Molecular characterization of defects of the nexin-dynein regulatory complex causing Primary Ciliary Dyskinesia (PCD)

• Applicant: Dr. Heike Olbrich
• Subject Area: Pediatric and Adolescent Medicine
• Term: from 2014 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 258092599

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic airway disease, randomization of left-right (LR) body asymmetry and male infertility due to defects of motile cilia/flagella function. Currently PCD diagnosis mainly depends on demonstration of ultrastructural defects of respiratory motile cilia. Mislocalization or absence of axonemal components like outer dynein arms, nexin-dynein regulatory complex (N-DRC) or radial spoke components result in ciliary immotility or aberrant ciliary beating pattern and thus disturbed mucociliary clearance. Effective ciliary beating is essential for proper mucociliary clearance of the airways and regulation of all involved ciliary components is required. The N-DRC, together with the radial spokes and the central pair, is responsible for modulation of ciliary movement and regulation of dynein arms. Inhibition of the sliding forces by the N-DRC converts the sliding forces into bending of the axoneme. Our group already described three genes that encode components of the N-DRC (CCDC164) or related to the N-DRC (CCDC39 and CCDC40). Loss-of-function mutations in CCDC39 and CCDC40 result in axonemal disorganization, inner dynein arm (IDA) and N-DRC defects. Interestingly, loss-of-function in CCDC164 causes defects of the N-DRC but does not affect axonemal organization and IDA. Within this proposal we aim to identify additional genes responsible for axonemal disorganization and IDA defects as well as isolated N-DRC defects in humans and to characterize the clinical PCD phenotype and diagnostic findings of these mutations by high-speed video microscopy, transmission electron microscopy and high resolution immunofluorescence analyses. Furthermore, we plan to analyze the composition and distribution of N-DRC and N-DRC related components in distinct cell types (respiratory, fallopian tubule and nodal cilia and sperm flagella). Since little is known about the assembly and docking of the N-DRC, we intend to characterize the N-DRC defects in lnks/Ccdc40 and Ccdc164 mutant mice. Additionally, we will analyze the interaction of N-DRC components and components responsible for proper docking of the N-DRC to microtubules by performing co-immunoprecipitation (Co-IP) experiments using recombinant epitope-tagged proteins of known and candidate N-DRC and N-DRC related components. To complement this approach, we will analyze immunoprecipitates of N-DRC wild-type and N-DRC defective cell lysates followed by mass spectrometry analyses to identify native N-DRC complexes. We expect to describe the N-DRC and N-DRC related components in more detail, which should help to explain different phenotypes observed in PCD. Taken together the proposed analyses will expand the knowledge about ciliary structure, ciliary beating regulation and PCD and will give additional insights into the pathogenesis of PCD and thus improving diagnosis and genetic counseling of affected individuals.

• DFG Programme: Research Grants