culture was used to inoculate 1.5 mL of LB media and grown until growth was exponential, which was verified by checking optical density (optical density of 0.5). Cephalexin (Alfa Aesar™) was then added at 60 μg/ml final concentration and the culture was incubated for two hours. Spaghetti-like OP50 were verified under a microscope and washed three times using 2 mL of M9 buffer followed by centrifugation at 400 g to gently pelletize the elongated bacteria.
Pharyngeal recordings and Analyses
Worms were loaded one-by-one inside the microfluidic chip recording channel and left to adjust for one minute prior to recording. All recordings were two minutes long. Records were analysed using NemAnalysis software (InVivo Biosystems) with the brute force algorithm turned off. Parameters were adjusted for each record in order to include the maximum numbers of clearly identifiable pharyngeal pumps. Results were exported from the software in sheet form and parameters were plotted and statistically analysed using MATLAB R2019a (MathWorks).
Development assay
Young adults were allowed to lay eggs on NGM plates seeded with control or spaghetti-like bacteria for two hours. Spaghetti-like bacteria were cultured as described above. Animals (10-20 worms) were removed for plates after two hours and the number of eggs laid was counted. After 3 days of incubation, animals that reached adulthood were counted in each trial, and results were compared across four trials.
Food ingestion assay
A drop of fresh cultured of control or spaghetti-like bacteria with 2 μM DiI dye (Sigma, CAS #41085-99-8) was placed on a NGM agar plate. Young adults were fed bacteria with DiI for 30 min. Worms were transferred into OP50 seeded NGM without dye for 5 min (Vidal-Gadea et al., 2012). Animals were placed in a thin-layered BDM-agarose plate for imaging under a Nikon SMZ18 stereomicroscope. Food occupation in the digestive tract was detected by fluorescence.
Primary culture of C. elegans embryo cells
C. elegans embryonic cells were generated as previously described (Strange, Christensen and Morrison, 2007). Worms were grown on 10 cm enriched peptone plate with NA22 E. coli. NA22 bacteria grow in very thick layers that provide abundant food source for large quantities of worms. The synchronized gravid hermaphrodites were bleached to release eggs and washed with sterile egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, pH 7.3, 340 mOsm, adjusted with sucrose). The isolated eggs were separated from debris by centrifugation in a 30% sucrose solution. Chitinase (1 U/ml, Sigma) digestion was performed to remove eggshells. The embryo cells were dissociated by pipetting and filtered through a sterile 5μm Durapore filter (Millipore). The cells were plated on glass coverslips coated with peanut lectin solution (SIGMA; 0.5 mg/ml) and cultured in L15 media (Gibco) supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin with 10% fetal bovine serum (FBS, Invitrogen) for 72-96 hrs.
Expression of pezo-1 in Sf9 insect cells
We generated a baculovirus construct consisting of an 8× histidines-maltose binding protein (MBP) tag over pezo-1 isoform G synthesized nucleotide sequence (one of the longest isoforms according to RNA sequencing, wormbase.org v. WS280). We infected Sf9 cells with the pezo-1 containing baculovirus for 48 hours. Infected cells were plated on glass coverslips coated with a peanut lectin solution (SIGMA; 0.5 mg/ml) for patch-clamp experiments.
Electrophysiology and mechanical stimulation
Primary cultured embryo cells labeled with Ppezo-1::GFP from strains VVR3, VVR69, or VVR70 were recorded in the cell-attached configuration of the patch clamp technique. Control and infected Sf9 insect cells were recorded in the whole-cell patch clamp configuration. For on-cell recordings, the bath solution contained 140 mM KCl, 6 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4; 340 mOsm, adjusted with sucrose). The pipette solution contained 140 mM NaCl, 6 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.3; 330 mOsm, adjusted with sucrose). Pipettes were made out of borosilicate glass (Sutter Instruments) and were fire-polished before use until a resistance between 3 and 4 MΩ was reached. Currents were recorded at a constant voltage (−60 mV, unless otherwise noticed), sampled at 20 kHz, and low pass filtered at 2 kHz using a MultiClamp 700B amplifier and Clampex (Molecular Devices, LLC). Leak currents before mechanical stimulations were subtracted offline from the current traces. Cells were mechanically stimulated with negative pressure applied through the patch pipette using a High-Speed Pressure Clamp (ALA Scientific) automated using a MultiClamp 700B amplifier through Clampex (Molecular Devices, LLC). Cell-attached patches were probed using a square-pulse protocol consisting of −10 mmHg incremental pressure steps, each lasting 1 s in 10 s intervals. Cells which giga-seals did not withstand at least six consecutive steps of mechanical stimulation were excluded from analyses. Isteady was defined as the maximal current in the steady state. Deactivation was compared by determining the percentage of Isteady left 100 ms after the mechanical stimuli ended.
