Experimental preparations
A colony of the Central American wandering spiders, Cupiennius salei (Keys.) was maintained at 22 ± 2°C under a 13/11 h light/dark cycle. Adult spiders were used for experiments and the experimental protocols were approved by the Dalhousie University Committee on Laboratory Animals. For patellar preparations, legs were autotomized and placed on a Sylgard-coated dish. We dissected the anterior side of the patella and removed the muscle tissue to provide access to sensory neurons. For dissection of the CNS, spiders were anesthetised with CO2 and for Western blot analysis the CNS was quickly dissected and placed in cold spider saline [33]. For in situ hybridization and immunocytochemistry, the tarsal leg segments were removed, and the anesthetized spider was perfused with 4% paraformaldehyde that was injected directly into the heart. The sternum plate was then removed, the CNS extracted and kept in 4% paraformaldehyde overnight.
Identification and analysis of putative mechanotransduction channels from C. salei transcriptome
Details of the C. salei transcriptome preparation, sequencing and assembly have been described previously [36, 37]. Twelve putative MET channel sequences were identified using the transcriptome walking algorithm [36]. Data analysis and programming were done using the C++ language with Microsoft Visual Studio. The sequences were analyzed by the InterProScan 5 server (http://www.ebi.ac.uk/Tools/pfa/iprscan5/), which scans the sequence against the protein signatures of several databases. Pairwise identity matrices were created using a Sequence demarcation tool with the MAFFT aligned sequences [64] and clustering the sequences with Neighbor joining tree [65].
Prediction of C. salei Piezo protein structure
We used the I-Tasser server [40] to generate 3D-structure predictions of the C. salei Piezo protein. The sequence was uploaded in FASTA format in two parts (residues 1-1300 and 1293-2537) and both were run without restraints allowing the software to select the templates. We used the PyMOL Molecular Graphics System, Version 2.1.0. (Schrödinger, LLC.) software to generate images of the homology models.
RNA Probe construction and in situ hybridization
Custom designed primers (Supplementary Table 1) were purchased from IDT (Coralville, IA, USA) and used to amplify the template DNA for the RNA probes using RT-PCR. Digoxigenin-labeled RNA probes were transcribed in vitro from the purified PCR product with T7 RNA polymerase (Roche Diagnostics, Laval, QC, Canada) [66]. Probe quality was confirmed by agarose gel electrophoresis. The probes were then stored at -80°C.
Whole-mount patellar preparations: Preparations were fixed for 1 hour in 4% paraformaldehyde, dehydrated in a series of methanol solutions in 0.1% Triton X-100 in PBS (PBST) and stored at ‑20°C.. The tissue was rehydrated through methanol series in PBST followed by a protocol previously described in detail [66]. In the end, 1:8,000 dilution of anti-Digoxigenin-Alkaline-Phosphatase, Fab-fragment (Roche) antibody reaction was visualized using NTB/BCIP [66]. The complete experimental protocols were repeated at least twice for each of the 12 antisense and sense (control) probes (Supplementary Table 1) using hybridization temperatures of 60°C and 63°C. In the end, the hypodermis bearing the sensory organs, was carefully detached from the cuticle, and mounted on a microscope slide in 70% glycerol. Previously confirmed probes were used as positive controls.
CNS preparations: The antisense and sense probes for CsPiezo (Supplementary Table 1) were tested on sections of the C. salei CNS. Paraformaldehyde fixed CNS was dehydrated in methanol series in PBST, stored at -20°C and then rehydrated in a series of methanol solutions in PBST. The CNS was then embedded into albumin-gelatin mixture, hardened with formaldehyde and glutaraldehyde as described earlier [67]. The blocks were sectioned by vibratome (Leica VT1000S, Leica Biosystems, Wetzlar, Germany), 30-40 µm thick horizontal sections were collected and processed as floating sections using the same protocol as for the whole-mount patellar preparations [66, 67]. Before the final dye reaction, the sections were placed on microscope slides and allowed to dry at 37°C. Warm glycerol/gelatin media was used to mount the coverslips on slides. Samples were photographed with an Axiovert microscope and Axioscope camera (Zeiss, Oberkochen, Germany) and the images were processed using Photoshop CC software (Adobe Systems, San Jose, CA, USA).
