CSC amiRNA mutants are independent isolates with reduced expression of both CaM-IP2 and CaM-IP3. (A) Southern blot of 5 μg of genomic DNA, digested with KpnI, probed with the APHVIII gene. Each amiRNA mutant has a different RFLP, indicating that the strains are independent isolates and that the amiRNA plasmid most likely inserted at different sites. (B) Relative transcript levels of wild-type and CSC amiRNA mutants. Transcript abundance was quantified by densitometry of Northern blot phosphorimages using Image J. S14 levels were used as a loading control, and wild-type levels were set to 100%. For CaM-IP2 amiRNA mutants, CaM-IP2 transcript levels (red) were ∼30–40% of wild-type levels. For CaM-IP3 amiRNA mutants, CaM-IP3 transcript levels (blue) were ∼30–50% of wild-type levels. Note that in CaM-IP2 amiRNA mutants, the CaM-IP3 transcript levels are also reduced, as well as the CaM-IP2 transcript levels in CaM-IP3 amiRNA mutants. RSP3 transcript levels (black) for all strains were near 100%.

CSC amiRNA mutants have reduced assembly and/or stability of the radial spokes. (A) Electron micrographs of cross- and longitudinal sections of chemically fixed axonemes isolated from CSC amiRNA mutants. Cross-sections show various numbers of doublet microtubules, which lack the radial spokes (red dots). In longitudinal sections, we observe both doublets with repeating pairs of radial spokes, as in wild type, and stretches of regularly repeating, single radial spokes (red arrows). Occasionally, a triplet of radial spokes was also observed (green arrowheads in 4A2 panel). (B) Histogram of the number of radial spokes missing in cross-sections of axonemes (n > 100 for each strain); ∼80% of cross-sections of wild-type axonemes (black bar) have spokes present on all nine doublets. In contrast, only ∼30% of cross-section for CSC amiRNA mutant sections have spokes present on all doublet microtubules. The majority of cross-sections for CSC amiRNA mutants have between one and five doublets that lack spokes. (C) Histogram indicating the specific doublet that lacked radial spokes in cross-sections (n > 20 for each strain). Doublet identity was determined by specific axonemal markers according to that established in Hoops and Witman (1983). In all strains, the distribution of doublets lacking radial spokes appeared random.

CSC amiRNA mutant cells have significantly slower swimming velocities than do wild-type cells; yet flagellar beat frequencies are nearly wild type. Swimming velocity (A) and beat frequency (B) were measured from swimming cells using high-speed video microscopy.

Reconstitution of the CSC onto CSC amiRNA mutant axonemes rescues high calcium–induced dynein activity. (A) Microtubule sliding velocity of 6E6pf18 and 4A2pf18 mutant axonemes. All sliding experiments represent the result of three to five independent experiments with a total n for each mutant and a condition of between 60 and 160. Sliding was measured under low (pCa8) or high calcium (pCa4, “+Ca”) conditions. For both double mutants, no increase in dynein activity is observed in pCa4 buffer. When the CSC is reconstituted (+CSC), a significant increase (p < 0.001, by Student's t test) in dynein activity is observed in pCa4 buffer. (B) Western blot of CSC reconstituted onto mutant axonemes. KI (after NaCl) extracts (E) isolated from pf14 axonemes were added to 6E6pf18 axonemes (A) at various ratios of equivalent protein (see Materials and Methods). Axonemes were then pelleted, and the supernatant and pellet were analyzed by Western blot using anti–CaM-IP2 antibodies. The first four lanes to the left are controls using either extract or axonemes alone mixed with buffer (B). When extracts and axonemes are mixed at a ratio of 1:1, all of the CaM-IP2 binds to 6E6pf18 axonemes. As the ratio of extract to axoneme is increased, CaM-IP2 is observed in the supernatant, indicating that binding of CaM-IP2 saturates.