For whole-cell recordings, the bath solution contained 140 mM NaCl, 6 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). The pipette solution contained 140 mM CsCl, 5 mM EGTA, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES (pH 7.2). For indentation assays, Sf9 cells were mechanically stimulated with a heat-polished blunt glass pipette (3–4 μm) driven by a piezo servo controller (E625, Physik Instrumente). The blunt pipette was mounted on a micromanipulator at an ~45° angle and positioned 3–4 μm above from the cells without indenting them. Displacement measurements were obtained with a square-pulse protocol consisting of 1 μm incremental indentation steps, each lasting 200 ms with a 2 ms ramp in 10 s intervals. Recordings with leak currents >200 pA, with access resistance >10 MΩ, and cells which giga-seals did not withstand at least five consecutive steps of mechanical stimulation were excluded from analyses.
Data and fits were plotted using OriginPro (from OriginLab). Sigmoidal fit was done with the following Boltzmann equation: where A2 = final value, A1 = initial value; Xo = center, and dX = time constant.
Data and Statistical analyses
Data and statistical analyses were performed using DataGraph 4.6.1, MATLAB R2019a (MathWorks), and GraphPad Instat 3 software. Statistical methods and sample numbers are detailed in the corresponding figure legends. No technical replicates were included in the analyses.
Results
pezo-1 is expressed in a wide variety of cells in the worm’s pharynx
To determine the expression of pezo-1 in C. elegans, we used a fluorescent translational reporter made by the TransgeneOme Project (Hasse, Hyman and Sarov, 2016). This fosmid construct contains pezo-1 native cis-regulatory elements, including introns, up to exon 17 and 3’ UTR sequences linked in-frame to the green fluorescent protein (GFP; Figure 1A). The position of the GFP with respect to the remainder of the gene creates an unnatural truncated version of the PEZO-1 protein. Hence, it likely expresses a non-functional protein that misses 16 exons containing the majority of the pezo-1 sequence (including the pore domain). GFP signals are present in all developmental stages and multiple cells (Supplementary Figure 1A-B), and it does not appear to be mosaic as similar expression patterns were observed in at least three independent transgenic lines. We imaged pezo-1::GFP worms at different focal planes to identify the different cells expressing GFP based on their morphological features (i.e., cell-bodies position, neurites extension and positions along the body, and branching). The strongest GFP signals that we identified come from the pharyngeal gland cells (Figure 1B, bright and fluorescence fields). These cells are composed of five cell bodies (two ventral g1s, one dorsal g1 and two ventral g2s) located inside the pharynx terminal bulb and three anterior cytoplasmic projections: two shorts that are superposed ending in the metacorpus and a long one reaching the end of the pm3 muscle. These cells are proposed to be involved in digestion (Albertson and Thomson, 1976; Ohmachi et al., 1999), lubrication of the pharynx (Smit, Schnabel and Gaudet, 2008), generation and molting of the cuticle (Singh and Sulston, 1978; Höflich et al., 2004), and resistance to pathogenic bacteria (Höflich et al., 2004). Additionally, we visualized, pezo-1::GFP in a series of cells surrounding the muscle of the corpus and the isthmus (Figure 1C) whose morphology and location match with those of the arcade cells; these were previously hypothesized to be support cells (Albertson and Thomson, 1976). We also recognized as putative pezo-1 expressing cells: glial cell AmSh, pharyngeal interneuron I3 (Figure 1D), and M3L/R somas and neurites (Figure 1E-F). These neurons are involved in pharyngeal pumping relaxation (Avery, 1993). The finger-like structure known as the pharyngeal sieve, at the junction between corpus and isthmus, also expresses pezo-1 (Figure 1F).