Generation of a polyclonal antibody against the CsPiezo and Western blot analysis
Peptide sequence NIKLVDEQPRKVSGEVDDVE representing residues 1483-1502 of C. salei Piezo protein was synthesized and used to generate a polyclonal antibody in rabbits by Biomatik (Cambridge, ON, Canada). Affinity purified antibody was used in all experiments. The CsPiezo antibody was tested in Western blot on C. salei CNS. Freshly dissected CNS was homogenized in RIPA lysis buffer using BioMasher single use homogenizers with the Powermasher Pestle motor (Diagnocine LLC, Hackensack, NJ, USA). The homogenate was incubated on ice for 20 minutes followed by centrifugation at 16,000 g at 4°C for 20 min. Standard SDS-PAGE and Western blotting methods were used [44]. Resulting membranes were probed with the CsPiezo antibody (1:1,000) in blocking solutions (Tris buffered saline with 0.1% Tween-20 and 5% skimmed milk powder) followed by incubation in 1:20,000 dilution of a peroxidase conjugated goat-anti-rabbit secondary antibody. Immunoreactive protein bands were visualized using Clarity Western ECL kit (BioRad, Mississauga, ON, Canada). The log molecular weights of the protein standards were blotted to extrapolate the weight of the band detected by the antibody. For control, peptide competition was performed by preabsorbing the CsPiezo antibody for 1 hour at room temperature in 5-fold excess of the peptide (by weight) before the Western blot protocol.
Immunocytochemistry
Patellar preparations: Similar whole mount preparations were prepared as for in situ hybridization. The tissue was permeabilized in 0.1% saponin in PBS and processed using standard protocol [44]. To cell nuclei were stained with 1 µg/mL of 4′,6-diamidino-2-phenylindolein (DAPI) in PBS. In the end, the hypodermis containing the leg nerves, and several mechanosensilla, was detached from the cuticle and mounted on a microscope slide in Mowiol mounting medium [68]. The primary antibodies were the polyclonal rabbit CsPiezo (1:2,000) and the monoclonal antibody against Drosophila synapsin (SYNORF 1, 1:100 dilution) [69] that has been tested previously in C. salei [39, 44, 68]. Secondary antibodies were Cy-3 conjugated goat-anti-rabbit IgG for the CsPiezo, and AlexaFluor 488 conjugated goat-anti-mouse IgG for anti-synapsin, both at 1:500 concentrations. For control, the CsPiezo antibody was preabsorbed in 5-fold excess of the blocking peptide for 1 hour before the samples were incubated in this antibody solution.
CNS preparation: The CNS dissection and fixation were performed as described for in situ hybridization followed by embedding into albumin-gelatin mixture, except that glutaraldehyde was omitted to avoid autofluorescence, and formaldehyde was used for hardening the block. Horizontal 60 µm sections were cut using a Leica vibratome and processed as floating sections. Similar immunolabeling protocol as described for patellar preparations was used with the CsPiezo as primary antibody and CY-3 as the secondary antibody. DyLight™ 488 Phalloidin (Cell Signaling Technology) was used to stain actin filaments and DAPI to stain the cell nuclei. The sections were mounted in Mowiol mixture on a microscope slide.
Digital image acquisition was done using a LSM 710 Meta laser-scanning confocal microscope (Zeiss). Digital images of 1 µm optical sections were captured processed using Zen software (Zeiss) and the final images were prepared with Adobe Photoshop CC.
Electrophysiology
We used two types of VS-3 preparations in electrophysiological experiments. In cuticular preparations, the hypodermis with the VS-3 neurons, is attached to the cuticle allowing mechanical stimulation of the slits as described in detail [31, 33]. For hypodermis preparations, the patellar hypodermis with the VS-3 neurons was detached from the cuticle, mounted on a coverslip, and placed in a recording chamber [44, 47]. Both preparations were superfused with spider saline. Neurons were observed using Axioscop 2FS Plus (Zeiss) microscopes. Sharp borosilicate glass microelectrodes were pulled with a P-2000 laser puller (Sutter Instrument, Novato, CA, USA) and filled with 3 M KCl. Recordings were made in discontinuous single electrode current- or voltage- clamp mode using SEC-10L amplifiers (npi electronic, Tamm, Germany) [44, 47, 68]. All experiments were controlled by an IBM-compatible computer using custom written software.
We used a custom-made borosilicate glass probe for mechanical stimulation [31, 33]. The probe was mounted on a P-841.10 piezoelectric stimulator driven by an E-505.00 LVPZT amplifier (Physik Instrumente, Auburn, MA). The stimulator was mounted on a micromanipulator (SD instruments, San Diego, CA) to position the probe tip underneath the VS-3 slits.
For voltage-clamp experiments, the voltage-activated Na+ currents were blocked by 1 µM TTX. We tested two putative blockers of mechanotransduction channels; 10 µM Ruthenium Red and a synthetic D-amino acid form analog of the tarantula toxin GsMTx4 (5 µM), generously provided by Thomas Suchyna (University of Buffalo, NY). These blockers were diluted in spider saline with TTX and manually added to the preparation chamber via a syringe and plastic tubing completely replacing the bath solution. Fresh solution was added every 10-15 min during an hour-long experiment.
Statistical analysis
Data from the electrophysiological experiments with Ruthenium Red and GsMTx4 was tested for normality using the d’Agostino-Pearson test. To test if the changes were statistically significant, t-test for paired samples was used.