All chemicals used were purchased from Sigma (St. Louis, MO, USA) unless otherwise noted. P. tetraurelia, 51-s (sensitive to killer) were maintained as described in Sasner and Van Houten .
In all construct designs, homologues in other organisms were used to find sequences in the Paramecium annotated genome using the dedicated database ParameciumDB (http://paramecium.cgm.cnrs-gif.fr/) . Genes with the highest homology were then used to design constructs for RNAi using genomic DNA (Additional file 1: Table S1). All inserts were ligated into the double T7-promoter vector, L4440 (Addgene, Cambridge, MA, USA). Off-target sequences were searched for using the ParameciumDB database (http://paramecium.cgm.cnrs-gif.fr/cgi/alignment/off- target.cgi). We found that both sequences of each paralog pair would be affected by the same RNAi sequence; BBS2 showed one off-target match of 23 nucleotides from a hypothetical protein; otherwise no other gene sequences in the genome would be targeted by our BBS RNAi plasmids.
Reverse transcription-PCR (RT-PCR)
This method was used as a check on the efficacy of the RNAi feeding, according to our procedures in Yano et al., . We consider the data from RT-PCR to be semi-quantitative and certainly not a suitable way to quantify the degree of mRNA reduction by RNAi. These experiments were repeated a minimum of three times. See Additional file 2: Figure S1 for a representative example.
Calmodulin primers were also used in RT-PCR as a check on the methodology. Concentrations of the cDNA used as template were undiluted, and diluted 10 fold, and 100 fold. Calmodulin primers used were 5′-CTGAAGCTGAACTTCAAG-3′ (forward) and 5′-CAGAATGATGGTTTCTAAATGA-3′ (reverse).
RNAi feeding method
We followed the methods for the BBS RNAi as previously described (http://paramecium.cgm.cnrs-gif.fr/RNAi/index.php) . Cells were fed HT115 bacteria transformed with the control (L4440) or with L4440 containing the RNAi insert of interest. After 2 h of incubation while shaking at 37°C, HT115 bacteria transformed with the L4440 plasmid or plasmid with BBS insert were induced to produce double stranded RNA by adding isopropylthio-β-galactoside (IPTG) (RPI Corp., Mt. Prospect, IL, USA) to a final concentration of 0.125 mg/ml and incubated for an additional 4 h. The induced culture was centrifuged at 3,439 × g for 10 minutes at 4°C (Beckman J2-21 centrifuge, Beckman Coulter, Brea, CA, USA) and the pellet was re-suspended in 100 ml of wheat grass medium. The 51-s Paramecium cells were washed in Dryl’s solution (1 mM Na2HPO4, 1 mM NaH2PO4, 1.5 mM CaCl2, 2 mM Na-citrate, pH 6.8) and approximately 50 to 100 paramecia were added to the induced culture. Additional stigmasterol, ampicillin, and IPTG were also added to the final concentrations of 8 μg/mL, 0.1 mg/mL, and 0.125 mg/mL, respectively. Cultures were maintained at 28°C. When required, additional induced bacteria, stigmasterol, ampicillin, and/or IPTG were added at 24 and 48 h after feeding. All experiments were carried out at 72 h of RNAi feeding.
The RNAi treatment of cells expressing FLAG-VGCC1c was somewhat different because large numbers of cells were required to harvest the cilia. Paramecia were fed bacteria for BBS8 RNAi or control RNAi as above. The expression of double stranded RNA was induced in 500 mL LB medium with the same concentration of IPTG for 4 h. The final pellets of bacteria were re-suspended in 1.5 L of wheat grass medium containing the same concentrations of stigmasterol (8 μg/mL), ampicillin (0.1 mg/mL), and IPTG (0.125 mg/mL). About 10,000 cells expressing FLAG-VGCC1c were added to the bacterial cultures with the BBS8 RNAi or control plasmids. For three consecutive days, induced bacteria and additional IPTG of 0.125 mg/mL were added to keep cells in log phase. Cells were harvested at 96 h of RNAi feeding for the ciliary membrane immunoprecipitation (IP).
