Flagellar CrPKD2 is cleaved. (A) Diagram of the CrPKD2 protein showing the regions used to generate the Loop1 and N- and C-terminal antibodies. The arrow marks the approximate location of the CrPKD2 cleavage site that generates fragments of 120 and 90 kD. (B) Immunoblots from whole cells (Cell) and flagella (F) of wild-type cells were probed with antibodies against Loop1 or the N or C termini of CrPKD2. The Loop1 antibody reacts with three bands (210, 120, and 90 kD; lane 1) in whole cells, but only the smaller two bands are present in flagella (lane 2). The C-terminal antibody does not react with the 120-kD N-terminal fragment (lanes 3 and 4), demonstrating that this fragment does not include the C terminus of the protein. The N-terminal antibody reacts with two bands (210 and 120 kD) in whole cells and the 120-kD form in the flagella (lanes 5 and 6). Thus, full-length CrPKD2 is cleaved into two fragments (120 and 90 kD) that are present in flagella. (C) Immunoblot of transformant expressing CrPKD2–GFP. GFP-tagging results in two bands that react with the GFP antibody (lane 1): a 240-kD band (the 210-kD full-length PKD2 + 30 kD GFP) and a 120-kD band (the 90-kD C-terminal fragment + 30 kD GFP). Only the smaller band is present in flagella (lane 2). Both bands also react with the C-PKD2 antibody (lane 3), as do the untagged CrPKD2 bands also present in these cells. (D) Immunoblots of bld1 and bld2 cells (mutants that have no flagella) and cell bodies of wild-type cells (WT CB) probed with the anti–C-PKD2 antibody. The flagellar form of CrPKD2 is present in the cell bodies, indicating that cleavage occurs in the cell body. The same blot was probed with α-tubulin as a loading control.

CrPKD2 is concentrated in flagellar membranes. (A) Electron micrographs of thin sections of isolated flagellar membrane vesicles. Flagellar membranes were isolated in 0.1% NP-40 and were sedimented (top) or further purified on an Optiprep gradient (bottom). (B) Immunoblots of the proteins (2.3 μg) in flagella (F) and vesicles from A purified without (1) or with (2) an Optiprep gradient were probed for an axonemal protein radial spoke protein 1, FMG-1, Loop1 PKD2, IFT 139 (IFT complex A), and IFT172 (IFT complex B). Black lines indicate that intervening lanes have been spliced out. (C) Coomassie blue–stained gel (CB) of equal protein loadings (11 μg) of flagellar fractions and immunoblots probed with FMG-1 and Loop1 PKD2 antibodies. FMG-1 and CrPKD2 are concentrated in flagellar membrane fractions. Some CrPKD2 remains associated with the axoneme even though the FMG-1 is released from the axoneme completely. F, flagella; S, flagellar proteins solubilized by freeze–thaw; A, axoneme extracted with 0.1 or 1% NP-40; M, corresponding flagellar membrane. (D) Electron micrograph of axonemes extracted with 1% NP-40 from Fig. 4 C. Bars: (A) 50 nm; (D) 500 nm.

CrPKD2–GFP FRAP is reduced in the flagella of fla10 cells at restrictive temperature. (A) An ∼2-μm segment of a fla10 flagellum containing CrPKD2–GFP is shown (box in DIC panel) before bleaching, after bleaching, and after 2 min of recovery. The rest of the flagellum is not visible because it is not illuminated. (B) The fluorescence in the bleached area increased in the flagellum of fla10 cells at nonrestrictive temperature after 1 and 2 min after photobleaching. In fla10 cells at restrictive temperature, recovery only reached 9.4 versus 19.3% at permissive temperature. (C) In control (pf18) cells at 22 and 32°C, fluorescence recovery is similar to that of fla10 cells at room temperature. Bar graphs represent mean ± SEM (statistics determined by t test).