By crossing pezo-1::GFP with a tph-1::DsRed marker carrying strain, we were able to identify pezo-1 expression in the pharyngeal NSML/R secretory, motor, and sensory neurons (Figure 1G). Importantly, these serotoninergic neurons have been proposed to sense food in the lumen of the pharynx through their proprioceptive-like endings and trigger feeding-related behaviors (i.e., increased pharyngeal pumping, decreased locomotion, and increased egg laying) (Albertson and Thomson, 1976; Avery, Bargmann and Horvitz, 1993). In addition to the pharyngeal cells, we observed expression of pezo-1 in the ventral nerve cord (VNC, Figure 1D) neurons, striated muscles (Supplementary Figure 1C), coelomocytes (Supplementary Figure 1D-E), spermatheca (Supplementary Figure 1B and 1E), vulval muscles (Supplementary Figure 1F), and various male neurons including the ray neurons (Supplementary Figure 1G). Importantly, the expression pattern reported by our pezo-1 fosmid construct matches very well with the gene expression atlas for C. elegans neurons (Taylor et al., 2020), except for the M3 neurons. The strong and varied pezo-1 expression in the pharynx along with the function of the cells expressing it, led us to investigate the potential contribution of PEZO-1 to pharyngeal function.
Serotonin stimulation reveals different pharyngeal pump parameters
To analyze the contribution of pezo-1 to pharyngeal pumping in C. elegans, we used the ScreenChip™ system (InVivo Biosystems) that allows measuring electropharyngeogram recordings (EPG; Figure 2A) (Raizen and Avery, 1994) by loading single-live worms inside a microfluidic chip. Figure 2A-B summarizes the pharynx anatomy, electrical properties measured during an EPG, and the neurons involved in pharyngeal function. For instance, the excitation event (E spike) precedes the pharyngeal contraction and is modulated by the pacemaker neuron MC (Figure 2B, top), whereas the repolarization event (R spike) leads to pharyngeal relaxation and correlates to the activity of the inhibitory M3 motorneurons (Figure 2B, middle). Every 3-4 pumps, there is relaxation of the terminal bulb (isthmus peristalsis) which is modulated by the motorneuron M4 (Figure 2B, bottom) (Avery and Horvitz, 1989). The main EPG events are regulated by the pharyngeal proprioceptive neuron NSM. Importantly, M3 motorneurons (potentially) and the NSM proprioceptive neuron express pezo-1 (Figure 1E-G).
Analyses of the EPG records allow determination of different pharyngeal pumping parameters including frequency, duration, and the time interval that separates two pumping events (hereafter referred to as the interpump interval). We used serotonin to increase pharyngeal activity since, in the absence of food or serotonin, the pumping events are infrequent. Serotonin mimics food stimulation by activating the MCL/R and M3L/R neurons (Niacaris, 2003). First, we established a serotonin dose-response profile of the WT strain pharyngeal pumping parameters (N2; Figure 2C-G). Serotonin increases pharyngeal pumping frequency in a dose-dependent manner, with concentrations above 5 mM increasing the likelihood of reaching 5 Hz (Figure 2C). We averaged the EPG recordings at each serotonin concentration and found a clear difference in pump durations between 0- and 5-mM. Concentrations equal or higher than 5 mM evoke similar pump durations (~100 ms; Figure 2D). Interestingly, analyses of the pump duration distribution profile under serotonin stimulation revealed that pharyngeal pump duration fits into two categories: fast (~80 ms) and slow (100-120 ms; Figure 2E; gray rectangles).
We observed that the fast and slow categories displayed an inverse relationship with respect to serotonin concentration (Figure 2E; arrows). We hypothesize that slow (100-120 ms) pumps are the result of the serotoninergic activation of M3L/R while the fast (~ 80 ms) pumps originate from a mechanism that is partly independent from the serotonin pathway. Unlike pump duration, we observed only one category for interpump intervals around 95-120 ms for 5-to 20-mM serotonin concentrations (Figure 2F-G). Interestingly, we did not observe inter pump intervals faster than 90 ms, regardless of the serotonin concentration. The inter pump intervals results support the idea that there is a minimum refractory period between two pumps. This set of analyses allowed us to establish a suitable model for evaluating the role of pezo-1 function in vivo.