Fluorescence imaging and ciliary measurements
Cells were imaged using the DeltaVision microscope system and SoftWoRx® Pro software (Applied Precision/GE Healthcare, Issaquah, WA, USA). Images were taken using 20×, 60× or 100× oil emersion objectives on an inverted Olympus IX70 microscope with a Photometrics Coolsnap HQ camera (Photometrics, Tucson, AZ, USA). Lenses used were the UPlanApo 20×/0.80 oil; PlanApo 60×/1.40 oil; PlanApo 100×/1.40 oil. Optical z-sections were 0.5 μm thick. For cilia length measurements, the entire course of a curved cilium in different z-sections was traced using the deconvolved images and softWoRx3.3.6 software in multiple segment mode. Care was taken to match up the segments of the cilia that crossed optical sections. Mann–Whitney U-tests of both the raw and normalized data were used to determine significant differences, with no differences in the outcomes. These experiments were repeated three times.
Scanning electron microscopy
We used scanning electron microscopy to examine 200 mL of cells grown for 72 h in RNAi bacteria. Cells were washed twice in Dryl’s solution using a table top centrifuge to remove debris (Damon/IEC Clinical centrifuge, Needham Hts, MA, USA). Pelleted cells were then treated with 1% osmium tetroxide in 10 mM sodium cacodylate for one minute. Cells were again collected by brief centrifugation and immersed immediately in fresh 2% gluteraldehyde in 10 mM sodium cacodylate buffer. After 10 minutes, cells were centrifuged and rinsed in the same buffer for one hour at room temperature (RT). Cells were then collected by brief centrifugation, placed on 13-mm glass cover slips which had been coated with 0.1% poly-L-lysine (high molecular weight) and rinsed in PBS (137 mM NaCl, 2.7 mM KCl, 10.4 mM sodium phosphate dibasic, 1.7 mM potassium phosphate monobasic, pH 7.4). Cells were allowed to settle for 15 minutes and were then rinsed, stacked, and dried at critical point. Cover slips were glued to an aluminum chuck using graphite cement and allowed to dry. The chuck was then sputter coated and imaged using a JEOL 6060 scanning electron microscope (JEOL USA, Inc., Peabody, MA, USA). These experiments were repeated twice.
Assays of behavior in response to ionic stimuli
All solutions used to test behavior in ionic stimuli contained a base buffer of 1 mM citric acid, 1 mM Ca(OH)2, and 1 mM Tris base. Salts were added from 100 mM stock solutions prepared to desired concentrations (see below) and pH was adjusted to 7.0 using 100 mM Tris Base. After 72 h of growth in RNAi bacteria, approximately 200 cells were removed from their culture and allowed to acclimate in resting buffer (4 mM KCl in the base buffer above) for 30 minutes. Individual cells were transferred to testing solutions in glass depression slides and timed for length of backward swimming; 10 to 20 cells were tested per solution. The experiments were repeated 3 to 10 times. The following solutions were used with the base buffer above: 30 mM KCl; 8 mM BaCl2; 25 mM TEA with 10 mM NaCl; and 25 mM TEA with 5 mM MgCl2. In some cases, backward swimming durations were normalized to the control backward swimming in order to combine data from many BBS-depleted lines. Mann–Whitney U-tests performed on the raw data and normalized data showed no difference in significance outcomes. These experiments were repeated a minimum of three times; we often used the swimming in TEA solutions with Na+ or Mg2+ as indicators of whether the RNAi for BBS7, 8, or 9 was working.
Deciliation and recovery of motility
Cells were deciliated using trituration in an ethanol solution and observed for recovery of motility. After culturing in 100 mL of RNAi bacteria for 72 h, the cells were collected by centrifugation (Damon/IEC Clinical Centrifuge), washed and re-suspended in Dryl’s solution. Cells were centrifuged again and re-suspended in 4 mM potassium chloride (KCl) buffer as for ionic stimulation. Cells that swam upward in the tube were collected after 5 minutes using a Rainin Pipetman (Mettler Toledo, Columbus, OH, USA) and placed in the same KCl buffer. A sample was removed to confirm that all cells were motile. We rapidly added 100% ethanol to the cells for a final concentration of 5%, sampled cells again to determine motility, and began triturating with a Pasteur pipet to sheer off the cilia. After each course of trituration, the cells were examined to determine how many were no longer motile. These experiments were repeated three times.
