Stocks, cultures and chemicals
Cells (stock 51s P. tetraurelia, sensitive to killer) were grown in wheatgrass medium inoculated with Klebsiella pneumoniae or Aerobacteraerogenes (adapted from ). Allchemicals were obtained from Sigma-Aldrich (St Louis, MO, USA) unless otherwisenoted.
Sequence analysis and construct design
BLAST searches in the Paramecium annotated genome were completed using thehuman sequence for TMEM67 (Q5HYA8) for MKS3 and the humanIFT88 (NP_783195) and mouse Tg737 (NP_033402) sequences forIFT88 orthologs. Searches identified GSPATG00015939001 as a potentialortholog for MKS3, which was used to create the RNAi construct. Fivepotential orthologs (GSPATG00038505001, GSPATG00021390001, GSPATG00011771001,GSPATG00022644001 and GSPATG00039556001) were identified for IFT88. Theconstruct to target IFT88 mRNA was designed from GSPATG00038505001. Homologyof these genes to those in other organisms is shown in Additional file 1: Tables S1 and S2.
All constructs were created from genomic DNA, which was collected by organicextraction. Briefly, 100 μl of cells were mixed 1:1 with denaturing buffer(Promega, Madison, WI, USA), mixed 1:1 with phenol:chloroform:isoamyl alcohol(25:24:1) and centrifuged for 5 minutes at 12,000 × g(Centrifuge 5424; Eppendorf, Hauppauge, NY, USA). The aqueous phase was removed,mixed 1:1 with chloroform:isoamyl alcohol (24:1) and spun again. The DNA wasprecipitated 2:1 with cold isopropanol for 20 minutes at -20°C and spun for 10minutes at 4°C (Centrifuge 5424). Pellets were rinsed twice with 75% ethanol,dried and resuspended in water.
FLAG-tag of MKS3
To localize MKS3p, we added the coding sequence for a threefold repeated FLAGsequence (DYKDDDDK) to the 5′ end of the genomic DNA sequence forGSPATG00015939001 in the pPXV plasmid using the restriction enzymes ApaI and SacI(USB/Affymetrix, Cleveland, OH, USA). These cut sites were created using largeprimers to add them to either end of the sequence: forward(5′-gcggggcccatgctaatttatatcg-3′) and reverse(5′-cgcgagctctcatattagaaaccttttgtc-3′). PlatinumPfx Polymerase(Invitrogen/Life Technologies, Grand Island, NY, USA) was used per the vendor’sinstructions to amplify the sequence. A total of 75 ng of genomic DNA was usedin each PCR: 94°C for 5 minutes; five cycles of 94°C for 1 minute,40°C for 1 minute and 68°C for 3 minutes; five cycles of 94°C for 1minute, 48°C for 1 minute and 68°C for 3 minutes; ten cycles of 94°Cfor 1 minute, 58°C for 1 minute and 68°C for 3 minutes; seventeen cycles of94°C for 1 minute, 65°C for 1 minute and 68°C for 3 minutes; and onecycle of 68°C for 15 minutes (Techne TC-4000 Thermal Cycler; KrackelerScientific, Albany, NY, USA). The products were cleaned using the PrepEasy GelExtraction Kit (Affymetrix). The resulting DNA was treated with restriction enzymes,cleaned again using the PrepEasy Gel Extraction Kit and ligated into thepPXV-5′-3xFLAG plasmid using the Ligate-IT Kit (Affymetrix). The mixture wasthen transformed into OneShot competent cells (Invitrogen/Life Technologies), and theresulting colonies were screened for positives. Positive clones were sequenced at theVermont Cancer Center DNA Analysis Facility (University of Vermont, Burlington, VT,USA).
Approximately 200 μg of pPXV-3xFLAG-MKS3 was linearized with NotI(Affymetrix) overnight at 37°C and then cleansed using an organic extractionmethod modified from that described earlier. This procedure required two washes inphenol:chloroform:isoamyl alcohol (25:24:1) followed by two washes ofchloroform:isoamyl alcohol (24:1). The final pellet was resuspended in50 μl of MilliQ water (EMD Millipore, Billerica, MA, USA), and theconcentration was checked using a spectrophotometer (Agilent Technologies, SantaClara, CA, USA). The sample was spun at 16,000 × g(Eppendorf Centrifuge 5424) for 10 minutes to pellet debris. The top 45 μlwas carefully removed and placed in a fresh RNase/DNase-free 1.5-ml Eppendorf tubeand again dried in a speed vac. The final pellet was resuspended in MilliQ water toobtain a concentration between 3 and 9 μg/μl and stored at 4°Cuntil injection.
