radial spokes and DRC, determining the stoichiometry of CSC components relative to each other and the radial spokes, identi- fying additional CSC interactors (potentially including polypep- tides comprising the DRC or dynein arms), and determining the effects of calcium on the CSC modulation of dynein activity. Materials and methods C. reinhardtii strains Strain A54-e18 (nit1-1, ac17, sr1, mt+), the wild-type strain for motility and axoneme structure, was obtained from P. Lefebvre (University of Minnesota, St Paul, MN). The central pair–defective strain pf18, the radial spoke–defective strains pf17 and pf14, and the dynein-defective strains pf2, pf3, ida1, ida4, and pf28 were obtained from the C. reinhardtii Genetics Center (Duke University). The pf30pf28, pf14pf28, and pf14pf30 strains were obtained from W. Sale (Emory University, Atlanta, GA). Gen- eration of the double mutants pf18pf28 and pf18ida1 have been previ- ously described (Smith, 2002b). The double mutant pf18ida4 was selected from nonparental ditype tetrads. All cells were grown in constant light in Tris acetate phosphate media (Gorman and Levine, 1965). Preparation of ﬂagella, ﬂagellar extracts, and sucrose gradients Flagella were severed from cell bodies by the dibucaine method (Witman, 1986) and isolated by differential centrifugation in NaLow (10 mM Hepes, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, and 30 mM NaCl). Axonemes were isolated by adding NP-40 (Calbiochem) to ﬂagella for a ﬁnal concentration of 0.5% (wt/vol) to remove ﬂagellar membranes. Axonemes were initially extracted in NaHigh (10 mM Hepes, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, and 0.6 M NaCl) at a concentration of 6 mg/ml on ice for 20 min. The axonemes were pelleted, resuspended in NaHigh, and immediately pelleted again. The supernatant was discarded, and the pellet was extracted with KI (10 mM Hepes, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, 30 mM NaCl, and 0.5 M KI) at a concentration of 12 mg/ml for 30 min on ice. This extract is referred to as the KI extract or high salt extract. KI extracts were dialyzed against NaLow buffer and clariﬁed by centrifugation at 12,000 relative centrifugal force for 10 min. For some experiments, the clariﬁed extracts were loaded onto 5–20% sucrose gradients and subjected to ultracentrifugation at 35,000 rpm for 16 h in an SW41Ti rotor (Beckman Coulter). 0.5-ml fractions were collected from the bottom of the tube and prepared for SDS-PAGE. Immunoprecipitation Immunoprecipitation was performed according to Wargo et al. (2005) with the following modiﬁcations. 150 μl KI axonemal extract was incu- bated with 40 μg of afﬁnity-puriﬁed anti-CaM or anti-IP2 antibody. After four washes with TBST150 (150 mM NaCl, 50 mM Tris-HCl, and 0.5 mM EDTA, pH 7.5), beads were resuspended in 90 μl TBST150 and 50 μl of 5× SDS-PAGE sample buffer. For experiments performed in high calcium, a ﬁnal concentration of 1 mM CaCl2 was included in the TBST150 buffer, and all washes were performed with TBST150 + 1 mM CaCl2. For immuno- precipitation performed in low calcium conditions followed by a ﬁnal high calcium wash, the beads were washed as before with TBST150, and then the beads were suspended in 100 μl TBST150 with 2 mM CaCl2. The beads were rotated for 5 min at RT and brieﬂy spun, and the supernatant was transferred to a new tube. 100 μl TBST150 was added to the remaining beads, and all samples were prepared for SDS-PAGE gels. For immuno- precipitation using sucrose gradient fractions, 50–80 μg of antibody was used with 1,200 μl of pooled sucrose gradient fractions. The sample was mixed for 5–8 h and washed with four 1-ml vol of TBS-T150. Mass spectrometry and sequence analysis Gel bands were excised from Coomassie-stained gels. These bands were analyzed by matrix-assisted laser desorption/ionization–time of ﬂight mass spectrometry with postsource decay conducted at the University of Massachusetts Medical School or liquid chromatography/liquid chromatography–electrospray ionization mass spectrometry performed at the Harvard Microchemistry and Proteomics Facility. Comparisons of pep- tide masses with translated genomic or EST sequences were made using the BLAST algorithm at the National Center for Biotechnology Information and the Chlamydomonas genome database (version 3.0; http://genome .jgi-psf.org/Chlre3/Chlre3.home.html). Searches of the ﬂagellar pro- teome (Pazour et al., 2005) were performed using http://labs.umassmed .edu/chlamyfp/index.php. 524 JCB • VOLUME 179 • NUMBER 3 • 2007 The coding sequence for each polypeptide was conﬁrmed by RT-PCR. RNA was isolated from wild-type C. reinhardtii cells before and after pH shock–induced deﬂagellation according to Wilkerson et