pezo-1 mutants display higher pumping frequency than WT worms
To determine whether pezo-1 has a functional role in pharyngeal pumping, we engineered LOF and GOF mutants. A putative LOF mutant obtained by deleting 6,616 bp from the pezo-1 locus (hereafter referred to as pezo-1 KO; Figure 3A, top). Previous works demonstrated that the substitution of R2456H (located at the pore helix) of the ortholog human Piezo1 gene increases cation permeability (GOF) and causes hemolytic anemia (Zarychanski et al., 2012; Albuisson et al., 2013; Bae et al., 2013). Moreover, a conservative substitution of Lys for Arg at position 2456 in the human Piezo1 channel exhibits a pronounced decreased inactivation when compared to the WT or R2456H channels (Bae et al., 2013). Hence, we engineered a putative GOF mutant strain obtained by substituting the conserved Arg 2373 with Lys (hereafter referred to as pezo-1 R2373K or GOF; Figure 3A, bottom). Parenthetically, the R2373K numbering position is based on isoform G – one of the longest isoforms according to RNA sequencing (wormbase.org v. WS280). We also included in our analysis two mutants known to alter pharyngeal function, eat-4(ad572) and avr-15(ad1051). EAT-4 is a glutamate-sodium symporter involved in postsynaptic glutamate reuptake. eat-4(ad572) affects the neurotransmission efficiency of all glutamatergic pharyngeal neurons (I2L/R, I5, M3L/R, M4, MI, NSML/R) (Lee et al., 1999). AVR-15 is a glutamate-gated chloride channel expressed in the pharyngeal muscle pm4 and pm5 (both synapsed by M3L/R) and involved in relaxation of the pharynx. Its mutant allele ad1051 lengthen pump duration by delaying relaxation of the pharynx in a similar fashion as laser ablation of M3L/R neurons (Dent, Davis and Avery, 1997). With these strains, we sought to determine if altering PEZO-1 function would affect the worm’s pharyngeal phenotype.
At 2 mM concentration of exogenous serotonin (to elicit pharyngeal activity), both pezo-1 KO and R2373K mutants displayed significantly higher pumping frequencies than WT and similar to avr-15(ad1051) (Figure 3B). On the other hand, the eat-4(ad572) mutant displayed lower pumping frequency at this serotonin concentration. To further assess the pezo-1 mutants’ pharyngeal altered function, we analyzed the pump duration distributions from the EPG records. pezo-1 KO distribution is similar to the WT (Figure 3C, red vs. black) whereas the R2373K mutant profile is reminiscent of the avr-15(ad1051), as both mutant strains displayed a narrower distribution around 100 ms pump events (Figure 3C, blue and green vs. black). Moreover, the R2373K mutant lacked fast pump events between 50 to 80 ms (Figure 3C, blue bracket), similar to the WT features observed at high serotonin concentrations (≥ 5 mM, Figure 2E), and the eat-4(ad572) mutant and the avr-15(ad1051) mutant at 2 mM serotonin concentration (Figure 3C, yellow and green brackets). The analysis of distribution of interpump intervals revealed that pezo-1 KO and R2373K mutants, although different, both spend less time resting between pumps (95-120 ms) than the WT (≈ 140 ms) (Figure 3D, red and blue brackets). This enhancement in function resembles the WT activity measured at 5-to 20-mM serotonin concentrations (Figure 2F-G) and could account for the increase in frequency shown in Figure 3B. The close resemblance between the PEZO-1 GOF and the avr-15(ad1051) mutants’ pharyngeal pumping parameters suggests a potential link between PEZO-1 and pharyngeal relaxation through M3L/R neurotransmission.
