Abstract
Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 2659-2680 |
| Number of pages | 22 |
| Journal | Molecular biology of the cell |
| Volume | 30 |
| Issue number | 21 |
| DOIs | |
| State | Published - Oct 1 2019 |
Bibliographical note
Funding Information:We thank LeeAnn Higgins, Todd Markowski, Bruce Witthun, and Alan Zimmerman from the Center for Mass Spectrometry and Proteomics at the University of Minnesota (UMN) for expert assistance with iTRAQ labeling, mass spectrometry, and spectral counting. This center is supported by multiple grants including the National Science Foundation major research instrumentation grants 9871237 and 0215759 as described at https://cbs.umn.edu/cmsp/about. We also thank Matt Laudon and the Chlamydomonas Resource Center (https://www.chlamycollection.org/) for strains. This center is supported by the National Science Foundation Living Stock Collections for Biological Research program (National Science Foundation grants NSF-0951671 and NSF-00017383). The Porter laboratory also acknowledges the dedicated assistance of multiple UMN undergraduates including Aimee DeCathelineau, Jasjeet Sekhon, Jared Rieck, Shada Ahrar, and Alexandria Schauer and Clare Palmer from Wesleyan University. Expert assistance with TEM and image analysis was also provided by Amanda Bednarz, Thomas Giddings, and David Mastronarde at the Boulder Laboratory for 3D Fine Structure (University of Colorado). We also thank Chen Xu (Brandeis University) and Daniel Stoddard (UT Southwestern Medical Center) for dedicated training and maintenance of EM facilities. The UTSW cryo-electron microscope facility is funded in part by a CPRIT Core Facility Award (RP170644). Richard Linck (UMN), Toshiki Yagi (Prefectural University of Hiroshima), Win Sale (Emory University), Ritsu Kamiya (Tokyo University), Paul Lefebvre (UMN), and Pinfen Yang (Marquette University) generously supplied antibodies as listed in Supplemental Table S3. Preliminary reports of this work were presented at the American Society for Cell Biology meetings and the International Conference on the Cell and Molecular Biology of Chlamydomonas. This work was supported by National Institutes of Health grants to M.E.P. (GM-055667), D.N. (GM-088122), and E.F.S. (GM-112050).
Publisher Copyright:
© 2019 Lin, Le, et al.