The Inner Dynein Arms 12 Interact with a "Dynein Regulatory Complex" in Chlamydomonas Flagella

We provide indirect evidence that six axonemal proteins here referred to as "dynein regulatory complex" (drc) are located in close proximity with the inner dynein arms 12 and 13. Subsets of drc subunits are missing from five second-site suppressors, pf2, p f3, suppI3, suD:4, and suppz 5, that restore flagellar motility but not radial spoke structure of radial spoke mutants. The absence of drc components is correlated with a deficiency of all four heavy chains of inner arms I2 and 13 from axonemes of suppressors pf2, pf3, suppi3, and supp:5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, p f3, and supp:5. Recombinant strains carrying one of the mutations p f2 , p f3, or suppy5 and the inner arm mutation ida4 are more defective for I2 inner arm heavy chains than the parent strains. This evidence indicates that at least one subunit of the drc affects the assembly of and interacts with the inner arms I2. T HE movement of Chlamydomonas flagella is generated by at least six types of dynein including the outer dynein arm, the inner dynein arm I1, and two types of inner dynein 12 and I3 (25). Outer dynein arm and inner dynein arm I1 are formed by three and two distinct heavy chains, respectively, and consist of the same subunits along the axoneme (14, 27). In contrast, the inner dynein 12 and 13 are each formed by two identical heavy chains and consist of different heavy chains depending on their location along the axoneme (25). Each dynein also comprises a distinct set of intermediate and light chains characterized by molecular weight ranging from 140,000 to 8,000 (27, 35). Two light chains, which are associated with inner arms I2 and I3, were referred to as actin and Caltractin/centrin, respectively, on the basis of physical and chemical properties of the two proteins (13, 22, 29). The role of actin and caltractin/centrin in other cytoskeletal structures is to transmit tensile stress and provide Ca++-dependent regulatory mechanisms, respectively (28, 30). In contrast, the function of the same proteins within the inner arm structure has not been identified. To investigate a possible function of actin and caltractin/centrin in the regulation of inner arm movement, we intended to determine whether the ATPase activity or stability of inner arms 12 and I3 is Ca ++ sensitive in vitro. Moreover, we aimed to identify other axonemal proteins that interact with actin and caltractin/centrin in vivo. To reach these goals we analyzed complexes formed by actin, caltractin/centrin, and 12 and 13 inner arm heavy chains that were isolated from the outer dynein mutant pf28 (18). We also quantitatively analyzed 12 and 13 inner arm deficiency in inner arm defective mutants (9, 16) and suppressors of flagellar paralysis that generate bending of flagella similar to those of inner arm mutants (3, 11). These second-site suppressors are missing different subsets of six axonemal polypeptides and suppress flagellar paralysis of radial spoke mutants without restoring the radial spoke structure (11). We found that the suppressors are defective for 12 and I3 inner arm subunits to different extents. We also found that the polypeptides missing from the suppressors interact at least with inner arms I2 in wild-type strains. Therefore, we referred to these polypeptides as "dynein regulatory complex" (drc). z The absence of drc components combined with the deficiency of inner arms 12 generate an extensive deficiency of 12 inner arms in recombinant strains carrying both suppressor and inner arm mutations. This and other evidence described here suggests that actin and caltractin/centrin are associated with I2 and I3 inner arms, probably forming a linkage between the drc and each of I2 and 13 inner arms. Materials and Methods Strains and Culture of Chlamydomonas Cells Chlamydomonas strains were obtained from the Chlamydomonas Genetics Center (Duke University, Durham, NC) and the laboratory of David Luck (Rockefeller University, New York). Nomenclature and phenotype of the strains used for analysis of axonemal components are listed in Table I. The mutant suppf5 was characterized at the beginning of this study and was isolated as a motility mutant following a mutagenesis of the wild-type strain 137c with nitrosoguanidine (8). It has slower motion than a wild-type strain and lacks four of the six axonemal polypeptides that compose the drc (Fig. 1). These four polypeptides are identical to those missing in the mutant pf3 (Table II). The motility of the mutant pf3, however, is slower than that 1. Abbreviation used in this paper: drc, dynein regulatory complex, 9 The Rockefeller University Press, 0021-9525/92/09/1455/9 $2.00 The Journal of Cell Biology, Volume 118, Number 6, September 1992 1455-1463 t455 on A ril 3, 2017 D ow nladed fom Published September 15, 1992