PEZO-1 determines pharyngeal pumping in response to hyperosmolarity
Mechanical stimuli come in many forms including stretching, bending, and osmotic forces (Cox, Bavi and Martinac, 2019). To further understand the functional role of pezo-1, we evaluated pharyngeal pumping parameters after challenging worm strains with different osmolarities. To this end, we diluted the standard solution used for worm experiments (M9 buffer) to different osmolarities (150, 260, and 320 mOsm). Low osmolarity solutions would be equivalent to swallowing food containing few solutes (150 mOsm) whereas high osmolarities would represent a “gulp” with large amounts of solutes (320 mOsm). Noteworthy, the higher the osmolarity, the shorter the mean pumping frequency of WT worms (Figure 4A). Our results indicate that the larger the number of solutes in solution, the longer they would reside in the pharynx before moving to the intestine. Notably, at 320 mOsm, both pezo-1 KO and GOF mutants displayed a significantly higher frequency than WT (Figure 4A). On the other hand, at 260 and 150 mOsm we did not measure significant differences between WT and the pezo-1 mutants. Similar to human Piezo2 KO and GOF mutations (associated with joint contractures), we demonstrated that lack of or enhanced PEZO-1 function modulated pharyngeal pumping frequencies in the same fashion (at high osmolarities). Next, we further examined the EPG parameters at high osmolarity (320 mOsm). Analyses of the distribution of pump durations and length of the mean interpump intervals revealed that both pezo-1 mutants had more frequent fast pumps (80-120 ms, Figure 4B, gray rectangles) and spent less time resting between pumps than the WT (Figure 4C). Interestingly, high osmolarity (320 mOsm) revealed a close resemblance between PEZO-1 GOF and the avr-15(ad1051) mutants’ pharyngeal pumping parameters (frequency and duration, Figure 4D-E) suggesting a potential link between PEZO-1 and M3L/R function. Altogether, our results suggest that PEZO-1 is required for fine tuning pharyngeal function in response to osmolarity changes.
PEZO-1 function is involved in food sensation
To determine the impact that PEZO-1 function has on food intake, we recorded pharyngeal pumping of WT and pezo-1 strains in response to different food stimuli. It has been hypothesized that food quality and feeding preferences displayed by worms is linked to the bacteria size (Shtonda and Avery, 2006). To this end, we measured worms pharyngeal pumping while feeding them the conventional food used in the laboratory for maintenance (Escherichia coli strain OP50). Additionally, we varied the dimensions of OP50 using the antibiotic cephalexin; an antibiotic that prevents the separation of budding bacteria, generating long spaghetti-like filaments of bacterium as observed under a microscope and elsewhere (Martinac et al., 1987) (Supplementary Figure 2A). A similar method was previously described using the antibiotic aztreonam and has been shown to affect pharyngeal pumping (Gruninger, Gualberto and Garcia, 2008; ben Arous, Laffont and Chatenay, 2009).
WT and pezo-1 mutants are able to ingest spaghetti-like bacteria and reached adulthood in three days, similar to worms fed with control bacteria (Supplementary Figure 2B-C). Notably, feeding worms with control or spaghetti-like bacteria revealed different pharyngeal traits between the pezo-1 mutants and the WT worms. When fed with control E. coli, both pezo-1 mutants (KO and GOF) have higher mean frequencies, shorter mean pump durations, narrower pump duration distributions, and fastest mean interpump intervals than the WT worms (Figure 5A-C, E-F). On the other hand, feeding worms with spaghetti-like E. coli elicits opposite effects on the pezo-1 mutants pharyngeal pumping parameters. For instance, spaghetti-like E. coli decreases pezo-1 KO mean frequency, while keeping the mean pump duration and distribution similar to WT (Figure 5A-B, D). Furthermore, this modified diet significantly increases the mean interpump interval of the KO in comparison to the WT and the GOF mutant (Figure 5E-F). Unlike the KO and WT, the R2372K pezo-1 mutant displays high frequency, short pumps (mean and distributions; Figure 5A-B, D) and short mean interpump interval durations (mean and distributions; Figure 5E-F). Altogether, our results indicate that PEZO-1 regulates the pharynx response to food physical parameters, such as length and shape of the ingested bacteria.
pezo-1 encodes a mechanosensitive ion channel
The PEZO-1 protein sequence shares 60-70% similarity with mammalian PIEZO channel orthologs. However, whether PEZO-1 responds to mechanical stimuli has not yet been established. To address this major question, we generated three different pezo-1 strains expressing the pezo-1::GFP, pezo-1::GFP KO, and pezo-1::GFP R2373K mutation. pezo-1::GFP cells were patch-clamped using the cell-attached configuration while applying constant negative pressure (−70 mmHg) and steps of positive and negative voltages to the pipette (Figure 6A-C). The current vs. voltage relationship is characterized by a reversal potential of + 9.31 mV (Figure 6D), indicating that PEZO-1 mediates a slight cation selective conductance like the mouse and Drosophila’s orthologs (Coste et al., 2012).
