RESUMEN
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.
Asunto(s)
Proteínas Algáceas/metabolismo , Axonema/metabolismo , Cilios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Proteómica/métodos , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Axonema/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cilios/genética , Cilios/ultraestructura , Microscopía por Crioelectrón/métodos , Dineínas/genética , Tomografía con Microscopio Electrónico , Flagelos/genética , Flagelos/ultraestructura , Microscopía Fluorescente/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Grabación de Cinta de Video/métodosRESUMEN
The nexin-dynein regulatory complex (N-DRC) plays a central role in the regulation of ciliary and flagellar motility. In most species, the N-DRC contains at least 11 subunits, but the specific function of each subunit is unknown. Mutations in three subunits (DRC1, DRC2/CCDC65, DRC4/GAS8) have been linked to defects in ciliary motility in humans and lead to a ciliopathy known as primary ciliary dyskinesia (PCD). Here we characterize the biochemical, structural, and motility phenotypes of two mutations in the DRC2 gene of Chlamydomonas Using high-resolution proteomic and structural approaches, we find that the C-terminal region of DRC2 is critical for the coassembly of DRC2 and DRC1 to form the base plate of N-DRC and its attachment to the outer doublet microtubule. Loss of DRC2 in drc2 mutants disrupts the assembly of several other N-DRC subunits and also destabilizes the assembly of several closely associated structures such as the inner dynein arms, the radial spokes, and the calmodulin- and spoke-associated complex. Our study provides new insights into the range of ciliary defects that can lead to PCD.