Preparing FLAG pPXV plasmid construct for microinjection
We prepared an N-terminal FLAG pPXV plasmid for expression of three FLAG sequences at the N terminus of the BBS proteins, SK1a (GSPATP00031195001) and VGCC1c (GSPATP00017333001) and a C-terminal FLAG pPXV plasmid with three FLAG sequences for PKD2 (GSPATP00005599001). The pPXV plasmid (courtesy of Dr. W. John Haynes, University of Wisconsin, Madison, WI, USA) with the 3× FLAG or 3× FLAG with insert was extracted using the WizardTM Plus Mini-Prep (Promega, Madison, WI, USA) and linearized with Not I restriction enzyme (New England BioLabs, Inc., Ipswich, MA, USA). The linearized plasmid was purified, re-suspended at a concentration of 5 to 10 μg/μl in sterile H20, and 5 to 9 pl was injected into the macronucleus of approximately 20 wild type cells. Individual cells were placed into depressions containing 500 μL of inoculated culture fluid and allowed to recover and divide at RT for 24 to 48 h in a humidification chamber. From each depression, 5 to 7 cells were removed and placed in 10 mL of inoculated culture fluid. Each depression was maintained as a separate cell line at 15°C and the cells were re-fed by transferring 5 to 7 cells to fresh culture fluid every 4 days. Cell lines were tested for the presence of the plasmid using PCR with extracted genomic DNA as a template.
Immunostaining and deconvolution microscopy image analysis
Collection of 100 mL of cultured cells by centrifugation and washing in Dryl’s solution was followed by permeabilization in PHEM solution (60 mM piperazine ethanesulfonic acid (PIPES), 25 mM hydroxyethyl piperazineethanesulfonic acid (HEPES), 10 mM ethylene glycol tetraacetic acid (EGTA), 2 mM MgCl2 and 0.1% Triton X-100, pH 6.9) and fixation for 60 minutes in freshly made 4% paraformaldehyde in PHEM. The fixed cells were washed three times with blocking buffer (2 mM sodium phosphate monobasic, 8 mM sodium phosphate dibasic, 150 mM sodium chloride, 1% Tween20, 1% BSA, 10 mM EGTA and 2 mM MgCl2; pH 7.4) by centrifugation and incubated for 1 h at RT with primary antibodies: monoclonal anti-FLAG M2 (Sigma) and Tetrahymena rabbit anti-centrin-1 (gift from Dr Mark Winey, University of Colorado Boulder, Boulder, CO, USA), or rabbit anti-folate binding protein (FBP) ; FBP gene GSPATP00025147001[GENBANK: AAS57871]. The cells were collected and washed three times by light centrifugation with 1 mL PBS-T (2 mM sodium phosphate monobasic, 8 mM sodium phosphate dibasic, 150 mM sodium chloride, 1% Tween20; pH 7.4) per wash and incubated for 1 h at RT with 100 μL PBS containing 1:10,000 dilution of secondary antibodies: Alexa fluor® 568-labeled goat anti-mouse and Alexa fluor® 488 goat anti-rabbit (Molecular Probes/Invitrogen, Carlsbad, CA, USA). Cells were washed five times with PBS-T solution and suspended in Vectashield® mounting medium (Vector Labs, Burlingame, CA, USA) for imaging using the DeltaVision® restoration microscopy system (Applied Precision/GE Healthcare, Issaquah, WA, USA) (see fluorescence microscopy and cilia lengths). These experiments were repeated at least three times.
Whole cell extract (WCE) preparation for immunoprecipitation
The WCE protocol was adapted from previous publications [14, 33]. Cells expressing FLAG-tagged BBS8 or BBS9 or control cells with the pPXV vector were grown in four 1.5 L wheat grass cultures at 22°C. The cells from the cultures were collected once densities were between 8,000 and 12,000 cells per mL. Cells were washed twice in 200 mL HM Buffer (20 mM Maleic Acid, 20 mM Trizma Base, 1 mM EDTA, pH 7.8), once in 200 mL LAP200 Buffer (50 mM HEPES, 200 mM KCl, 1 mM EGTA, 1 mM MgCl2, pH 7.8) and then in 100 mL LAP200 with protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin (RPI Corp., Mt. Prospect, IL, USA) and 1 μg/mL pepstatin A (RPI Corp.) in addition to 100 μL protease inhibitor cocktail. Cells were then homogenized and the protein concentration was determined using a Pierce protein assay (Thermo Scientific/Pierce, Rockford, IL, USA). Equal concentrations of test and control protein were solubilized by adding Triton X-100 to a final concentration of 1%. Cell lysates were rocked on ice at 4°C for one hour and insoluble proteins were removed by centrifugation at 31,000 × g (Beckman J2-21, Beckman Coulter, Brea, CA, USA) for 20 minutes and then 100,000 × g (Beckman L8-80 M Ultracentrifuge, Beckman Coulter, Brea, CA, USA) for 1 h, both at 4°C.
