Strains and culture conditions.
Stock d4-2 of Paramecium tetraurelia
, the wild-type reference strain, was used in all RNAi experiments. The mutant nd7-1 (31
), which carries a recessive monogenic mutation preventing trichocyst discharge, a dispensable function under laboratory conditions, was used for transformations. Cells were grown at 27°C in wheat grass infusion, BHB (L'arbre de vie, Luçay Le Mâle, France), inoculated with Klebsiella pneumoniae
and supplemented with 0.8 μg/ml β-sitosterol according to standard procedures (33
In order to visualize the cellular localization of Bug22p, the Bug22a paramecium gene, as well as the human (Hs) Bug22 cDNA, were cloned into the Paramecium expression vector pPXV-GFP. The human Bug22 cDNA was also cloned into the plasmid pD2EGFP for expression in HeLa cells. In all three constructs, the green fluorescent protein (GFP) sequence was fused at the N terminus of the BUG22 gene.
(i) pPXV-HsBUG22 expression vector for Paramecium.
The human BUG22 sequence was amplified by PCR from a cDNA library kindly provided by François Lacroute using the primers 5′-GGTGCTCGAGATGTTCAAAAACACGTTCCAG-3′ and 5′-GTGGGGTACCTCATTGCTTTGCCTTGTTCTG-3′ and cloned into the XhoI/KpnI sites of the Paramecium expression vector pPXV.
(ii) GFP-PtBUG22 fusion gene to be expressed in Paramecium.
The pPXV-GFP expression vector was used as previously described. The Paramecium (Pt) BUG22c gene was amplified by PCR with primers 5′-GTGGGGTACCGGAGGAGGAATGTTCAAGAACTCATTCCAAAG-3′ and 5′-CCACGGTACCTCATCCTTATTTTTAGATGGGTAAG-3′ and cloned into the KpnI site of pPXV-GFP.
(iii) GFP-HsBUG22 fusion gene to be expressed in Paramecium.
The human sequence of BUG22 was amplified by PCR with primers 5′-GTGGGGTACCGGAGGAGGAATGTTCAAAAACACGTTCCAGAG-3′ and 5′-CCACGGTACCTCATTGCTTTGCCTTGTTCTGAAC-3′ and cloned into the KpnI site of pPXV-GFP.
The sequences of genes to be knocked down were amplified by PCR and cloned into a modified Litmus 28i plasmid (New England Biolabs) in which the EcoRI site had been replaced by an SrfI site, using the method developed for PCRscript cloning by Stratagene (La Jolla, CA), and transferred to the feeding vector L4440 (37
) into the XhoI/HindIII sites of the polylinker between two T7 promoters.
(i) Constructs for RNAi of the Paramecium BUG22 genes.
Two constructs were used to knock down the two subfamilies of BUG22 genes. For BUG22a and -b knockdowns, BUG22a was amplified from positions 75 to 391 with the primers 5′-GGATAAGCAAAGTAATTTATGATTGG-3′ and 5′-CTTTATACTAATTACTTAATAATTCGATGC-3′. For BUG22c and -d knockdowns, BUG22d was amplified from positions 75 to 394 with the primers 5′-TCAAAGCCCTTGTAGATTTG-3′ and 5′-GTGTTTTAAGATGTACTTGATAATTAG-3′. A short sequence was also used for RNAi by direct cloning of a pair of complementary oligonucleotides corresponding to the sequence 5′-ATGTTCAAGAACTCATTCCAAAGTGGATTTCTTTCAATTTTATATTCAATAGGATCAAAGCC-3′.
(ii) Constructs for RNAi of human BUG22 transgenes in Paramecium.
The HsBUG22 sequence was transferred from the pPXV-HsBUG22 construct by digestion and cloning into the XhoI/HindIII sites of the L4440 vector.
(iii) Constructs for RNAi of the IFT172 genes in Paramecium.
One of the two close paralogous IFT172 genes (GSPATG00033191001) was amplified by PCR using the primers 5′-GCAGTCATTAGATCATCATAGG-3′ and 5′-CAATAACTTCACTTTCAGGAAC-3′ and cloned into the L4440 vector as the BUG22 sequences. Due to high sequence conservation, this single plasmid is supposed to knock down both paralogs.
