Animals

All experiments were performed in accordance with guidelines set by the Institutional Animal Care and Use Committee at the University of Iowa and University of Texas-Austin. Mice were housed under a standard 12-h light/dark cycle with access to food and water ad libitum. The Cabp1 KO mouse strain (RRID: MGI: 5780462) was described previously (Kim et al., 2014) and maintained on a C57BL/6J background. WT mice were age- and sex-matched C57BL/6J mice. Mice expressing Cre and a PIEZO2 fusion with enhanced green fluorescent protein (PIEZO2-GFP) under the endogenous Piezo2 promoter (Piezo2Cre) (Woo et al., 2015) were obtained from Jackson Laboratories (RRID:IMSR_JAX:027719). Floxed CaBP1 mice (Cabp1fl/fl) mice were obtained from MRC Harwell Institute (EM:05847) and bred with Piezo2Cre mice to generate the conditional Cabp1 KO mouse strain (Piezo2Cre/Cabp1fl/fl).

Immunofluorescence and confocal microscopy

Mice were anesthetized in a narcosis chamber with isoflurane and euthanatized by cervical dislocation and decapitation. Lumbar DRGs (L4–L6) were harvested from male or female mice (6–10 weeks old) and fixed using 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C. Fixed DRGs were then infused with 15% sucrose in PBS 24 h at 4°C. The fixed tissue was molded in Optimal Cutting Temperature compound (Sakura Finetek) and 15-mm-thick sections were obtained using a cryostat (Leica). Sections were mounted on charged slides and dried on a slide warmer for 5 min. Sections were then subject to rinsing, blocking, and antibody incubating at room temperature (RT). After rinsing in PBS and incubating in blocking buffer (10% normal goat serum in 0.2% Triton X-100, in PBS) for 30 min, the sections were incubated for 1 h in the following primary antibodies diluted 1:1,000 in blocking buffer: chicken polyclonal anti-NF200 (RRID: AB_2313552, Aves); rabbit polyclonal anti-CaBP1/caldendrin (UW72 (Haeseleer et al., 2000); these antibodies do not produce immunoreactivity in tissues of Cabp1 KO mice (Kim et al., 2014)); mouse monoclonal anti-parvalbumin (Sigma-Aldrich Cat# P3088, RRID:AB_477329); mouse monoclonal anti-calcitonin gene-related peptide (CGRP) (Sigma-Aldrich Cat# C9487, RRID:AB_1078377); mouse monoclonal anti-Green Fluorescent Protein GFP (Novus Cat# NB110-40670, RRID:AB_714984). Sections were washed three times with PBS for 10 min followed by incubation with secondary antibodies diluted 1:500 in blocking buffer: goat anti-Chicken Alexa Fluor 546 (RRID: AB_2534097, Thermofisher), goat anti-Chicken Alexa Fluor 647 (RRID:AB_2535866, Thermofisher), goat anti-Rabbit Alexa Fluor 488 (RRID:AB_2534096, Thermofisher), goat anti-Rabbit Alexa Fluor 555 (RRID:AB_2535849, Thermofisher), goat anti-Mouse Alexa Fluor 488 (RRID:AB 2576208, Thermofisher), goat anti-Mouse IgG2a, Alexa Fluor 488 (RRID:AB_2535771, Thermofisher) and goat anti-Mouse IgG1, CF647 (RRID:AB 10853957, Biotium). Isolectin B4 (IB4), FITC conjugate (Sigma-Aldrich Cat# L2895) was used to label small DRGNs. Sections were then washed three times with PBS for 10 min each. Coverslips (#1.5) were then mounted using Fluoromount-G (Southernbiotech) and allowed to cure for 24 h in the dark before imaging. DRGs from at least 3 mice were processed in 3 independent experiments. At least 3 sections per animal were analyzed using an Olympus Fluoview 3000 confocal microscope equipped with an UPlanXApo 20× objective. Images were captured using Olympus FLUOVIEW software (RRID: SCR_014215). Acquisition settings were standardized and optimized using the Hi-Low saturation mask to prevent signal saturation prior to collecting z-stacks. All confocal images are presented as the maximum Z-projections.

