Cell and culture media
The C. reinhardtii
wild-type strains CC-125 (mt+) and CC-124 (mt−), paralyzed flagella strain pf18
(CC-1297, mt−), temperature-sensitive flagellar assembly mutant fla10
allele, CC-1919, mt−), and arg−
strain CC-3681 (arg7
, mt−) were obtained from the Chlamydomonas Genetics Center. The arg2 pf1
double mutant was generated by K. Kozminski (University of Virginia, Charlottesville, VA). Cells were grown on solid media supplemented with 1.5% agar or in a liquid minimal medium, MI (Harris, 1989
), MI-N (MI medium without nitrogen to induce gametogenesis), or Tris-acetate-phosphate media (Harris, 1989
) at 22–23°C with a 14/10-h light/dark cycle and constant aeration.
Cloning of the CrPKD2 gene
The EST clone AV395567 (Asamizu et al., 1999
) corresponding to the predicted CrPKD2 protein C_590099 (Joint Genome Institute version 2 draft assembly of the C. reinhardtii
genome) was sequenced. It included the sequence from bp 1,223 of the cDNA through the 3′ UTR of CrPKD2
. We extended the cDNA to the 5′ UTR by sequencing the two RT-PCR products generated using two sets of primers: TCTTGGGAGTGCTGTATGAGCAGCGC (bp 318–343 of the cDNA) and CGGTGGTGTTGTACACGATGC (bp 1,696–1,716 of the cDNA); and AAGTAGCATGTCACATTAATGCATG (bp 1–25 of the cDNA) and CCGACACCGACTGGTTAAGGT (bp 1,125–1,145 of the cDNA), which were generated according to the C. reinhardtii
genome sequence database (http://genome.jgi-psf.org/chlre2/chlre2.home.html
). The mRNA was isolated and reverse transcribed as described previously (Huang et al., 2002
). The RT-PCR products were cloned into PCRII-TOPO (Invitrogen), generating plasmids pHK5 and pHK15, respectively. The intron coded by genomic DNA was not present in the PCR products.
Loop1 CrPKD2 antibodies were generated against a His-tagged fragment of CrPKD2 (aa 358–1,019) expressed in E. coli and purified by affinity chromatography using nickel–nitrilotriacetic acid agarose according to the manufacturer's instructions (QIAGEN). The purified protein was used for antibody production in rabbits at Pocono Rabbit Farm and Laboratory Inc. A peptide (AGEGDDKDDSPEVREEKRK, corresponding to aa 1,444–1,462 of CrPKD2) was synthesized and used to produce a second antibody by the same company. The N-PKD2 antibody was affinity purified from the Loop1 antiserum using a peptide containing aa 358–583 of CrPKD2, which was expressed in E. coli and purified using nickel–nitrilotriacetic acid agarose.
Flagellar isolation and flagellar membrane vesicle isolation
Flagella were isolated from C. reinhardtii
by pH shock as described previously (Witman et al., 1972
) and modified by Cole et al. (1998)
. The flagella from 40–48 liters of wild-type cells were resuspended in 6 ml HMDEK buffer (10 mM Hepes, pH 7.2, 5 mM MgSO4
, 1 mM DTT, 0.5 mM EDTA, and 25 mM KCl, including the protease inhibitors 1.0 mM PMSF, 50 μg/ml soybean trypsin inhibitor, 1 μg/ml pepstatin A, 2 μg/ml aprotinin, and 1 μg/ml leupeptin) for a protein concentration of ~3–4 μg/μl, and NP-40 (Calbiochem) was added to a final concentration of 0.1%. The mixture was incubated at 4°C for 30 min with shaking. The membrane vesicles were separated from the axonemes by centrifugation at 16,000 g
for 10 min at 4°C, the supernatant was centrifuged again to remove any remaining axonemes, and the membranes were harvested by centrifugation at 228,000 g
for 30 min (TLA 120.2 rotor, Optima Ultracentrifuge; Beckman Coulter). The pellet was resuspended in 1 ml of 0.1% NP-40 HMDEK buffer and centrifuged again to obtain the final pellet. Alternatively, membranes were further purified by density gradient sedimentation. For this, 1/3 volume of 60% iodixanol (Optiprep density gradient medium; Sigma-Aldrich) was added to the membrane-containing supernatant for a final concentration of 15%, and 0.9 ml of the mixture was added to a 1.5-ml tube. After underlaying with 0.3 ml of 30% iodixanol in HMDEK + 0.1% NP-40 buffer, the sample was centrifuged for 1 h and 11 min at 431,000 g
. A white band of membrane vesicles in the middle of the tube was collected and diluted 10 times with 0.1% NP-40 HMDEK buffer. The mixture was split and centrifuged as before, sedimenting the membrane vesicles. One membrane pellet was used for EM and the other was resuspended in 100–200 μl HMDEK buffer with 0.1% NP-40 for immunoblotting.