The flagella of CSC amiRNA mutants have reduced amounts of both CaM-IP2 and CaM-IP3. Western blots of flagella (8 μg) isolated from wild-type and CSC amiRNA mutants were probed with antibodies that recognize the CSC (CaM-IP2 and CaM–IP3), radial spokes (RSP1), inner dynein arm I1 (IC138), and central apparatus (PF20). CaM-IP2 amiRNA mutants 4A2 and 4D6 and the CaM-IP3 amiRNA mutants 6E6 and 1A3 have reduced amounts of both CaM-IP2 and CaM-IP3. 6E6 is the most efficient knockdown; no CaM-IP3 or CaM-IP2 is detectable by Western analysis. RSP1, IC138, and PF20 are present at near wild-type levels in each amiRNA mutant.

For all CSC amiRNA mutants, the remaining CSC subunits associate with the radial spokes. (A) Silver-stained gels of immunoprecipitates using anti-CaM antibodies and axonemal extracts. As previously reported (Dymek and Smith, 2007), anti-CaM antibodies precipitate central pair (circles), radial spoke (squares), and CSC proteins (labeled) from wild-type extracts. Only central pair and CSC proteins precipitate from radial spokeless extracts (pf14). In CSC amiRNA mutants only small quantities of CSC and radial spoke proteins are precipitated from axonemal extracts compared with wild type; in contrast, the amount of central pair proteins precipitated is the same as in wild type. For strain 6E6, which is the most efficient knockdown, only central pair proteins precipitate. Heavy chain (HC) from the precipitating antibody is present at 50 kDa. (B) Silver-stained gels of immunoprecipitates using anti-CaM-IP2 antibodies and axonemal extracts. As previously reported (Dymek and Smith, 2007), anti-CaM-IP2 antibodies precipitate CSC members and radial spoke proteins from wild-type extracts. Only the CSC is precipitated in pf14 extracts. Little if any protein precipitates from strain 6E6 axonemal extracts. For strains 4A2, 4D6, and 1A3, small amounts of CSC and radial spokes precipitate, indicating that the remaining CSC in the amiRNA mutants is associated with the radial spokes.

Radial spoke 2 is the missing spoke in CSC amiRNA mutant axonemes. (A–H) Slices through cryo-electron tomograms of axonemes from wild-type (WT; A and B) and amiRNA mutants 4A2 (C and D), 4D6 (E and F), and 6E6 (G and H), showing the three-dimensional (3D) structure in cross-sectional (A, C, E, and G) and longitudinal views (B, D, F, and H). By identifying A- and B-tubules (A_t, B_t) in cross-sectional slices, the proximal (prox) and distal (dist) end of the axoneme can be determined and thus radial spokes 1 and 2 (RS1/blue; RS2/yellow) assigned unambiguously. All three CSC amiRNA mutants show many axonemal repeat units with missing spoke 2 (red dots). Occasionally, repeat units have additional spokes (green) at locations different from the normal RS1 or RS2 position. No defects of RS1 were observed. Scale bars: 50 nm (G), 100 nm (H). (I) Histogram showing the analysis of 675 repeat units from six tomograms of 6E6 axonemes; approximately half of the repeat units were normal (yellow bar), one quarter were missing RS2 partly or completely (red bar), and the remaining repeats had either an additional spoke (green bar) or were too ambiguous to score (purple bar). (J and K) Graphical model and 3D visualization of a cryo-tomogram of a 6E6 axoneme, showing the 3D architecture of the axoneme and location of the radial spokes. The modeled microtubules (gray), spokes 1 (blue) and 2 (yellow), and occasional additional spokes (green) are shown both as partial overlaid on tomographic slices (J) or alone (K); transparent colors indicate ambiguous spokes (see also Supplemental Video 1).

All CSC amiRNA mutants exhibit defects in flagellar beating. (A) Montages of images from digitally recorded videos of wild-type, 4A2, and 6E6 cells. Wild-type cells swim forward with an asymmetric waveform, and the flagella are in sync through the entire beat. CSC amiRNA mutant cells swim forward with a few beats in sync, and then their flagella lose synchrony. (B) Traced diagrams of flagellar beats from wild-type, 4A2, 4D6, 6E6, and 1A3 cells. Colors correspond to traces taken from the same frame (see colored time sequence). Wild-type flagella complete a full asymmetric beat with flagella in sync. The two flagella for the CSC amiRNA strains are out of sync and do not exhibit complete recovery and effective strokes.