New insights into the nexin-dynein regulatory complex

Raqual Bower1, Douglas Tritschler1, Eileen O'Toole2, and Mary E. Porter1

1) GCD, Univ. of Minnesota, Minneapolis, MN, 55455, USA 2) Laboratory for 3D Fine Structure, Univ. of Colorado, Boulder, CO, 80309, USA

The DRC is a group of tightly bound axonemal polypeptides thought to mediate signals between the radial spokes and dynein arms (Gardner et al., 1994; Piperno et al., 1994). CryoET has revealed that the DRC is part of the nexin link and extends from the A-tubule to the neighboring B-subfiber (Heuser et al., 2009). To further characterize the DRC, we generated antibodies PF2/ DRC4. Western blot analysis confirmed that DRC4 is modified in sup-pf-3 by insertion of a TOC1 transposon. Truncated DRC4 subunits assemble into the axoneme, but other DRC subunits are missing (Huang et al., 1982). Sucrose gradient centrifugation indicates that DRC4 is part of a large complex in wild-type, but sediments as a smaller complex in sup-pf-3 extracts. These observations show that DRC4 plays a key role in the assembly of the linker domain. Other proteins that co-IP with DRC4 are being explored as potential DRC components. We have also characterized the IDA6 gene product as a DRC subunit. FAPs mapping near ida6 were tested for rescue of the ida6 motility defect. The predicted amino acid sequence of the rescuing subclone corresponds to an ~65 kD, highly conserved, coiled coil polypeptide. IDA6 antibodies reveal that IDA6 is enriched in flagella, and co-extracts and co-IPs with DRC4. IDA6 is reduced in drc mutants that lack DRC1 and DRC2, but it assembles at wild-type levels in pf2 and sup-pf-3. Image averaging of ida6 axonemes reveals structural defects in both the DRC and an associated DHC. These and other observations indicate that IDA6 encodes DRC2, that DRC2 is closely associated with DRC1 (see related abstract by Wirschell et al.), and that DRC1 and DRC2 are required for facilitating interactions with at least one inner arm DHC. Our studies further suggest that DRC2 and DRC4 are associated with distinct domains within the DRC.

e-mail address of presenting author: porte001@umn.edu