To remove all the flagellar membrane from the axoneme, the flagella were freeze–thawed twice to release the matrix and a small amount of membrane. Much of the membrane remained in the axonemal pellet. The axonemes were exacted twice with 0.1 or 1% NP-40 at room temperature for 30 min, and after centrifugation at 16,000 g for 10 min, the soluble fractions contained the membrane and the pellet contained axonemes. The membranes were sedimented at 280,000 g for 30 min.
Protein extraction, concentration determination, and immunoblot analysis
Preparation of the C. reinhardtii
whole cell extract, determination of the protein concentration, PAGE, and immunoblotting were performed as described previously (Huang et al., 2002
). Immunoblots were scanned and the relative protein concentrations were determined using ImageJ (National Institutes of Health).
GFP constructs and transformation with CrPKD2–GFP
To make the CrPKD2–GFP fusion, the C. reinhardtii
bacterial artificial chromosome clone 18K16 (https://www.genome.clemson.edu/cgi-bin/orders
) was cut with EcoRV and SpeI, generating an ~18-kb fragment, which included the entire CrPKD2
gene and its promoter. This fragment was subcloned into the EcoRV and SpeI sites of pBluescript II KS+ (Stratagene), generating a plasmid named pHK25. This plasmid was cut with HindIII, generating three fragments. One of these fragments, an 8-kb fragment containing the promoter and most of the genomic DNA encoding CrPKD2, was cloned into the HindIII site of the pBluescript KS+, generating pHK28. A second fragment, a 5.8-kb fragment containing the last two introns, exons, the 3′ UTR of CrPKD2
, and the KS+ vector, was religated to produce pHK26.
To tag the CrPKD2
gene, we cloned the GFP
gene into a unique EcoRI site in intron 11, flanked by the first intron of RBCS2 (Goldschmidt-Clermont and Rahire, 1986
). For this, the two ends of the intron were subcloned as follows: using the genomic DNA as template, the 3′ end of this intron was amplified with the primers CGGAATTC
AGTCGACGAGCAAGCC and GTGGATCC
TCCTGCAAATGGAAAC. EcoRI and BamHI sites (added sites in bold) were added to the primers and the PCR product was cut with EcoRI and BamHI and cloned into the same sites of pBluescript II KS+ vector (pHK31). Another set of primers was used to amplify the 5′ end of the first intron of the RBCS2: CAGGATCC
CCAGGTGAGTTCGACGAGCAAG including a BamHI site and CTCTAGAGAATTC
AAATGGAAACGGCGACG including XbaI and EcoRI sites. The PCR product was cut with BamHI and XbaI and cloned into pHK31 (BamHI and XbaI sites), generating pHK32. The BamHI fragment of the pCrGFP (Entelechon GmbH), which contains the C. reinhardtii
codon-adapted GFP ending with a stop codon, was inserted into the BamHI site of pHK32, generating pHK35. The sense orientation of this and following inserts was identified by restriction enzyme digests and confirmed by sequencing. The EcoRI fragment from pHK35 was inserted into the EcoRI site in intron 11 of the CrPKD2
gene of pHK26 in the sense orientation, generating pHK37. The 8-kb HindIII fragment that contains the promoter and 5′ end of CrPKD2
was inserted in the HindIII site of pHK37 in the sense orientation, generating pHK38.
To remove the stop codon at the end of GFP from pHK38, two primers, AGGTCGACTCTAGAGGATCCC and TGGATCCTTGTACAGCTCGTCCATGCCG, both containing a BamHI site, were used to amplify the GFP fragment. The PCR product, which did not have the GFP stop codon, was cloned into the TOPOII-PCR vector (Invitrogen), generating plasmid pHK34. The BamHI fragment of the pHK34 was exchanged with the BamHI fragment of pHK37 (which has a stop codon at the end of the GFP gene), generating plasmid pHK39. The HindIII fragment of pHK28 was inserted into pHK39 in the sense orientation to generate pHK41. Plasmids pHK38 and pHK41 were linearized with SpeI before transformation.