al. (1994). PolyA enrichment was performed using the Oligotex mRNA kit (QIAGEN) according to the manufacturer’s instructions. Reverse transcription was per- formed using either Superscript II or III (Invitrogen) according to the manu- facturer’s instructions. Subsequent PCRs were performed using primers based on the known or predicted coding regions as determined by EST, EST contig, or mass spectrometry data. Peptide and antibody production The CaM peptide and corresponding antibodies as well as the anti-C1a32 antibodies were generated as described previously (Wargo et al., 2005). For CaM-IP2, the ﬁrst 680 bp of the coding sequence was cloned into the pET30 vector and transformed into BL21 (DE3) pLysS cells. Protein ex- pression was induced by the addition of IPTG, and the expressed protein was puriﬁed from bacterial cell lysates using a Ni2+-resin column accord- ing to the manufacturer’s protocol (Novagen). For CaM-IP3 antibodies, a C-terminal peptide (CZTHAQDAVLEFARAHAAE) was synthesized and con- jugated to keyhole limpet hemocyanin (Aves Labs, Inc.). For both CaM-IP2 and -IP3, polyclonal antibodies were generated in rabbits against the puri- ﬁed protein or peptide at Spring Valley Laboratories. Antibodies were afﬁnity puriﬁed on Sulfolink columns (Pierce Chemical Co.) according to the manufacturer’s protocol. Columns contained puriﬁed expressed protein (CaM-IP2) or puriﬁed peptide (CaM-IP3). Gel electrophoresis, Western blots, and silver-stained gels For immunoblots, equivalent loads of ﬂagella (8 mg/ml), axonemes, ex- tracts, and/or extracted axonemes were subjected to SDS-PAGE using 7% polyacrylamide gels. Gels were transferred to polyvinylidene diﬂuoride (Immobilon P; Millipore). CaM Western blots were performed as previ- ously described (Wargo et al., 2005). For CaM-IP2 and -IP3 Western anal- yses, 7% polyacrylamide gels were subject to SDS-PAGE and transferred for 1 h to polyvinylidene diﬂuoride (Immobilon P; Millipore). Membranes were blocked for 1 h in 2% BSA (regular fraction V; A-7906; Sigma- Aldrich) in TBST (0.1% Tween and TBS, pH 7.5). For primary antibody incubations, afﬁnity-puriﬁed anti–CaM-IP2 or anti–CaM-IP3 antibodies were diluted 1:100 or 1:5,000, respectively, and anti-RSP2 or anti-RSP3 antibodies (provided by D. Diener, Yale University, New Haven, CT) were diluted 1:5,000 in TBST. Primary antibody incubations were conducted for 2 h at RT or overnight at 4°C. Membranes were washed three times for 5 min with TBST, incubated with anti–rabbit HRP secondary antibodies (GE Healthcare), and diluted 1:30,000 in TBST. After four 5-min washes with TBST, the ECL Plus Western Blotting kit (GE Healthcare) was used for chemiluminescent detection. Gels were silver stained according to the methods described in Wargo et al. (2005). For anti-IC138 Western blots, axonemes isolated from wild type, pf14, and pf18 were resuspended at 1 mg/ml in pCa8 or pCa4 buffer containing 1 mM ATP. For some samples, 50 μg of axonemes were treated with 4 μg of afﬁnity pure anti–CaM-IP2 or 4 μg of afﬁnity pure anti–C1a- 32 antibodies (Wargo et al., 2005). For samples that were treated with calf intestine alkaline phosphatase (Roche), the axonemes were incubated with 0.75 U of calf intestine alkaline phosphatase for 30 min at RT. 10 μg axonemal proteins were resolved on a 5% polyacrylamide gel and trans- ferred to nitrocellulose. Blots were incubated with anti-IC138 (1:10,000 in TBST) overnight at 4°C. After washing, blots were incubated for 45 min with anti–rabbit HRP in TBST, washed, and incubated with ECL Plus for detection. The IC138 antibodies were provided by W. Sale. Blot overlay RSP3-GST (provided by W. Sale) was expressed and puriﬁed on a GST- resin column (Novagen) according to the manufacturer’s protocol. The N terminus of CaM-IP2 was expressed and puriﬁed as described in Peptide and antibody production. For CaM-IP3, DNA sequence encoding the C-terminal 220 amino acids was cloned into pET30 and transformed into BL21 (DE3) pLysS cells. For CaM-IP4, 1.8 kb of DNA sequence encoding amino acids 1–580 was cloned into the pET30 vector. Expression was induced with IPTG, and proteins were puriﬁed from bacterial cell lysates using a Ni2+-resin column (Novagen). For SDS-PAGE, 15 μg CaM-IP2 (37 kD), 15 μg CaM-IP3 (32 kD), 15 μg CaM-IP4 (62 kD), and 25 μg RSP3-GST (110 kD) were resolved on a 12% polyacrylamide gel and trans- ferred for 40 min to nitrocellulose membrane. Membranes were blocked overnight at 4°C in 5% milk/TBST (0.1% Tween) and were incubated with 40 μg/ml RSP3-GST in 1% BSA/TBST for 2 h at RT. After three 5-min TBST washes, membranes were incubated with anti-RSP3 (1:5,000) in TBST for 1 h and with secondary antibody (anti–rabbit HRP; GE Healthcare) diluted