Mechanical stimulation of cells expressing WT PEZO-1 elicited mechano-dependent currents (Figure 7A-C, black traces and bar) with a half pressure activation (P1/2) corresponding to −59.1 ± 4.3 mmHg (mean ± SEM). Importantly, pezo-1::GFP cells expressing KO PEZO-1 did not feature mechanosensitive currents, even at larger negative pressure magnitudes (Figure 7A and C, red traces and bar). On the other hand, PEZO-1 R2373K displayed mechano-dependent currents (Figure 7A-C, blue traces and bar) with lower P1/2 than the WT channel (39.2 ± 2.2 mmHg, mean ± SEM), indicating that the GOF mutant requires less mechanical activation to open. Notably, the R2373K mutation introduced a latency for activation that was not detected in the WT (Figure 7A, blue traces and 7D). The decrease in mechanical threshold along with the slow activation were previously reported for the equivalent human PIEZO1 R2456K mutation in mammalian cell lines (Zarychanski et al., 2012; Albuisson et al., 2013; Bae et al., 2013; Romero et al., 2019). Future experiments are needed to understand the origin of these differences in activation. Unlike pezo-1 WT, approximately 50% of the mechano currents elicited from the pezo-1 R2373K expressing cells remained active even after the mechanical stimulus ended (Figure 7A, blue traces, and 7E). This slow deactivation is also reminiscent of the human PIEZO1 R2456K GOF phenotype previously characterized by Bae and collaborators (Bae et al., 2013). Overall, our results support that PEZO-1 is an ion channel gated by membrane tension and that a conservative mutation in the pore domain elicits similar activation and kinetic changes as its human counterpart.
To further validate that the pezo-1 gene encodes for a mechanosensitive ion channel, we heterologously expressed one of the longest isoforms of pezo-1 (isoform G; wormbase.org v. WS280) in Sf9 cells. Similar to mammalian PIEZO channels, PEZO-1 mediates indentation-activated currents (Figure 8). Uninfected Sf9 cells do not display mechanosensitive channel currents (Figure 8B-C). Importantly PEZO-1 displayed the properties described for mammalian PIEZOs in other cell types including (Coste et al., 2010; Wu et al., 2017) non-selective cation currents, as determined by the reversal potential (+ 6.74 mV; Figure 8D-E) and voltage-dependent inactivation (Figure 8D and 8F). Our results demonstrate that expressing pezo-1 in a naïve cell was sufficient to confer mechanosensitivity to Sf9 cells.
DISCUSSION
In 2010, Coste and collaborators reported that the C. elegans genome contained a single Piezo gene, pezo-1 (Coste et al., 2010). However, the functional role of pezo-1 remained elusive even after a decade of its discovery. Here, we showed that PEZO-1 is a mechanosensitive channel with a novel functional role in the worm pharynx by combining fluorescent reporters, genome editing, electropharyngeogram, behavioral, and patch-clamp measurements. We found that pezo-1 is highly expressed in neurons involved in pharyngeal pumping relaxation. In addition to its expression, several lines of evidence suggested that PEZO-1 modulated several discrete but reliable features of the pharyngeal function. Lack- or augmentation-of PEZO-1 function increased pharyngeal pumping frequencies when worms were challenged with 2 mM serotonin, hyperosmotic conditions, or fed with control bacteria. In the absence of functional PEZO-1, worms reduced pharyngeal function (i.e., low frequency and long pump intervals) when fed with spaghetti-like bacteria. Finally, we demonstrated that the pezo-1 gene encodes a mechanosensitive ion channel. Altogether, our results established that PEZO-1 is important for pharyngeal function regulation and food sensation.