Because we did not observe fluorescence in transformants that harbor pHK38 or 41, these cells were only used for immunoblots, and the overlap PCR method was used to make another construct in which GFP was fused to the end of CrPKD2 through a flexible linker. Using the left (GGAGAAAGCTTGTGTTTTGG, HindIII) and right primers (CGCGCCGGAGGCGCCCTGGCCGGAGGCGCCCTGGGGCGGGGTCTCATTCATCA) and pHK26 as the template to amplify the C terminus of CrPKD2, the PCR product containing the fragment from the HindIII site in the last intron to the stop codon of CrPKD2 was obtained. The nucleotide sequence GGCGCCTCCGGCCAGGGCGCCTCCGGCGCG, which corresponds to a flexible protein linker GASGQGASGA, was included in the right primer. Another set of primers (GGCGCCTCCGGCCAGGGCGCCTCCGGCGCGAAGGGCGAGGAGCTGTTCACC and CTCGGTACCCGCTTCAATACG, KpnI) was used to amplify GFP and the 3′ UTR of RBCS2 from the plasmid pCrGFP. The sequence encoding the protein linker was included in the left primer. Both PCR products were purified using a PCR purification kit (QIAGEN), and equal molar amounts of the products were used as templates to fuse these two PCR products together. For this purpose, primers (GGAGAAAGCTTGTGTTTTGG [HindIII] and CTCGGTACCCGCTTCAATACG [KpnI]) were used, and the new PCR product was cut with HindIII and KpnI and cloned into the pBluescript II KS+ vector (HindIII and KpnI sites). The resulting plasmid was named pHK49. The HindIII fragment from pHK28 was inserted into the HindIII site of pHK49 in the sense orientation, generating pHK52. pHK52 was linearized with SpeI before transformation.
To prepare cells (CC-3681, arg7
, and arg2 pf1)
for transformation, cell walls were removed with autolysin as described previously (Huang and Beck, 2003
). Linearized plasmids pHK38, pHK41, and pHK52, together with pCB412, which harbors an AGR7
gene as a selectable marker (gift from C.F. Beck, University of Freiburg, Freiburg, Germany), were introduced into the cells using the glass bead method. Transformants were selected on Tris-acetate-phosphate medium plates and tested for GFP by Western blot analysis. GFP antibody was obtained from Roche Applied Science (clones 7.1 and 13.1).
To construct the fla10 (mt−) and pf18 (mt−) strains expressing CrPKD2–GFP, the original CrPKD2–GFP transformant (mt−) was crossed to CC-125 (mt+) to produce an mt+ strain. This strain was crossed with fla10 and pf18, and the progeny were analyzed to obtain the desired phenotype.
Nucleic acid manipulations and transformation of CrPKD2 interference construct
To make the genomic and cDNA fusion construct for RNAi, primers (GGACATGGTTCGTAGCGTTTAATGCC [700 bp in front of the translation start site of CrPKD2
gene] and GTCGAC
CGACACCGACTGGTTAAGGT [corresponding to the cDNA 1,145–1,125]), including a SalI site, were used to amplify the promoter and 5′ region of the CrPKD2
gene using genomic DNA as a template. The 2.5-kb PCR product was cloned into TOPO TA vector with the 3′ SalI site near the EcoRV site of the vector, generating plasmid pHK3. Another set of primers (AAGTAGCATGTCACATTAATGCATG [corresponding to bp 1–25 of the cDNA] and GTCGAC
CGACACCGACTGGTTAAGGT [corresponding to the cDNA 1,145–1,125]), including a SalI site, were used to amplify the PKD2
cDNA fragment, which corresponds to the genomic fragment in pHK3. The PCR product was cloned into the TOPO TA vector, and a clone with the 5′ end near the EcoRV site was identified and named pHK15. The EcoRV and SalI fragment from the plasmid pHK15 was inserted into the same sites in pHK3, creating pHK19. The PvuII fragment of pSI103 (Sizova et al., 2001
), which includes the aphVIII gene driven by the HSP70A-RBCS2 fusion promoter, was inserted into the EcoRV site of the pHK19, generating pHK22 (). pHK22 was linearized with ScaI before transformation using the glass bead method. Transformants were selected on plates with 10 μg/ml paromomycin (Sigma-Aldrich) and tested for PKD2 levels by Western blot analysis. 95 transformants were screened and 15 clones were picked for further analysis. Eventually, four clones showed a stable reduction of CrPKD2. Two of these (Ri22 and Ri39) showed only a modest decrease, and only one of these is included. The other two (Ri45 and Ri46) showed a more substantial depletion of CrPKD2.
Immunofluorescence light microscopy, live cell imaging, and FRAP
Wild-type strains were prepared for immunofluorescence light microscopy using methanol fixation, and were stained with primary and Alexa fluor–conjugated secondary antibodies (Invitrogen) as described previously (Pedersen et al., 2003
), modified by the addition of 0.05% glutaraldehyde directly to the medium for primary fixation before fixing with methanol at −20°C. Images were recorded with a microscope (Eclipse TE2000; Nikon) equipped with a Plan Apo 100×, 1.4 NA objective lens and a forced-air–cooled camera (Cascade 512B; Photometrics). Photoshop (Adobe) was used to adjust brightness and contrast and crop images.