Immunoprecipitation with anti-FLAG M2 agarose beads
The protocol for IP of WCE was followed as described previously  with some modification: 5 to 6 mL of both control and test WCE were clarified by incubating each lysate with 30 μl of Protein A beads (Amersham/GE Healthcare, Pittsburgh, PA, USA). Anti-FLAG M2 agarose beads (Sigma-Aldrich, St. Louis, MO, USA) were prepared by washing eight times in cold LAP200 buffer containing 1% BSA and 1% TritonX-100 . These prepared beads were added to the clarified sample, incubated on ice while rocking for 2 h, and collected by centrifugation (Damon/IEC Clinical Centrifuge). Beads were washed five times in cold LAP200 buffer with 1% TritonX-100 followed by a final wash in cold LAP200 buffer without TritonX-100. An equal volume (30 to 60 μl) of 2× SDS sample buffer (62.5 mM Tris–HCl, 10% w/v glycerol, 2% SDS, 0.01 mg/mL bromophenol blue, pH 6.8) with 3% β-mercaptoethanol (BME) was added and the sample was boiled for five minutes, and centrifuged at 16,000 × g (Eppendorf centrifuge 5424, Hauppauge, NY, USA) for one minute. The supernatant was then loaded and separated by SDS-PAGE on a 7 to 18% gradient SDS gel. BenchMarkTM prestained protein ladder (Invitrogen/Life Technologies, Carlsbad, CA, USA) was loaded to ascertain the approximate molecular mass of the resolved protein samples. Experiments were repeated twice.
Whole cilia were isolated following Adoutte et al. and a total of 5.3 mg from control or test RNAi-treated cells was used for IP. The whole cilia were re-suspended in membrane buffer (10 mM Tris buffer, 50 mM KCl, 5 mM MgCl2, 1 mM EGTA, pH 7.4) with 1% Triton X-114, and then agitated for 1 h at 4°C. After centrifugation at 16,000 × g (Eppendorf centrifuge 5424) for 10 minutes at 4°C, the supernatant was clarified as previously described using protein A beads (Amersham/GE Healthcare). The clarified lysate was centrifuged at 16,000 × g for 10 min at 4°C and the supernatant was incubated with 20 to 30 μl of prepared anti-FLAG M2 beads (Sigma-Aldrich) for 1 h at 4°C. Beads were prepared by washing four times in membrane buffer with 1% Triton X114. Beads were collected by brief centrifugation and washed in membrane buffer with 1% Triton X-114 and 0.1% BSA three times and then in membrane buffer three times. Samples were prepared as in the WCE before separation by SDS-PAGE.
The proteins separated by SDS-PAGE were transferred to BioTraceTM nitrocellulose blotting membrane (PALL Life Sciences, Ann Arbor, MI, USA). Blots were incubated in blocking buffer comprising 0.5 g skim milk powder, 200 μl of Telost fish gelatin, and 300 μl of normal goat serum (Vector Labs) dissolved in 10 ml of TBS-T (15 mM Tris, 140 mM NaCl, 0.1% Tween, pH 7.5)] at RT for 1 h with rocking. Blots were probed using the following primary antibodies: 1:2500 rabbit anti-FLAG M2 or 1:2000 mouse anti-tubulin. Secondary antibody was either alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated goat-anti-mouse or anti-rabbit at 1:10,000 dilution and developed accordingly (all antibodies from Sigma-Aldrich, St. Louis, MO, USA).