C. elegans feeding relies on the ability of its pharynx to contract and relax. The pharynx is a tube of electrically coupled muscle cells that continuously pump throughout the worm’s life (Mango, 2007). Several ion channels have been identified to be crucial for the pharyngeal muscle action potential, including acetylcholine receptors, T- and L-type Ca2+ channels, glycine receptors, and K+ channels (Avery and You, 2012). Although the pharyngeal muscle is capable of pumping (albeit at low frequencies) without nervous system input, higher pumping frequencies are controlled by pharyngeal motor neurons, namely MCL/R and M3L/R (Avery and You, 2012). Nevertheless, the role of the nervous system in the control of rhythmic pharyngeal pumping is not completely understood. It is known, however, that the pharynx responds to a variety of neuromodulators (Avery and Horvitz, 1989). We found that pezo-1 is expressed in proprioceptive/mechanosensory neurons NSML/R and M3L/R (both important for the pharyngeal nervous system) and in the pharyngeal interneuron I3 (Avery and Horvitz, 1989). Unlike NSML/R and M3L/R, the function of I3 has not been established (Avery, 1993; Avery and Thomas, 1997). Our results suggest that PEZO-1 is not essential for pharyngeal muscles, but fine tunes the role of the nervous system controlling the pharynx function. This is reminiscent of the novel role of mammalian PIEZO1 and PIEZO2 mediating neuronal sensing of blood pressure and the baroreceptor reflex (Zeng et al., 2018).
NSML/R and M3L/R, both pezo-1-expressing neurons, have been postulated to sense bacteria in the pharynx lumen via their proprioceptive endings and secrete serotonin in response to this mechanical stimulus (Avery, 1993; Avery and Thomas, 1997). Laser ablation of NSML/R in unc-29 mutants leads to subtle changes in pharyngeal pumping rate; however, this was done while simultaneously ablating other pharyngeal motor neurons (M1, M2L/R, M3L/R, M5, and MI) (Avery, 1993). This approach could exert antagonistic effects on pumping rate yielding a steady pharyngeal activity. Using the Screenchip™ system allowed us to reveal the potential roles of extrapharyngeal neurons expressing pezo-1 (NSML/R and M3L/R). Our results determined that activation of PEZO-1 inhibited serotonin-dependent fast pumping rate in the absence of food. They further demonstrated that PEZO-1 modulated the feeding behavior of worms confronted to various food consistencies (control and spaghetti-like bacteria). This led us to hypothesize that PEZO-1 is involved in food sensation and modulates pharyngeal pumping rate. Hence, similar to the mammalian ortholog PIEZO2, PEZO-1 is expressed in proprioceptive endings and involved in stretch reflexes (Woo et al., 2015; Chesler et al., 2016). Nevertheless, it remains to be determined if mammalian PIEZO channels play a role in food sensation and/or the swallowing reflex.
Humans sense various organoleptic food qualities such as visual aspects (color and shape), odorants through smell, and texture and flavor through tasting. In nematodes, there is a lack of understanding of what is sensed as food. Worms are able to filter particles from fluid in a size-dependent manner (Fang-Yen, Avery and Samuel, 2009; Kiyama, Miyahara and Ohshima, 2012) and feeding is facilitated by attractive odors or suppressed by repellents (e.g., diacetyl, isoamyl alcohol, quinine) (Gruninger, Gualberto and Garcia, 2008; Li et al., 2012). Others have demonstrated that worms prefer to feed from active (i.e., bacteria reproducing rapidly and emitting high levels of CO2) rather than inactive bacteria (Yu et al., 2015). We determined that pezo-1 KO worms “choke” when presented with spaghetti-like bacteria, whereas WT and GOF strains increase pharyngeal pumping when ingesting this elongated food. Therefore, we propose that the pharynx itself might be a sensory organ, as worms modify their pumping parameters when they sense solutions of different osmolarities or food with different textures and/or consistencies. We further hypothesized that worms are able to perceive changes in texture and adjust their pumping frequency by a mechanism requiring PEZO-1. Since pezo-1 is not essential for C. elegans when cultured in standard laboratory conditions (e.g., monoaxenically on E. coli OP50), we wonder if in its natural biotic environment this mechanosensitive ion channel plays a crucial role, as it does in humans and Drosophila. Given that worms grow in microbe-rich and heterogenous environments (feeding from prokaryotes of the genera Acetobacter, Gluconobacter, and Enterobacter) (Schulenburg and Félix, 2017), they might encounter bacteria of different dimensions and textures that will make pezo-1 function more relevant to the worm’s ability to discriminate the food on which it grows best.
