We are studying the inner and outer dynein arms found on the doublet microtubules of Chlamydomonas flagella. These are the enzymes that make flagella move. Both transverse and longitudinal sections have been cut and imaged in the CTEM. Image averaging techniques have been developed to give reliable views of both inner and outer dynein arms. We are comparing the inner dynein arms from wild type flagella with those from several mutants with altered flagellar function. Image subtraction has been used to determine the statistically significant differences between dyneins from cells with different genotypes, allowing us to map into the flagellum the material that is absent when a particular gene is mutant. These structural differences in both transverse and longitudinal orientation have been correlated with the biochemical phenotype of the same mutants, using FPLC and high resolution one-and two-dimensional gel electrophoresis to identify polypeptides that are missing in each mutant. From this work we have learned that there are at least three types of inner arms on flagellar doublets of Chlamydomonas, and that their arrangement within the longitudinal repeating unit of the axoneme is quite complex (Mastronarde et al., 1992). The pattern is clearly inconsistent with previous models of inner arm structure. We are now working to define more precisely the location of each polypeptide in the structure by analyzing a battery of new dynein mutants obtained by insertional mutagenesis. We are also analyzing mutant strains defective in the genes encoding the dynein regulatory complex and have identified a structure that lies at the junction between the radial spokes and one of the inner dynein arms that is missing or altered in several mutants of the regulatory complex.
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