Approximately 20 cells which had recently undergone autogamy were placed underhigh-temperature silicon oil to immobilize them. Approximately 5 to 50 pg of theplasmid was injected into the macronucleus of each cell using a pulled capillary anda Narishige micromanipulator (Narishige International USA, East Meadow, NY, USA).Individual injected cells were transferred to 750 μl of inoculated culturefluid in depression slides and incubated in a humidifying chamber at RT for 2 days,allowing the cells to recover and divide. Cells were then transferred to test tubeswith inoculated culture fluid and maintained at 15°C as individual clones.Genomic DNA was extracted from the clone cultures as described previously (seeSequence analysis and construct design text section) and tested by PCR usingplasmid-specific primers: the forward primer for the plasmid pPXV(5′-taagatgaatggaatataatg-3′) and a reverse primer(5′-gaaaacccaagccaatcaatac-3′), which was sequence-specific forMKS3. DNA (1 μl, approximately 400 ng) was used in eachPCR: one cycle at 95°C for 5 minutes followed by 30 cycles at 95°C for 1minute, 40°C for 1 minute and 72°C for 3 minutes followed by one15-minute cycle at 72°C.
Localization, visualization and analysis of FLAG-MKS3p
We tested small cultures of individual clones to ascertain whether the cellsexpressed the protein and where it was localized. A 10-ml culture of injected cellswas added to 50 ml of inoculated culture fluid and grown at 22°C forapproximately 48 to 72 hours. The cells were immunostained and imaged as describedbelow. Images were analyzed for colocalization using softWoRx Pro software (AppliedPrecision, Issaquah, WA, USA). Experiments were repeated five times.
To isolate pellicle membrane and whole cilia membrane, wild-type (stock 51s P.tetraurelia) cells expressing FLAG (control) or FLAG-MKS3 (Test) weremaintained in large cultures (3 to 6 L of culture fluid) at 22°C until adensity of 8,000 to 12,000 cells/ml was achieved. For pellicular membrane, cells wereharvested as described previously . Inseparate experiments, cilia were separated from cell bodies and collected aspreviously described  up to the point ofseparation of the ciliary membrane from the axoneme. Protein concentrations weredetermined using a bicinchoninic acid protein assay (Thermo Scientific, Pittsburgh,PA, USA) and equalized between the test and control. Samples were separated on a 12%SDS-PAGE gel after adding 1 μl of β-mercaptoethanol and boiling for 5minutes. One hundred micrograms of pure pellicular membrane and 400 μg ofwhole cilia were loaded, along with 10 μl of a Pierce Biotechnologythree-color prestained protein molecular weight marker (Thermo Scientific). Proteinswere transferred onto nitrocellulose membrane (Pall Gelman Versapor; KrackelerScientific, Albany, NY, USA) and blocked for 1 hour using 5% nonfat dry milk, 2%Telost gelatin from fish, 3% normal goat serum (Vector Laboratories, Burlingame, CA,USA), in Tris-buffered saline Tween 20 (TBS-T) (15 mM Tris-Cl, 140 mM NaCl,0.1% v/v Tween 20, pH 7.5). Blots were probed with a 1:2,500 dilution ofrabbit Anti-FLAG M2 clone or 1:10,000 mouse anti-tubulin in the blocking buffer.Blots were incubated overnight while rocking at 4°C. Buffers were removed, theblots were rinsed three times in TBS-T and then incubated for 1 h in 1:10,000goat anti-rabbit or anti-mouse alkaline phosphatase (AP)-conjugated secondaryantibody. Blots were rinsed again four times in TBS-T for 15 minutes for each washand developed using nitroblue tetrazolium/5-bromo-4-chloro-3′-indolyl phosphateAP (Moss, Inc, Pasadena, MD, USA).