For quantifying the number of NF200-positive DRGNs expressing either CaBP1, PIEZO2-GFP, or CaBP1 and PIEZO2-GFP, confocal Z-stack images of DRGs from CaBP1 KO and PIEZO2-GFP mice immunolabeled for CaBP1, PIEZO2-GFP and NF200 were processed using Amira software (Thermo Fisher). For each Z-stack, the NF200 channel was used to create a binary mask. This binary mask was then used to subtract any signal in the CaBP1 and PIEZO2-GFP channels that was not within NF200 immunofluorescence. The number of DRGN somas of the subtracted images with immunofluorescence for either CaBP1, PIEZO2-GFP, or CaBP1 and PIEZO2-GFP were counted using ImageJ (NIH).

Behavioral assays

All behavioral tests and analyses were conducted by an experimenter blind to genotype. Von Frey, Hargreaves, and cold plantar assays were performed on the same cohorts of male or female mice (8–12-week-old). Each assay was replicated at least three times with the same cohort of mice. Prior to the behavioral assays, mice were handled and adapted to the environment for at least 3 consecutive days. For each assay, animals were acclimatized to the testing room for 30 min prior to the experiment on the day of testing. This was accomplished by placing the mice on platforms with Plexiglass covers for at least 30 min.

The von Frey assay was conducted utilizing Ugo Basile Dynamic Plantar Aesthesiometer (RRID:SCR_021751) as previously described (Mickle et al., 2015). The assay was conducted using 8 von Frey filaments (0.04, 0.07, 0.16, 0.4, 0.6, 1.0,1.4, 2 g; Stoelting Co, Wood Dale, IL) which were applied to the plantar surface of each hind paw 5 times starting from the lowest filament strength (0.04 g) and moving up to the highest (2 g). The withdrawal responses were recorded based on the number of times the mouse withdrew the paw out of 5 trials for each filament applied, which was represented as % withdrawal response.

The Hargreaves and cold plantar assays were conducted on the plantar test apparatus (IITC Life Sciences, Woodland Hills, CA) as previously described (Mickle et al., 2015). In the Hargreaves assay, the heat stimulus was applied using a 100-W projector lamp (50°C), with an aperture diameter of 6 mm, applied from underneath the glass floor (maintained at 30°C) onto the middle of the tori of each hind paw. The latency of paw withdrawal was recorded with a cutoff value of 25 s. At least 7 min was allowed between testing the paws on a single mouse, and an interval of at least 15 min was allowed between trials on any single paw for both Hargreaves and cold plantar assay. Each mouse was tested at least 2 times per paw to obtain the average for paw withdrawal latency.

The cold plantar assay utilizes a cold stimulus delivered by applying a cut-off syringe filled with crushed dry ice (for temperature ranges of 5–12°C) to the glass underneath the paw as previously described in (Brenner et al., 2012). The paw withdrawal latency was measured using the same setup as for the Hargreaves assay. Each mouse was tested at least 4 times per paw and a maximum time of 20 s was set up as cutoff to avoid potential tissue damage.

The tape assay was modified from a protocol described previously (Ranade et al., 2014; Murthy et al., 2018; Michel et al., 2020). Cohorts of female mice (12 weeks) were acclimatized to the assay room for 30 min 2 consecutive days before, as well as on, the day of the experiment. For the assay, the mice were separated into individual arenas and allowed to explore for 5 min. To test mechanical sensitivity on the hairy skin, a circular adhesive tape (white, 10 mm, Tough-Spots) was placed on the upper back of mice. The mice were then returned to their respective arenas and the following tape-directed responses were recorded as a “bout”: wet-dog shake, trying to reach tape with snout, bursts of biting tape, bursts of pulling tape with front paws, bursts of scratching tape with hind leg. The behaviors were recorded with an area scan camera (Basler acA1300 – 60gm) and EthoVision XT16 software. Recording was stopped when a mouse successfully managed to remove the tape or at the end of the 5-min trial. The following parameters were analyzed: the interval between placing the animal in the cage and the first bout (response latency), total number of bouts throughout the trial, bouts per minute and tape riddance (% of cohort). Data analysis and scoring was done manually by researchers blinded to mouse genotype.

