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. © 2010 Wiley‐Liss, Inc.