RNAi by feeding construct
Constructs for RNAi were created from genomic DNA using the following primers:MKS3 forward, 5′-gaaaacccaagccaatcaatac-3′ and reverse,5′-ggtcgacaatctgaaggataag-3′; and IFT88 forward,5′-caattaaggaaaaccacctg-3′ and reverse,5′-aaaactaacaggattgtcatct-3′. All PCR conditions began with an initialstep at 95°C for 5 minutes and ended with a final stage at 72°C for 20minutes. The MKS3 RNAi construct was amplified by 30 cycles at 95°C for1 minute, 52°C for 1 minute and 72°C for 2 minutes. The IFT88construct was amplified by five cycles at 95°C for 1 minute, 47°C for 1minute and 72°C for 2.25 minutes; followed by twenty-five cycles at95°C for 1 minute, 50°C for 1 minute and 72°C for 2.25 minutes(Techne Thermal Cycler; Bibby Scientific, Burlington, NJ, USA). The final PCRproducts were analyzed on 0.75% or 1.0% agarose gel (Invitrogen/Life Technologies)and visualized with ethidium bromide. Resulting PCR products were cloned directlyinto pCR2.1-TOPO vector (Invitrogen/Life Technologies), transformed into OneShotcells (Invitrogen/Life Technologies), and sequenced. Correct sequences were cut fromthe pCR2.1-TOPO vector and ligated into the double-T7 promoter vector L4440 (AddGene,Cambridge, MA, USA) using the Ligate-IT Kit (USB/Affymetrix) as per the kitinstructions. Escherichia coli strain Ht115 (DE3), which lacks RNaseIII, wastransformed with 50 ng of plasmid DNA for either MKS3 orIFT88. As a control, Ht115 cells were transformed with L4440 with no insert.Bacterial cultures were maintained with tetracycline (12.5 μg/ml) andampicillin (AMP) (100 μg/ml).
RNAi by feeding
Overnight cultures of Ht115(DE3) transformed with RNAi or control plasmids were usedto inoculate 50 ml of LB-AMP (100 μg/mL) and grown until the 595-nmoptical density reached 0.3 to 0.4, at which point isopropylβ-D-1-thiogalactopyranoside (IPTG) (RPI, Mount Prospect, IL, USA) was added to afinal concentration of 0.125 mg/ml. Cultures were incubated with shaking for 3hours at 37°C to induce the production of double-stranded RNA. Paramecia thathad recently undergone autogamy were collected by centrifugation and resuspended in10 ml of Dryl’s solution (1 mM Na2HPO4,1 mM NaH2PO4, 1.5 mM CaCl2, 2 mMNa-citrate, pH 6.8) to purge bacteria from their surfaces and food vacuoles.
The induced bacteria were collected by centrifugation at4,000 × g (Beckman J2-21 centrifuge, JA-14 rotor;Beckman Coulter, Brea, CA, USA) at 4°C and resuspended in 100 mL of wheatculture medium containing an additional 8 μg/mL stigmasterol,0.125 mg/mL IPTG (RPI), and 100 μg/ml AMP. Approximately 50 to 100 ofthe purged paramecia were added to the control culture. In the case of theMKS3 and IFT88 RNAi cultures, 4,000 and 8,000 cells were addedto 100 ml, respectively. Cultures were maintained at 28°C, and after 24hours, an additional 0.125 mg/ml of IPTG (RPI) and 800 μg ofstigmasterol were added. Growth rates of cultures were determined by counting cellsat 24, 48 and 72 hours of growth. All experiments were repeated a minimum of threetime and all cultures were harvested or observed after approximately 72 hours ofgrowth unless noted otherwise.
Cultured cells (100 ml) were collected by centrifugation (Damon IEC DivisionClinical Centrifuge, Needham Heights, MA, USA) and rinsed twice in 100 ml ofDryl’s solution. The cell volume was reduced to approximately 100 μlin a 1.5-ml Eppendorf tube before 1 ml of PHEM and 0.1% or 0.5% Triton X-100)was added. Cells were undisturbed for 1 to 4 minutes, then spun at250 × g (Damon IEC Division Clinical Centrifuge) andthen the supernatant was removed and the pellet (cells) was mixed with 1 ml offixation buffer (2% or 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield,PA, USA), 2 mM NaH2PO4•H2O, 8 mMNa2HPO4, 150 mM NaCl, pH 7.5). Samples wereundisturbed for 10 minutes or rocked for 1 hour at room temperature (RT) and washedthree times in 1 ml of blocking buffer (2 mMNaH2PO4•H2O, 8 mMNa2HPO4, 150 mM NaCl, 10 mM EGTA, 2 mMMgCl2, 0.1% Tween 20, 1% or 3% bovine serum albumin (BSA),pH 7.5).