The hanging-wire assay and the inverted screen test was modified from a previously described protocol (Florez-Paz et al., 2016; Ma et al., 2023). Cohorts of WT and Cabp1 KO; male and female mice (12–13 weeks) were acclimatized to the assay room for 15 min before the experiments. For the hanging-wire assay, a 90 cm long, 2-mm-thick metal wire was suspended 40 cm above soft bedding and secured between two vertical poles. The wire was adjusted to minimize slack and prevent vibrations during an assay trial. The suspension time for each mouse was measured in 3 trials with 1-minute recovery periods and the average across the 3 trials was reported. Trials were concluded after 30 s, regardless of whether a mouse had yet to fall from the wire. For the inverted screen test, the mice were placed individually on a 22 cm × 30 cm metal grid screen which was then rotated 180 degrees to position the mice 40 cm above soft bedding. Suspension time was recorded in 6 trials, with 1-minute recovery periods. If a mouse climbed to the top, the screen was rotated again to ensure the animal remained inverted. The average time across all trials was reported, with a 90 s trial cutoff.

Western blot analysis of mouse DRGs

Five lumbar DRGs were harvested from each mouse (4–10-week-old males or females), flash frozen in liquid nitrogen, and lysed in N-PER Neuronal Protein Extraction Reagent (Thermo Fisher Cat# 87792) with Protease Inhibitor Cocktail and 1 mM PMSF. Nupage LDS Sample Buffer (4×) (Thermo Fisher Cat# NP0007) was added to lysates which were then incubated at 65°C for 10 min. The samples were loaded into a 10 well 4–20% Tris-Glycine gel (Invitrogen Cat# XP04200BOX), run in Tris-Glycine SDS Running buffer (Novex Life technologies Cat# LC2675), and transferred overnight using Tris-Glycine Transfer Buffer (Novex Life technologies Cat# LC3675). Blots were incubated in blocking buffer consisting of 5% fat free milk in tris-buffered saline with 0.05% Tween (Thermo Fisher Cat# J77500.K8; TBST) for 30 min at RT prior to incubation for 2 h or overnight in primary antibodies diluted in blocking buffer at 4°C: rabbit anti-UW72 1:1,000 or mouse anti- β-Actin 1:3,000 (Sigma-Aldrich Cat# A5316, RRID:AB_476743). After primary antibody labeling, the blots were wash 4 times with TBST and incubated with secondary HRP antibody ECL anti- rabbit (1:4,000, Cytiva Cat# GENA934, RRID:AB_2722659) and ECL anti-mouse IgG (1:3,000, Cytiva Cat# NA9310-1 ml, RRID:AB_772193) for 1 h at RT. The blots were then washed 4 times with TBST and developed with Chemiluminescent reagent (Thermo Fisher Cat# 34580) prior to exposure in an iBright 750 imager (Thermo scientific). Densitometric analyses were done using Image Studio Lite software. Following subtraction of background signals, the signal corresponding to GFP-PIEZO2 and caldendrin was normalized to that for β-Actin.

