Figure 4. Binding of CaM-IP2, -IP3, and -IP4 to CaM is calcium sensitive. (A) Silver-stained gel of CaM immunoprecipitation from radial spokeless axonemal extracts (pf14) followed by treatment with CaCl2 (top). After pre- cipitation (CaM-IP), the protein A beads were washed twice with 2 mM CaCl2 (Ca2+1 and Ca2+2), and the resulting extract was loaded onto the gel. Proteins remaining (lane R) associated with the beads after the CaCl2 wash were eluted with sample buffer. CaM-IP2, -IP3, and -IP4 (asterisks) are extracted from the beads. In contrast, the central pair proteins (dots) are not extracted with CaCl2. Western blots using anti-CaM antibodies (bottom) reveal that CaCl2 extracts very little CaM from the beads. These results indicate that binding of CaM-IP2, -IP3, and -IP4 to CaM is calcium sensitive. (B) Silver-stained gel of immunoprecipitation experiments per- formed in 1 mM CaCl2 using anti–CaM-IP2 antibodies and wild-type, spokeless (pf14), and central pairless (pf18) axonemal extracts. The CaM- IP2 antibodies precipitate the CaM-IP2, -IP3, and -IP4 complex (asterisks) and spokes (triangles), indicating that they do not dissociate from each other or the spokes in high calcium buffer conditions. The association of CaM-IP2, -IP3, and -IP4 with CaM is altered in DRC mutants Our data are consistent with the localization of a CaM-containing complex at the base of the spokes. Previous studies have indi- cated that the components of the dynein regulatory complex (DRC) localize to the doublet microtubules near the spokes (Piperno et al., 1992; Gardner et al., 1994; Nicastro et al., 2006). Therefore, we hypothesized that the CaM-IP2, -IP3, and -IP4 complex may be disrupted in mutants lacking subsets of DRC components. To investigate this possibility, we precipitated the complex from axonemal extracts isolated from DRC mutants (pf2 and pf3) using either our anti-CaM or anti–CaM-IP2 anti- bodies. The anti-CaM antibodies precipitate substantially less CaM-IP2, -IP3, and -IP4 from pf2 and pf3 axonemal extracts compared with that precipitated from wild-type extracts even though axonemes isolated from these mutants appear to have wild-type levels of CaM as judged by Western blotting (Fig. 5). These results suggest that the assembly of CaM-IP2, -IP3, and -IP4 is defective in DRC mutants. However, when we precipitate the complex using our anti–CaM-IP2 antibodies, the precipi- tates from pf2 and pf3 are indistinguishable from that of wild type (Fig. 5). These results indicate that CaM-IP2, -IP3, and -IP4 assemble normally in DRC mutants but that their associa- tion with CaM is disrupted. Anti–CaM-IP2 antibodies restore dynein activity to mutant axonemes Localization of the CaM-IP2, -IP3, and -IP4 complex at the base of the radial spokes, potentially near DRC components, suggested a possible role in modulating dynein-driven micro- tubule sliding. Based on this localization, we hypothesized that regulation of dynein activity by this complex would occur downstream of regulatory signals derived from the radial spokes or central apparatus. To test this hypothesis, we conducted the microtubule sliding assay in the presence of the anti–CaM-IP2 antibody. We initially tested whether the antibodies possessed any function-blocking activity using wild-type axonemes and a microtubule sliding assay (see Materials and methods). The anti–CaM-IP2 antibodies had no effect on microtubule sliding velocity (Fig. 6 A). We then examined dynein activity in mutant axonemes. The first mutant tested was the central pairless strain pf18; axonemes from this mutant have reduced dynein activity com- pared with wild-type (Smith, 2002b). To our surprise, the anti– CaM-IP2 antibody significantly increases microtubule sliding velocity (P < 0.001 by t test; Fig. 6 A). In addition, the increase in velocity in central pairless axonemes is dependent on the concentration of the antibody, with maximal effect at 0.2 μM CaM-IP2 antibody (Fig. 6 B). Importantly, the increase in dy- nein activity is not observed upon the addition of anti–CaM-IP3 or anti-C1a32 antibodies (Wargo et al., 2005), further indicating that the effect is specific for binding of the anti–CaM-IP2 anti- body. Based on these results and the hypothesis that the com- plex affects dynein activity downstream of regulatory cues from the radial spokes, we predicted that addition of our antibodies to radial spokeless axonemes would also increase dynein activity. Indeed, addition of the CaM-IP2 antibodies to radial spokeless axonemes (pf14) significantly increases dynein activity (P < 0.001 by t test; Fig. 6 D). The simplest interpretation of these results is that the CaM-IP2, -IP3, and -IP4 complex is involved in modulating the activity of the dynein arms. To determine whether specific subforms of axonemal dynein are the targets for modulation by this complex, we re- peated the sliding microtubule experiments using mutants that lack specific dynein subforms. The CaM-IP2 antibodies have no effect on the sliding velocities of a mutant lacking the outer dynein arms (pf28; Fig. 6 C). Double mutants lacking both the REGULATION OF DYNEIN ACTIVITY • DYMEK AND SMITH 519