Diagram of the mating process in C. reinhardtii. (A) When starved for nitrogen in the light, mt+ and − vegetative cells differentiate into gametes. When mixed, the gametes adhere by their flagella and become activated, resulting in the loss of their cell wall, assembly of mating structures (visible in mt+ gametes), and cell fusion to form a dikaryon. The two nuclei fuse in the dikaryon, producing the diploid zygote. (B) Flagellar adhesion initiates a signal cascade involving a calcium influx and an increase in cAMP in the cell body. IFT is important for the first steps of this pathway after flagellar adhesion.

CrPKD2 is a member of the TRPP2 family. (A) Secondary structure of CrPKD2. CrPKD2 contains a coiled-coil domain at the N and C termini, an EF hand domain, six transmembrane domains (numbers denote amino acid positions), and acidic amino acid cluster domains. The pore region (P) is shown, between aa 1,342–1,377, by comparison to human and D. melanogaster PKD2 (Tsiokas et al., 1999; Venglarik et al., 2004). Not drawn to scale. (B) The EF hand domain of CrPKD2 contains the six conserved residues (coordination vertices) involved in calcium binding (positions 1, 3, 5, 7, 9, and 12).

Flagellar CrPKD2 increases during gametogenesis. (A) C. reinhardtii vegetative cells (V) and gametes (G) stained with N-PKD2 or C-PKD2 (green) and acetylated α-tubulin (red) antibodies. Bars, 5 μm. (B) 10 μg of flagellar protein from CC125 (mt+) vegetative cells and cells undergoing gametic differentiation in nitrogen-deficient medium was probed on immunoblots with antibodies against the Loop1 PKD2 epitope and an intermediate chain of outer arm dynein, IC2, as a loading control. The amount of CrPKD2 in the flagella increased during the course of gametic differentiation.

Some of the CrPKD2–GFP moves in the flagellar membrane. (A) Fluorescence micrograph of the flagellum used to generate the kymographs of CrPKD2–GFP. Only one flagellum was illuminated. The cell body is outlined with a dotted line. (B) Kymograph of CrPKD2–GFP generated using the video (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200704069/DC1). (C) The lines corresponding to those seen in the kymograph were used to measure the anterograde velocity of CrPKD2–GFP. (D) Two CrPKD2–GFP particles are shown (above the solid line) moving in the anterograde direction at ∼1.6 μm/s. The dotted lines mark particles that did not move.

Flagellar CrPKD2 increases when IFT is blocked. (A) Wild-type and fla10 cells were shifted to 32° for 1 or 1.5 h, and immunoblots of isolated flagella were probed with antibodies as listed. Note the rise in CrPKD2 when IFT proteins disappear in flagella from fla10 cells. (B) Cells were treated with 20 mM NaPPi to induce flagellar resorption, and flagellar proteins were probed on immunoblots as in A. CrPKD2 did not increase in these flagella during resorption under this condition.

CrPKD2 is involved in mating. (A) The construct used for RNAi included 1,000 bp of the CrPKD2 cDNA (5′ UTR and first two exons) appended antisense to the corresponding genomic fragment, driven by the native promoter. AphVIII is a selectable marker gene driven by the HSP70A-RbcS2 fusion promoter (Sizova et al., 2001). (B) Immunoblot analysis shows a reduced amount of CrPKD2 in RNAi transformants Ri45 and Ri46 compared with wild-type cells. An HSP70B antibody was used as a loading control (Schroda et al., 1999). (C) Histogram showing the decrease in mating efficiency that occurs in RNAi transformants with reduced levels of CrPKD2. Bars represent the mean ± SEM from three independent experiments. (D) Protein tyrosine kinase activity was assayed in vitro in flagellar proteins isolated from mated gametes. Samples were incubated for 30 min and analyzed on immunoblots probed with antibodies against the phosphotyrosine residue of CrPKG (α-pTyr) or α-tubulin as loading control. Phosphorylation of CrPKG is reduced in flagellar extracts of gametes of the RNAi46 strain.