Transfection of N2A cells

Mouse neuro-2a (N2A; CCL-131) cell line was obtained from the American Type Culture Collection (ATCC). PIEZO1 knock-out mouse N2A (Piezo1−/− N2A) cells were a gift from Dr. Gary R. Lewin (Moroni et al., 2018). N2A and Piezo1−/− N2A cells were cultured in Dulbecco's Modified Eagle's medium (DMEM), 5% penicillin–streptomycin, and 10% fetal bovine serum (FBS) and maintained at 37°C, with 95% relative humidity and 5% CO2. N2A and Piezo1−/− N2A cells were transfected using Lipofectamine 2000 (ThermoFisher Scientific), according to the manufacturer's instructions. Piezo1−/− N2A cells were grown in 6-well plates to 80% confluency and co-transfected with cDNAs encoding: mouse PIEZO2 variant 14-pcDNA3.1(+) (0.25 µg/ml), GFP-pMO (a pcDNA3.1-based vector with the 5' and 3' untranslated regions of the β-globin gene, 50 ng/ml), and GFP-tagged mouse caldendrin variants (GFP-Caldendrin-L or GFP-Caldendrin-S, Genbank # KJ364651.1, 150 ng/ml) or CaBP1 (GFP-CaBP1-S, Genbank # NM_013879.2, 150 ng/ml). N2A cells were transfected in the same conditions and cDNAs omitting the PIEZO2 construct. The GFP-caldendrin and CaBP1 constructs were generated by cloning with C-terminal EGFP tag into HindIII, NotI sites of pRNA2 vector. After 48 h, the cells were plated on Poly-L-Lysine (Sigma-Aldrich)-treated glass coverslips in 24-well plates for electrophysiological recordings.

Co-immunoprecipitation (Co-IP) assay

Human embryonic kidney cells transformed with SV40T-antigen (HEK293T, ATCC CRL-3216) were maintained in DMEM supplemented with 10% FBS and 5% penicillin–streptomycin in a humidified incubator with 5% CO2 at 37°C. The cells were plated in 35 mm dishes and transfected using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions. 35 mm plates of HEK293T cells were transfected with cDNAs encoding: HA-PIEZO2 variant 14-pcDNA3.1(+) (1.5 µg) (Szczot et al., 2017), and GFP (0.5 µg) or GFP-Caldendrin (0.5 µg) or GFP-CaBP1-S (0.5 µg) described for N2A transfections. Negative control groups were transfected with GFP-caldendrin or GFP-CaBP1-S alone.

Twenty-four hours later, transfected cells were lysed in lysis buffer (50 mM Tris-HCl pH7.4; 150 mM NaCl; 0.5% Triton X100; 0.5% n-Dodecyl-β-Maltoside detergent, Thermo Fisher Cat# 89902), Protease Inhibitor Cocktail (Roche complete, EDTA-free, Cat# 05056489001) and 1 mM phenylmethylsulfonyl fluoride (RPI, SKU# P20270). Plastic pestles were used to homogenize the pellet in a 1.5 ml microfuge tube and lysates were incubated on ice for 10 min. Lysates were cleared by spinning down samples at 14,000 × g at 4°C for 10 min. The lysate was collected and 80% of the lysate was incubated in 30 µl of bed volume of Pierce Anti-HA Magnetic Beads (Thermo Fisher Cat# 88836) per sample and incubated at 4°C for 1 h with rotation. The beads were pelleted using a magnet washed three times with 1 ml of PBS prior to resuspending in 50 µl of 1× Nupage LDS Sample Buffer and incubating at 65°C for 10 min prior. The samples were processed for western blot using anti-rabbit UW72 1:1,000, anti-rabbit GFP 1:1,000 (Thermo Fisher Scientific Cat# A-11122, RRID:AB_221569), and goat anti-mouse β-Actin 1:3,000 (Sigma-Aldrich Cat# A5316, RRID:AB_476743). Western blot detection and densitometry was performed as described for DRG lysates. For quantitative analysis, background-subtracted signals corresponding to GFP-caldendrin or GFP-CaBP1-S in the total input sample was divided by that for β-Actin. This value was used to normalize the signals corresponding to the GFP-tagged proteins Co-IP'ed with the anti-HA antibodies.

