Hiroko Takazaki

Partially Functional Outer-Arm Dynein in a Novel Chlamydomonas Mutant Expressing a Truncated γ Heavy Chain

ABSTRACT The outer dynein arm of Chlamydomonas flagella contains three heavy chains (α, β, and γ), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose γ heavy chain is truncated at about 30% of the sequence. While the previously isolated γ chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain α and β heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the γ heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the α heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the β heavy chain). Thus, the outer-arm dynein lacking the γ heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein.

Three outer arm dynein heavy chains of Chlamydomonas reinhardtii operate in a coordinated fashion both in vitro and in vivo.

Outer arm dynein (OAD) in cilia and flagella contains two to three nonidentical heavy chains (HCs) that possess motor activity. In Chlamydomonas, flagellar OAD contains three HCs, α‐, β‐, and γ‐HCs, each appearing to have a distinct role. To determine the precise molecular mechanism of their function, cross‐sectional electron micrographs of wild‐type and single HC‐disruption mutants were compared and statistically analyzed. While the α‐HC mutant displayed an OAD of lower density, which was attributed to a lack of α‐HC, the OAD of β‐ and γ‐HC mutants not only lacked the corresponding HC, but was also significantly affected in its structure, particularly with respect to the localization of α‐HC. The lack of β‐HC induced mislocalization of α‐HC, while a disruption of the γ‐HC gene resulted in the synchronized movement of α‐HC and β‐HC in the manners for stacking. Interestingly, using cryo‐electron microscopy, purified OADs were typically observed consisting of two stacked heads and an independent single head, which presumably corresponded to γ‐HC. This conformation is different from previous reports in which the three HCs displayed a stacked form in flagella observed by cryo‐electron tomography and a bouquet structure on mica in deep‐etch replica images. These results suggest that γ‐HC supports the tight stacking arrangement of inter or intra α‐/β‐HC to facilitate the proper functioning of OAD.