Abstract
In eukaryotic cells, cilia and flagella are highly conserved organelles that use dynein motor proteins to generate bending waves required for locomotion or propelling tluid. Defects in these organelles have been associated with many human diseases such as polycystic kidney disease and infertility. The core stmcture of cilia and tlagella, the so called axoneme, has a 9 + 2 arrangement of microtubules in which nine outer doublets surround a central microtubule pair (Porter et al. 2000). Dynein activity has to be precisely regulated to produce coordinated inter-doublet sliding that is transformed into bending by connections, like the ncxin link, that restrict this sliding motion (Satir 1968). One of the key regulators of motor activity is the I1 complex, which consists of at least II proteins (Bower et al. 2009). However, the detailed structure and mechanisms of this complex have not yet been determined. Therefore, we are using cryo-elcctron tomography and image processing to determine the three-dimensional (3D) structure and subunit composition of the I1 complex in wild-type and mutant flagella of Chlamydomonas. Cryo-electron tomography has two advantages: 1) it allows us to reconstruct the 3D structure of axonemes, and 2) rapid freezing ("cryo") preserves biological specimen in a near-to-native state (Dubochct et al. 1988). Our data illustrate the wild-type II structure in the PF2GFP mutant and morphological defect in the 6F5 mutant in unprecedented detail and molecular resolution. By comparing WT and mutant structure and taking the known biochemical differences into account we are able to localize some of the II components in 3D for the first time. These techniques will establish a foundation for further research of II subunit interactions and localization, which may have implications in human ciliary disease.