In all cases, plasmid DNA was prepared using the plasmid midi kit according to the protocol of the manufacturer (Qiagen, Courtaboeuf, France). The sequences of Paramecium
genes were retrieved from Paramecium
) and verified by resequencing after cloning.
Transfection. (i) HeLa cells.
HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Gibco) at 37°C in 5% CO2. The day prior to transfection, HeLa cells (1 × 105) were plated on a 24-well plate containing presterilized coverslips in DMEM supplemented with 10% FBS. The cells were transfected with 0.5 μg of Hs-Bug22-GFP using Lipofectamine 2000 (Invitrogen) according to the manufacturer's directions.
(ii) Paramecium transformation.
nd7-1 cells were transformed by microinjection into their macronuclei of filtered and concentrated plasmid DNA (5 μg/μl) containing a mixture at a ratio of 10:1 of the plasmids of interest (GFP fusion genes and HsBug22p expression) and a plasmid directing the expression of the ND7
). Microinjection was done under an inverted Nikon phase-contrast microscope, using a Narishige micromanipulation device and an Eppendorf air pressure microinjector. Successfully transformed cells were screened for the ability to discharge trichocysts on picric acid stimulation and further analyzed.
The polyclonal GTL3 anti-Bug22p antibody was purchased from Aviva Systems Biology (San Diego, CA). The monoclonal antibody 20H5, raised against Chlamydomonas
), was used to label centrioles in HeLa cells. The monoclonal antibody ID5 (38
), which essentially labels basal bodies and a few microtubule arrays (10
), was used to visualize the basal-body pattern. The polyclonal anti-GFP antibody was purchased from Interchim (Montluçon, France). The polyclonal anti-Paramecium
ciliary tubulin (9
) and anti-ciliary rootlet (34
) antibodies were homemade antibodies.
The secondary antibodies used in immunofluorescence were Alexa Fluor 488 or 594 anti-mouse or -rabbit IgG antibodies from Invitrogen, the antibody used in Western blot experiments was an anti-rabbit IgG-alkaline phosphatase (AP) conjugate (Promega), and the antibodies used in immunoelectron microscopy were colloidal-gold-conjugated anti-rabbit immunoglobulins (Gar G5 and Gar G10; Aurion).
Protein extraction, SDS gels, and Western blots.
Paramecium protein extracts were prepared from a pellet of 2 × 105 cells homogenized in a potter with tight clearance in 10 mM Tris HCl, 1 mM EDTA, 250 mM sucrose containing a protease inhibitor cocktail (Set1; Calbiochem). Low-speed pellets and supernatants were separated by centrifugation at 10,000 × g. Total protein extracts of bacteria were obtained by sonication of a bacterial pellet in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na deoxycholate). Gel electrophoresis was performed using Criterion XT gels at neutral pH and 12% Bis-Tris (Bio-Rad) according to the manufacturer's recommendations. The primary antibodies used were anti-GTL3 (1:500) and anti-GFP (1:750). The secondary antibody was anti-rabbit–AP (1:500). Western blots were revealed using the 5-bromo-4-chloro-3-indolylphosphate (BCIP)-nitroblue tetrazolium (NBT) liquid substrate system from Sigma.
Immunofluorescence microscopy. (i) HeLa cells.
After 24 h of culture, cells were washed with phosphate-buffered saline (PBS), fixed for 6 min at −20°C in 100% methanol, and blocked in 3% bovine serum albumin (BSA) and 0.1% Tween 20 in PBS for 30 min. The cells were incubated at room temperature with either the 20H5 (1:1,000) or the GTL3 (1:200) antibody for 1 h and then washed three times in 1% BSA and 0.1% Tween 20 and incubated for 1 h with the secondary antibody before three additional washes.