Why pezo-1 loss- and gain-of-function mutations cause similar behavior phenotypes? Our data show that both pezo-1 mutants (KO and GOF) increase the pumping frequency of the pharynx in different settings: serotonin exposure, high osmolarity, and ingestion of control bacteria. While it may seem counterintuitive at first, there are several scenarios in which too little or too much mechanosensation can be detrimental for animal’s behaviors. For instance, the mec-4 gene encodes for the DEG/ENaC ion channel subunit of the mechanoelectrical transduction channel complex in touch. Like our pezo-1 mutants that lack control of pharyngeal pumping function, LOF (u253, a deletion) and GOF (e1611, missense mutation A713V) alleles of the mec-4 gene render touch-insensitive worms (Driscoll and Chalfie, 1991; Hong, Mano and Driscoll, 2000; O’Hagan, Chalfie and Goodman, 2005). Similarly, deg-1, a DEG/ENaC ion channel subunit expressed in ASH neurons, LOF (u443, eliminates the 3’ end of deg-1) and GOF (u506, missense mutation A393T) alleles decrease the worm’s ability to respond to nose touch (Savage et al., 1989; García-Añoveros, Ma and Chalfie, 1995; Geffeney et al., 2011). In humans, PIEZO2 LOF (premature stop codon) and GOF (missense mutation I802F) alleles caused joint contractures, skeletal abnormalities and alterations in muscle tone (Coste et al., 2013; Chesler et al., 2016; Yamaguchi et al., 2019). Only when feeding worms, the spaghetti-like bacteria, we were able to uncover a difference between the LOF and the GOF mutants. Hence, we hypothesize that lacking the function of PEZO-1 significantly slows down pharyngeal function when passing down the lengthy bacteria from the pharynx to the gut.
Several requirements must be met for a channel to be considered mechanically gated (Arnadóttir and Chalfie, 2010). Accordingly, we found that pezo-1 is expressed in the proprioceptive neuron NSM, knocking out pezo-1 inhibits worm’s pharyngeal function when challenged with hyperosmolarity or elongated bacteria, engineering a single point mutation in the putative pore domain (R2373K) elicited similar activation and deactivation delays that are reminiscent of the gating behavior reported for the human PIEZO1 R2456K (Bae et al., 2013), and expression of pezo-1 confers mechanosensitivity to, otherwise naïve, Sf9 cells. We propose that PEZO-1 is a mechanosensitive ion channel directly gated by bilayer tension given that the time it takes to reach half of the steady state currents ranges between 3.5 to 15 ms upon application of negative pressure. These are faster than activation times reported for the Drosophila phototransduction cascade, one of the quickest second messenger cascades (Hardie, 2001). These combined efforts highlight the versatile functions of the PIEZO mechanosensitive channel family as well as the strength of the model organism C. elegans to reveal physiological functions.
Our findings revealing PEZO-1 as a mechanosensitive ion channel that modulates pharyngeal function raise several important questions. How does pezo-1 modulate pumping behavior electrical activity? Does pezo-1 equally enhance or inhibit the function of the pharyngeal hypodermal, gland and muscle cells, and neurons expressing this channel? Could pezo-1 phenotypes be exacerbated if the gene function is nulled in a cell-specific manner? Does PEZO-1 require auxiliary subunits and/or cytoskeleton for gating? Regardless of the answers, the plethora of physiological roles that this eukaryotic family of mechanosensitive ion channels play is outstanding. More experimental insight will be needed to grasp the full implications of pezo-1 in the physiology of C. elegans.
Competing interests
The authors declare no competing financial interests.
Author contributions
Conceptualization, VV; Methodology, VV and JRMM; Investigation, JRMM, LOR, and JL; Writing, VV and JRMM; Funding Acquisition, VV; Supervision, VV.
Acknowledgements
The authors thank Dr. Julio F. Cordero-Morales, Dr. Andrés G. Vidal-Gadea, and Dr. Christopher E. Hopkins for critically reading the manuscript, and Dr. Rebeca Caires, MSc Briar Bell, and MBBS Soumi Mazumdar for technical assistance. C. elegans (N2, DA572, and DA1051) and E. coli strains (OP50 and NA22) were obtained from the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). LX960 was provided by Dr. Kevin Collins (University of Miami). This work was supported by the American Heart Association (16SDG26700010 to VV) and the National Institutes of Health (R01GM133845 to VV) and the Neuroscience Institute at UTHSC (Research Associate Matching Salary Support to JL).