Silverstained gels and mass spectrometry analysis
After electrophoresis, the SDS-PAGE gel was stained using directions of the FASTSilverTM kit (G-Biosciences, St. Louis, MO, USA). From the BBS8 and 9 IP, eight regions of each of the silver stained gels were removed (see Additional file 3: Figure S2). The same regions were removed from the control lanes and sliced into small pieces. The molecular mass ranges covered the masses of the BBS proteins (88 kd to 38 kd). Before the trypsin digest, gel slices were destained with 500 μL of destain solution (30 mM K3Fe(CN)6 and 100 mM sodium sulfate, pentahydrate (EMD Chemicals, Billerica, MA, USA)) at RT for 15 minutes with occasional vortexing. Gel slices were each rinsed twice in 1 mL HPLC water for 5 minutes, then were covered in 100 μL 100 mM NH4CO3. This was removed after incubating at RT for 10 minutes, and samples were incubated at 37°C for 10 minutes in 500 μL 50% MeCN in 50 mM NH4CO3. This was removed, 500 μL was again added and the samples were incubated at 37°C for 30 minutes. Samples were dehydrated using 100 μL 100% MeCN and incubated at RT for 10 minutes. Samples were spun briefly and the solution removed. Gel pellets were allowed to dry completely and were then incubated in 30 to 60 μL of 12.5 ng/μL trypsin (Promega, Madison, WI, USA) in 50 mM NH4CO3 at 37°C. After 10 to 15 minutes, samples were given an additional 30 μL of 50 mM NH4CO3 if needed and then incubated at 37°C overnight. The following morning, the solution was removed and saved. The gel slices were vortexed in 150 μL of 50% MeCN and 2.5% formic acid and spun for 10 minutes at 16,000 × g (Eppendorf centrifuge 5424). The supernatant was removed and added to the trypsin sample removed earlier for each sample. The gel slices were lastly dehydrated in 100 μL of 100% MeCN for 5 minutes at RT, then centrifuged at 16,000 × g for 5 minutes. Each supernatant was again removed and added to the other collected supernatants for each sample. These collected digested peptides were then dried completely in a SpeedVac, re-suspended in 8 μL of 2.5% MeCN and 2.5% formic acid, and 4 μL was transferred to a 100 μL glass deactivated tube with polymer feet (Agilent Technologies, Santa Clara, CA, USA). Each tube was then capped in a glass autosampler vial.
Tryptic peptides in an autosampler vial were loaded using a Micro-autosampler (ThermoElectron, Waltham, MA, USA) onto a microcapillary column packed with 12 cm of reversed-phase MagicC18 material (5 μm, 200 Å; Michrom Bioresources, Inc., Auburn, CA, USA). After a 15-minute isocratic loading at 2.5% MeCN and 0.5% formic acid, peptides were eluted with a 5 to 35% MeCN (0.1% FA) gradient over 60 minutes. Ten mass spectrometry (MS/MS) scans followed each survey scan for the entire run (75 minutes). Mass spectra were acquired in a LTQ-XL linear ion trap mass spectrometer (Thermo Electron).
The raw MS/MS data were searched against the Paramecium tetraurelia forward (target) and reverse (decoy) proteome databases (http://aiaia.cgm.cnrs-gif.fr/download/fasta/) , using the Sequest algorithm with a precursor mass tolerance of 2 Da. A static increase in 71.0 Da of cysteine residues for acrylamide adduction was required and differential modification of 16.0 Da on methionine residues was permitted. The top matches were filtered using a unique delta correlation score (dCn2) of 0.16 and Xcorr (cross-correlation) values of 1.8, 2.4 and 2.8 for single-, double- and triple-charged ions, respectively. At the protein level, only proteins for which two unique peptides were assigned to a given Genoscope annotation identification were retained. The number of protein entries having an identical peptide match is listed for each peptide in the column ‘redu’ for redundancy. The estimated false-discovery rates were calculated based on the number of reverse database assignments after the above filtering was applied. Specifically the false discovery rate equals the number of peptides assigned to a reverse database entry times two (to account for unknown false positives) divided by the number of peptides assigned to the forward database. The estimated false discovery rate of proteins identified by more than two unique peptides for the BBS8 WCE IP sample was 0.36% and for the BBS9 WCE IP sample was < 0.00%. The tryptic peptides were analyzed twice for each BBS IP.