Figure 6. CaM-IP2 modulates dynein-driven microtubule sliding. (A) Microtubule sliding veloc- ities of wild-type and pf18 axonemes. The CaM- IP2 (IP2) antibodies have no effect on the sliding velocity of wild-type axonemes. In contrast, slid- ing velocities of pf18 axonemes increase upon addition of the CaM-IP2 antibody. This increase is not observed in the presence of C1a-32 (C1a; Wargo et al., 2005) or CaM-IP3 (IP3) antibodies. (B) Microtubule sliding velocities of pf18 axo- nemes incubated with varying concentrations of CaM-IP2 antibody. The increase in dynein activity is dose dependent, with a maximal in- crease in velocity at 0.2 μM CaM-IP2 antibody. (C) Microtubule sliding velocities of mutants lack- ing the outer dynein arms (pf28), outer dynein arms and the central apparatus (pf18pf28), and outer dynein arms and spokes (pf14pf28) in the presence (IP2; patterned bars) and absence (black bars) of the CaM-IP2 antibody. (D) Slid- ing velocities of a mutant lacking the central pair and I2 inner arm heavy chains (pf18ida4) increase upon the addition of anti–CaM-IP2 antibodies. Velocities of a mutant lacking the central apparatus and the I1 inner dynein arm (pf18ida1) are signiﬁcantly higher than veloci- ties of pf18 (P < 0.001 by t test). However, slid- ing velocities of pf18ida1 axonemes incubated with CaM-IP2 antibodies are signiﬁcantly lower than those of pf18 incubated with CaM-IP2 anti- bodies (P < 0.001 by t test). Sliding velocities of a radial spokeless mutant (pf14) increase upon the addition of anti–CaM-IP2 antibodies (IP2). However, the addition of CaM-IP2 antibodies to axonemes that lack the radial spokes and inner dynein arm I1 (pf14pf30) have no effect on slid- ing velocity. All bars represent the mean of >70 measurements ± SEM (error bars) from three or more independent experiments. (E) Summary of microtubule sliding experiments. The CaM-IP2, -IP3, and -IP4 complex (purple) is localized to the base of the spoke (blue). the base of the radial spokes, and plays an important role in modulating dynein-driven microtubule sliding. This discovery represents an important step toward defining a molecular mech- anism for control of ciliary and flagellar motility. The identities of CaM-IP2, -IP3, and -IP4 suggest a role in regulating motility Three polypeptides precipitated by anti-CaM antibodies form a distinct complex with CaM and were tentatively named CaM-IP2, -IP3, and -IP4. Based on the amino acid identities of these polypeptides and searches of the C. reinhardtii f lagellar proteome, we determined that CaM-IP2, CaM-IP3, and CaM- IP4 correspond to FAP91, FAP61, and FAP251, respectively (Pazour et al., 2005). Potential mammalian homologues for each of these proteins were found in searches of sequence databases, indicating that this complex is not unique to C. reinhardtii. Most notably, CaM-IP2 (FAP91) is most similar to AAT-1, a protein originally identified as a testis-specific protein that interacts with AMY-1 (a c-myc–binding protein), an AKAP, and two regulatory (RII) subunits of PKA (cAMP-dependent protein kinase; Yukitake et al., 2002). Based on gel overlay assays, we have shown that CaM-IP2 binds to an AKAP (RSP3; Gaillard et al., 2001); however, none of the members of this complex ap- pear to be AMY-1 or RII homologues. AAT-1α, a small splice variant of AAT-1, has been localized to mature sperm and ap- pears concentrated in the sperm midpiece region (Matsuda et al., 2005). Matsuda et al. (2005) suggest that AAT-1α may be asso- ciated with mitochondria, but the precise localization of AAT-1α within sperm has not yet been determined. In addition to AAT-1α, Matsuda et al. (2005) report the presence of several splice variants of AAT-1 expressed in vari- ous tissues, some of which have cells that possess cilia/flagella and some of which do not. The authors propose that AAT-1 may participate in additional cellular functions as well as sperm development. Northern blots of RNA isolated from C. reinhardtii cells before and after deflagellation reveal that C. reinhardtii REGULATION OF DYNEIN ACTIVITY • DYMEK AND SMITH 521