The movement of the GFP-tagged CrPKD2 was recorded as described in the previous paragraph using an argon ion 488 laser controlled by a Mosaic System (Photonics) for illumination and photobleaching experiments.
For photobleaching, fla10
cells were immobilized with 0.02-M LiCl (Dentler, 2005
) and embedded in 0.75% low-melt agarose. First, a DIC picture was taken of the flagellum, and the GFP fluorescence of the selected region was recorded for 2 s using laser illumination at 32 mW (Fprebleach
). Next, the selected region of the flagellum was bleached for 3 s with the laser at 300 mW. GFP fluorescence of the selected region was recorded again for 2 s using the laser at 32 mW (Fbleached
). After 1 or 2 min of recovery, the GFP fluorescence of the selected region was recorded for 2 s at low power (Frecovery
). Finally, another DIC picture of the flagellum was taken. By comparing the two DIC pictures of the same flagellum, videos were selected for further analysis in which the flagellum did not move and the focus did not change. The mean fluorescence of each parameter, Fprebleach
, and Frecovery
, were used to calculate recovery: % recovery = (Frecovery
. For performing the experiment at 32°C, cells were incubated at 32°C in a water bath and observed for a maximum of 10 min at room temperature on the microscope. Alternatively, cells were observed in a glass-bottom Petri dish (WillCo Wells B.V.) in a stage-mounted dish heater (DH-35; Warner Instruments). Data were analyzed using MetaMorph (Universal Imaging Corp).
Flagellar length measurements
fla10 cells were maintained at 22 or 32°C to induce flagellar resorption. Wild-type cells were induced to resorb their flagella with 20 mM NaPPi. Aliquots of these cells were fixed with 1% glutaraldehyde. Cells were imaged as described in the previous section and the lengths of individual flagella were measured using the MetaMorph software package.
Measurement of mating efficiency and flagellar protein tyrosine kinase activity
Gametes were generated by resuspending vegetative cells in MI-N medium at a density of 1–2 × 107
cells/ml. The cells were incubated under continuous light for 16–24 h and gametes (CC-125 and RNAi strains) were adjusted to the same cell density. The gametes to be assayed were mixed with a twofold excess of gametes of the opposite mt (CC-124) to ensure that mating efficiency of the test strains was not limited by depletion of the mating partner. After completion of the mating reaction in 1 h, the cells were fixed by addition of glutaraldehyde, and the number of the quadriflagellate zygotes and biflagellate cells were counted by phase-contrast microscopy (Martin and Goodenough, 1975
; Beck and Acker, 1992
). The percentage of cells that had mated was then calculated according to the method of Beck and Acker (1992)
To assay flagellar protein tyrosine kinase activity, gametes of CC-125 or Ri46 were mixed with the same number of CC-124 gametes, and flagella were isolated 3 min after mixing. The concentration of the flagellar protein was measured using the Amido black method (Huang et al., 2002
), and equivalent amounts of protein were used to assay the protein tyrosine kinase activity by immunoblotting for phosphorylated CrPKG, the substrate of protein tyrosine kinase (Wang and Snell, 2003
Axonemes and membrane pellets were treated sequentially at room temperature for 1 h each with 2.5% glutaraldehyde in HMDEK, 1% osmium tetroxide in HMDEK, and 1% uranyl acetate in water with brief rinses in between. The pellets were dehydrated through ethanol and propylene oxide and embedded in epoxy resin according to standard procedures. The final steps of dehydration and the initial steps of resin infiltration were performed at −20°C for the membrane samples. Silver sections were observed with an electron microscope (1230; JEOL) equipped with a digital camera (Orca HR; Hamamatsu Photonics).
RNA isolation and Northern blot hybridization
Gametes were deflagellated using the pH shock method and vigorously aerated under light. Total RNA was isolated from cells before deflagellation and after 10 or 15 min of flagellar regeneration. RNA was blotted and hybridization was performed as described previously (von Gromoff et al., 1989
Online supplemental material
Table S1 lists the lengths of flagella measured from cells induced to resorb their flagella. Fig. S1 shows a diagram of the CrPKD2
gene and the amino acid sequence of the protein. Fig. S2 shows a phylogenetic tree of TRP channels. The Northern blots in Fig. S3 demonstrate that only one 6-kbp transcript is present encoding CrPKD2. Additional immunoblots of the CrPKD2 fragments from cells expressing CrPKD2–GFP are shown in Fig. S4. Video 1 shows the movement of CrPKD2 in the flagella of fla10
gametes at permissive temperature. The online version of this article is available at http://www.jcb.org/cgi/content/full/jcb.200704069/DC1