The role of ODA5, ODA8 and ODA10 in axonemal outer arm dynein assembly

Paurav Desai, Judy Freshour, Jenny Rowe, Anudariya Batbold, and David R. Mitchell

Cell & Developmental Biology, SUNY Upstate Medical Univ, Syracuse, NY 13210

Outer arm dynein assembly defects are the most common causes of Primary Ciliary Dismotility in humans, but many of the loci linked to this disorder have not been identified. The Chlamydomonas ODA5, ODA8 and ODA10 loci are hypothesized to encode interacting proteins needed for binding of outer row dynein onto axonemal doublet microtubules (Kamiya, JCB 107:2253-2258, 1988; Wirschell et al. MBC 15:2729-2741, 2004), but only ODA5 has been previously characterized (Wirschell et al., 2004). We cloned ODA8 by RFLP analysis, identified the mutation in one allele, and rescued the mutant phenotype by transformation with an HA epitope tagged gene. ODA8 is an LRR protein, homologous to vertebrate LRRC56, which is conserved among organisms with motile cilia/flagella and expressed in vertebrate cell types that display such organelles. Using this tagged protein we show that ODA8 is an axonemal protein and that it does not co-fractionate with outer arm dynein. Assembly of ODA8 protein into flagella is greatly reduced in the presence of either the oda5 or oda10 mutations. Together with the previously published data, our results support the hypothesis that these three loci encode proteins that interact on the doublet surface. Studies on in vitro binding of outer arm dynein onto axonemes show that wild type dynein does not require ODA8 to bind to axonemes. Dynein from oda8 and wild type cytoplasm have different axonemal binding characteristics, indicating a possible role for ODA8 in the cytoplasm. To complete the characterization of these loci we are currently using AFLP analysis to clone the ODA10 locus. Supported by National Institutes of General Medical Sciences grant GM44228 to DRM.

e-mail address of presenting author: desaip@upstate.edu