Preparation of mouse DRGNs for electrophysiology

At the University of Texas-Austin, WT and Cabp1 KO mice were anesthetized in a narcosis chamber with isoflurane and euthanatized by cervical dislocation and decapitation. 15–20 DRGs were harvested from 3 different WT and Cabp1 KO female mice (4–6-weeks old) and stored in 2 ml of Neurobasal media supplemented with 2.5% L-glutamine (ThermoFisher, Cat #25030081), 2% N-21 (Millipore Sigma, Cat #SCM081), and penicillin–streptomycin (ThermoFisher, Gibco, Cat #15140122) and shipped over night with ice packs to the University of Tennessee Health Sciences Center. Upon receipt, the DRGs were incubated with 1 mg/ml collagenase B (Sigma) in HBSS at 37°C and 5% CO2 and then, after 1 h, were dissociated in a medium without serum. The cell suspension solution was centrifuged for 8 min at 800 × g. The pellet was resuspended in DMEM (Gibco) complete media containing 1% penicillin–streptomycin (Gibco), 1% MEM vitamin solution (Gibco), 1% L-glutamine (Gibco), and 10% horse serum (Gibco). Cells were plated on Poly-L-Lysine (Sigma-Aldrich)-treated glass coverslips in 24-well plates. All mouse DRGNs were kept at 37°C, with 95% relative humidity and 5% CO2. Cells were used in experiments after 24 h.

Electrophysiological analysis of MA currents

Patch-clamp recordings were performed on cells plated on glass coverslips. For whole-cell recordings of mechano-activated currents in the voltage-clamp mode, 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), while the pipette solution contained 140 mM KCl, 6 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). Pipettes were fabricated from borosilicate glass capillaries (Sutter Instruments) and fire-polished to a resistance between 3 and 5 MΩ before use. Mechanical stimulation was performed during voltage-clamp recordings with cells held at −60 mV. Recordings were sampled at 100 kHz and low pass filtered at 10 kHz using a MultiClamp 700 B amplifier and Clampex (v10.4.2.0; Molecular Devices, LLC). Leak currents before mechanical stimulation were subtracted offline from the current traces and data were digitally filtered at 2 kHz with ClampFit (v10.4.2.0; Molecular Devices, LLC). Recordings with leak currents >200 pA, with access resistance >10 MΩ, and cells with giga-seals that did not withstand at least six consecutive steps of mechanical stimulation were excluded from analyses.

For indentation assays, DRGNs, N2A, and transfected Piezo1−/− N2A 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 the cells. 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. The threshold of mechano-activated currents for each experiment was defined as the indentation step that evoked the first current deflection from the baseline. Only cells that did not detach throughout stimulation protocols were included in the analyses. The piezo servo controller was automated, using a MultiClamp 700B amplifier, through Clampex (Molecular Devices, LLC).

Data were plotted using OriginPro (2018 v:b9.51.195; OriginLab Corp.). The time constant of inactivation τ was obtained by fitting a single exponential function (X) between the peak value of the current at the end of the stimulus:f(x)=∑i=1nAi*e−tTi+C where A = amplitude, τ = time constant, and the constant y-offset C for each component i.

All boxplots show mean (square), median (bisecting line), bounds of box (75th to 25th percentiles), and outlier range with 1.5 coefficient (whiskers).

Experimental design and statistical analyses

No statistical method was used to predetermine the sample size. The experiments were not randomized. For electrophysiology, the investigator was blind to genotype and treatment. All attempts at replication were successful. Experiments were performed at least three times on different days with different/independent preparations. Statistical analyses for electrophysiology were performed using GraphPad InStat software (version 3.10; GraphPad Software Inc.) and OriginPro. We used the Kolmogorov and Smirnov method to determine normality of data, as well as Bartlett's test to determine differences between standard deviations. Specific tests are described in figure legends.

For behavioral studies, GraphPad Prism 9 software (RRID: SCR_002798, GraphPad Software) was used to generate graphs and statistical analysis. All data sets were first tested for normality by Shapiro–Wilk normality test. When data sets were normally distributed, the data was analyzed by unpaired t test or ANOVA with Tukey's multiple comparison test with a single pool variance. If the data was not normally distributed, the data was analyzed by Mann–Whitney test. For two-way ANOVA tests with main effects only, Sidak's multiple comparisons test was performed with individual variances calculated for each comparison. Data was presented as mean and SEM and p ≤ 0.05 was considered statistically significant.

Data availability

Data supporting the findings of this manuscript are available from the corresponding author upon request. The source data for western blot images can be accessed in the Figshare data repository: https://figshare.com/s/a9a0635790418de1cbe9.