Immunostaining of cells was carried out as previously described (28
) using the anti-ciliary tubulin (1:500), the monoclonal ID5 (1:350), or the anti-ciliary rootlet (1:400) antibody. For both HeLa cells and paramecia, appropriate fluorescent secondary antibodies were applied at 1:500 dilutions, nuclei were stained with Hoechst-33258, and slides were mounted in Cityfluor (London). Preparations were observed under a Zeiss Axioskop 2-plus fluorescence microscope equipped with a Roper Coolsnap-CF intensifying camera with GFP filters. Images were processed with Metamorph software (Universal Imaging).
For morphological observations, cell pellets were processed as previously described (5
) using 1% or 2% glutaraldehyde for fixation. For postembedding immunolocalization, cell pellets were processed as described previously (18
), using either 2% paraformaldehyde, 0.15% glutaraldehyde, or 3% paraformaldehyde and 0.5% glutaraldehyde for fixation, and embedded in LR White (London Resin). Thin sections were collected on nickel grids and treated with double-distilled water or 0.1 M NH4
Cl in 0.1 M PBS and then saturated and processed with 3% BSA in PBS. Primary polyclonal antibodies were diluted 1:50 to 1:75 (anti-GTL3) and 1:10, 1:20, or 1:500 (anti-GFP). After being washed, the sections were incubated with, respectively, 1:40, 1:75, or 1:100 dilutions of 5-nm (GTL3 labeling) or 10-nm (GFP labeling) gold particles. The sections were examined with a Philips CM10 or a Jeol transmission electron microscope.
RNAi by feeding.
RNAi gene knockdown was performed according to the method of Galvani and Sperling (13
). HT115, an RNase III-deficient strain of Escherichia coli
with an isopropyl-β-d
-thiogalactopyranoside (IPTG)-inducible T7 polymerase, was transformed by the desired constructs into Litmus or L4440 plasmids. Wild-type paramecia were incubated in double-stranded-RNA (dsRNA)-expressing bacteria and were transferred daily into fresh feeding medium. Control cells were fed with bacteria carrying the complete coding region of the ND7
Two methods were used for swimming analysis. First, we measured the swimming speed of cells under dark-field microscopy using a Zeiss Axioskop 2-plus microscope with a 10× objective by recording tracks during 650-ms pauses. Second, we used a Zeiss Steni 2000-C dissecting microscope with 1-min time-lapse acquisitions at 7 frames per second with a Roper Coolsnap-CF camera and Metamorph software (Universal Imaging). The speed means and standard errors were calculated by measuring the lengths of the tracks using ImageJ and subtracting a mean length of the paramecia for the first method and using automated analysis of the swimming tracks currently under development for the second method (J. Ferracci, O. Arnaiz, and J. Cohen, unpublished data).
High-speed video microscopy.
All of the observations were made under an inverted microscope (Axiovert 200; Carl Zeiss S.A.S., Le Pecq, France) using a 100× objective on 5 μl of a suspension of paramecia immobilized between a cover glass (diameter, 50 mm; thickness, 0.13 to 0.16 mm) from Menzel-Glaser and a 22- by 22-mm coverslip, with a few hundred calibrated 10-, 15-, or 20-μm polystyrene beads (Fluka), during overall periods shorter than 5 min to avoid changes in ciliary activity. A final dilution of ca. 1/10 of India ink was added to the medium to be visualized as particles that materialized the fluid flow during the acquisitions. Cilium movements were digitally recorded with a camera (PixeLink A741; Ottawa, Canada) at a rate of 355 images per second. A movie was constituted of 1,800 images with a definition of 256 by 192 pixels. The size of a pixel was 0.13 by 0.13 μm2. In each sequence, we selected a cilium that we were able to follow during an entire cycle of beating. For this, the video sequences were played back frame by frame using Streampix software (Norpix Inc., Montreal, Canada). The angle of beating was calculated by trigonometric calculations in the triangle made by the anchoring point and the two extreme positions of the cilium extremities during the power stroke.
Cells to be assayed were first equilibrated for a few minutes in resting solution (2 mM Na citrate, 1 mM NaH2
, 1 mM Na2
, 1.5 mM CaCl2
) and then individually transferred to test solutions that consisted of either 30 mM KCl or 5 mM tetraethylammonium, 10 mM NaCl in resting solution (19
). Observation of immediate swimming behavior was done during the first minute after transfer under the dissecting microscope.