complex might predictably include defects in radial spoke assembly. In fact, CaM-IP3 and -IP4 map to linkage group III near the pf5 mutation, and the pf5 mutant is defective for radial spoke assembly (Huang et al., 1981). We have sequenced the genes encoding CaM-IP3 and -IP4 from genomic DNA iso- lated from the pf5 mutant and found no mutations in either gene (unpublished data). In addition, we have screened several avail- able insertional mutant libraries for mutations in the members of this complex and found no candidate mutants. Finally, we have made numerous attempts at knocking down expression levels by RNA-mediated interference for both CaM-IP2 and -IP3 and have not obtained a stable mutant with reduced transcript levels for either gene. Therefore, the mutant phenotype for any mem- ber of this complex remains unknown. The CSC regulates dynein-driven microtubule sliding Using a microtubule sliding assay, we have demonstrated that the CSC plays a role in regulating dynein-driven microtubule sliding. This conclusion is based on the observation that the ad- dition of antibodies raised against CaM-IP2 specifically restores dynein activity to axonemes that lack either the central apparatus or the radial spokes. Importantly, the increase in dynein activity is not observed upon the addition of antibodies raised against CaM-IP3 or C1a32, and the CaM-IP2 antibody effect is satu- rable. Therefore, the increase in dynein activity is not caused by particular buffer or rabbit serum components that may remain after affinity purification; rather, the increase in dynein activity is specific for binding of the anti–CaM-IP2 antibody. The challenge now is to determine the mechanism by which the CSC regulates dynein activity. One possibility is that the CSC directly mediates regulatory signals from the radial spokes to the dynein arms. A second possibility is that the CSC mediates regulatory signals via the DRC. The DRC is com- prised of seven polypeptides that were discovered through ge- netic and biochemical analyses of mutants that, when combined with central apparatus or radial spoke defect mutations, restored motility without restoring the missing structures (Huang et al., 1982; Piperno et al., 1992). The polypeptides comprising the DRC have been localized near the base of the spokes (Gardner et al., 1994; Nicastro et al., 2006). However, little is know about their identities or their function in regulating dynein (Rupp and Porter, 2003). In these experiments, we have discovered that the two mutants that define the DRC, pf2 and pf3, assemble wild-type levels of CSC components. However, only a small fraction of the CSC is associated with CaM. Our working hypothesis is that CaM association or dissociation with the CSC plays a role in modulating dynein activity. To determine whether the CSC in- teracts with DRC components or specific subunits of dynein arms will require the generation of additional reagents targeted to these polypeptides. Regardless of the mechanism for CSC modulation of dy- nein activity, our data indicate that specific dynein subforms are affected (summarized in Fig. 6 E). Our antibodies either fail to increase dynein activity or only modestly increase dynein ac- tivity in mutants lacking the outer dynein arms. In addition, our antibodies do not increase dynein activity in double mutants lacking the I1 inner dynein arm. In contrast, the CaM-IP2 antibodies fully restore dynein activity to mutants that lack the I2 (fraction a, c, and d) inner arm heavy chains. Collectively, these results indicate that the I1 inner dynein arm as well as the outer dynein arms may be targets of regulation by the CSC. In light of these results, it is interesting to note that in recent structural experiments, Nicastro et al. (2006) have ob- served a structural linkage between the inner dynein arm I1 and the outer dynein arms. In addition, work from several labora- tories have indicated that inner dynein arm I1 is an important regulatory target for modulating dynein-driven microtubule sliding and that this modulation includes phosphorylation of the I1 IC138 intermediate chain (Habermacher and Sale, 1996, 1997; King and Dutcher, 1997; Yang and Sale, 2000; Hendrickson et al., 2004). Some I1-defective mutants can act as weak sup- pressors of paralysis for particular central apparatus–defective mutants (Porter et al., 1992). Importantly, some I1-defective mutants are deficient in their phototaxis response (King and Dutcher, 1997; Okita et al., 2005). Our results indicate that the overall phosphorylation state of IC138 does not change in either radial spokeless or central pairless mutants upon addition of the CaM-IP2 antibodies. Therefore, the increase in dynein activ- ity we observe upon addition of our antibodies may involve a mechanism that does not alter IC138 phosphorylation. Alter- natively, addition of these antibodies may ultimately result in changes in the phosphorylation of specific IC138 residues that we cannot detect by Western blotting. An additional challenge is to determine what role the binding of Ca2+ to CaM plays in CSC-mediated changes in dynein activity. We have previously shown that high calcium buffer increases dynein activity in central pairless mutants but not spokeless mutants and that CaM is a key calcium sensor (Smith, 2002a). In the present study, we report the discovery of a CaM-containing complex that exhibits calcium-sensitive CaM binding. What we do not know is whether Ca2+-CaM release from the CSC accounts for the increased dynein activity of central pairless mutants in high calcium conditions. Interestingly, anti- bodies generated against one member of this complex increase dynein activity in both central pairless and spokeless axonemes in low calcium conditions. Is there a functional relationship be- tween antibody binding to the CSC and high calcium condi- tions? Our observations that the CaM-IP2 antibody increases dynein activity in mutants in which high calcium has no effect (pf14) suggests that the antibody acts downstream of high cal- cium in a signal transduction pathway. A second possibility is that high calcium affects additional axonemal targets that are missing in these mutants, and these possibilities are not mutu- ally exclusive. For example, CaM is a component of both the central apparatus (Wargo et al., 2005) and radial spokes (Yang et al., 2001), and several calcium-binding proteins, including centrin, are associated with the dynein arms (for a complete list of both known and putative calcium-binding proteins in flagella, see Pazour et al., 2005). To fully elucidate the mechanism by which the CSC par- ticipates in signal transduction pathways that modulate dynein activity will require precisely localizing the CSC relative to the REGULATION OF DYNEIN ACTIVITY • DYMEK AND SMITH 523