Primary antibodies for the immunostaining for localization were as follows:FLAG-MKS3: mouse anti-FLAG, M2 clone at a 1:300 dilution (Sigma-Aldrich) andanti-centrin at a 1:1,000 dilution (anti-Tetrahymena centrin, gift from MarkWiney, University of Colorado, Boulder, CO, USA). For ciliary measurements, we usedmouse anti-α-tubulin at a dilution of 1:200 (Sigma-Aldrich). For visualizationof basal bodies, we used anti-centrin at a dilution of 1:1,000. For cortical unitvisualization, we used anti-2F12 at a dilution of 1:200 (gift from Jean Cohen,Gif-sur-Yvette, France). For the visualization of the kinetodesmal fibers (KDFs), weused anti-KDF at a 1:400 dilution (gift from Janine Beisson, Centre deGénétique Moléculaire, Gif-sur-Yvette, France) andanti-Glu-α-tubulin at a 1:500 dilution (Synaptic Systems, Göttingen,Germany). Primary antibodies in 100 μl of blocking buffer were mixed withthe cells and rocked at RT for 1 hour. Cells were washed three times in blockingbuffer or wash buffer (2 mM NaH2PO4•H2O,8 mM Na2HPO4, 150 mM NaCl, 0.1% Tween 20, 1% BSA,pH 7.5). The cells were mixed with 100 μl of blocking buffer with a1:200 dilution of secondary antibodies. Secondary antibodies (MolecularProbes/Invitrogen, Grand Island, NY, USA) included Alexa Fluor 488 or 555 goatanti-mouse and Alexa Fluor 488 or 568 goat anti-rabbit. After 30 minutes to 1 hour ofincubation while rocking, cells were washed three to five times with blocking or washbuffer and, to the final 20 μl of cells, one drop (approximately15 μl) of VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA,USA) was added. Tubes were wrapped in aluminum foil and stored at 4°C untiluse.
Imaging of the immunostained cells was done using a DeltaVision RestorationMicroscopy System (Applied Precision), consisting of an inverted Olympus IX70microscope (Olympus America, Center Valley, PA, USA) and a Kodak CH350E camera(Rochester, NY, USA). Prepared cells (7 μl) were placed under a glasscoverslip and imaged at 20°C to 22°C using either a PlanApo 60× or100×/1.40 oil-immersion lens objective and deconvolved and analyzed usingsoftWoRx Pro software.
Colocalization of FLAG-MKS3 and centrin (basal bodies) was analyzed using softWoRxPro software or ImageJ software . Elevencells were analyzed for the colocalization of these two proteins. To examine thestaining patterns and calculate the number of basal bodies with FLAG-MKS3 staining,15 μm × 15 μm grids were chosen from both theventral and dorsal surfaces of each of three cells. Basal bodies within that gridwere counted, and we noted whether they had FLAG-MKS3 staining. A total of 463 basalbodies were analyzed on these three cells.
Scanning electron microscopy
RNAi cultured cells (200 ml) were collected by brief centrifugation at800 × g (Damon IEC Division Clinical Centrifuge), washedtwice in Dryl’s solution and fixed as described by Lieberman et al.. After critical point drying,coverslips were glued onto an aluminum chuck using colloidal graphite cement andallowed to dry in a desiccator overnight. The samples were sputter-coated and storedin a desiccator until imaged using a JSM-6060 scanning electron microscope (JEOL USA,Peabody, MA, USA).
Transmission electron microscopy
RNAi cultured cells (100 ml) were collected by brief centrifugation at800 × g (Damon IEC Division Clinical Centrifuge) andwashed twice in 100 ml of Dryl’s solution, then approximately100 μL of the cell pellet was removed and placed in 1.5-ml Eppendorf tubes.One milliliter of Fixation Solution A (1% gluteraldehyde (Electron MicroscopySciences), 0.05 M sodium cacodylate, pH 7.2) was added, rocked for 30minutes on ice and washed three times for 10 minutes under the same conditions. Cellswere resuspended in postfix Solution B (1% gluteraldehyde (Electron MicroscopySciences), 0.05 M sodium cacodylate buffer, 1% osmium tetroxide, pH 7.2)and again washed and rinsed as described above. Cells were preembedded in 2% agarosegel (Invitrogen/Life Technologies) in 0.05 M sodium cacodylate buffer andallowed to set, then sliced into 1 mm × 1 mm blocks.Blocks were placed in glass vials with 50% ethanol and rocked on a specimen rotatorfor 30 minutes during each of the following washes: ethanol at concentrations of 50%,70% and 90% and two times at 100%, with both of the latter in propylene oxide. Cellswere left overnight on a specimen rotator in 1:1 propylene oxide and Spurr’ssolution (Electron Microscopy Sciences). The next day, samples were placed in freshSpurr’s solution for 6 hours and placed in flat embedding molds with freshSpurr’s solution at 60°C for 48 hours. Sections were cut to 90-nmthickness, placed on copper 200-mesh grids and contrasted on droplets of 2% uranylacetate in 50% ethanol for 6 minutes followed by lead citrate (120 mM sodiumcitrate, 2.66% lead nitrate and 0.65% sodium hydroxide in water) for 4 minutes.Sections were imaged using a JEM-1210 electron microscope (JEOL USA). These studieswere repeated three times.
Glutathione S-transferase pull-down and mass spectrometry analysis
The coiled-coil domain of MKS3 was expressed with a glutathioneS-transferase (GST) tag for use in a GST pull-down assay. The construct wascreated by amplifying positions +2,183 to +2,273 of GSPATG00015939001 using thefollowing forward and reverse primers, respectively:5′-gcgggatccatgaattttgtcgatctc-3′ and5′-gcggaattctgatggattttctccatg-3′. The PCR product was treated with BamHIand EcoRI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and cleanedusing gel purification and the PrepEase Gel Extraction Kit (Affymetrix, Santa Clara,CA, USA), then ligated into a pGEX-2TK plasmid vector (GE Healthcare Life Sciences,Pittsburgh, PA, USA) using the Ligate-IT Rapid Ligation Kit (Affymetrix). ThepGEX-2TK plasmid vector had already been opened using the same restriction enzymes,treated with 1 U of calf intestinal alkaline phosphatase at 37°C for 5 minutesto remove the phosphate groups, followed by heat inactivation with 5 mMNa2-ethylenediaminetetraacetic acid at 72°C for 20 minutes. TheGST-MKS3 coiled-coil domain and GST were expressed in BL-21 cells and bound toglutathione sepharose beads (GE Healthcare Life Sciences) as described previously. After beads were collected frombacterial cell lysates, they were washed in a 1 M MgCl2 buffer toremove bacterial proteins from the GST and GST-MKS3 proteins. Protein-bound beadswere stored at 4°C in phosphate-buffered saline for up to 2 weeks.
Stock 51s P. tetraurelia cells were cultured and harvested as describedpreviously  for whole-cell extract (WCE).Glutathione sepharose beads (GE Healthcare Life Sciences) were prepared by washingthree times in LAP200 buffer (50 mM HEPES, 200 mM KCl, 1 mM EGTA,1 mM MgCl2, pH 7.4) buffer with 1% Triton X-100. Washed beads(200 μl) were added to 20 ml of WCE. This precleared WCE was thensplit in half and incubated with 200 μl of glutathione sepharose beadsattached to either GST or GST-MKS3. Beads in the supernatant were allowed to rock onice at 4°C for 1 hour. Control and test beads were recovered and washed threetimes in LAP200 buffer with 1% Triton X-100. Samples were run on a 7% to 14% gradientacrylamide gel and silver-stained, then gel slices were trypsin-digested as describedpreviously .
Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) ona linear ion trap LTQ XL Linear Ion Trap Mass Spectrometer (Thermo Fisher Scientific,Asheville, NC, USA). Half the material was loaded onto a100-μm × 120 mm capillary column packed with MAGIC C18(5-μm particle size, 20-nm pore size; Michrom Bioresources, Auburn, CA, USA) ata flow rate of 500 nl/min. Peptides were separated by a gradient of 5% to 35%CH3CN/0.1% formic acid for 30 minutes, 40% to 100%CH3CN/0.1% formic acid for 1 minute and 100% CH3CN for 10minutes.
Product ion spectra were searched using the SEQUEST search engine on ProteomeDiscoverer 1.4 (Thermo Fisher Scientific) against a curated P. tetraureliadatabase with sequences in forward and reverse orientations. The 13 raw files fromcontrol samples and the 13 raw files from test samples were searched as onecontiguous input file, and a single result file was generated for each. The databasewas indexed to allow for full trypsin enzymatic activity, two missed cleavages andpeptides between the molecular weights of 350 to 5,000 Da. Search parameters setthe mass tolerance at 2 Da for precursor ions and 0.8 Da for fragment ions.The result files were then searched against Scaffold version 4.0.5 software (ProteomeSoftware, Portland, OR, USA). Cross-correlation (xcorr) significance filters wereapplied to limit the false-positive rates to less than 1% in both data sets. Thexcorr values were as follows: (+1): 1.8, (+2): 2.7, (+3): 3.3 and (+4): 3.5. Otherfilters applied were a minimum peptide cutoff of 2 as well as DeltaCN >0.1.Ultimately, the confidence parameters resulted in 0% false discovery rate at theprotein and peptide level for both the control and test results.