Date of Completion

4-24-2015

Embargo Period

4-22-2018

Keywords

Cilia, Dynein, Cytoplasmic Assembly, Dynein Arms

Major Advisor

Dr. Joseph LoTurco

Associate Advisor

Dr. Randall Walikonis

Associate Advisor

Dr. Marie Cantino

Associate Advisor

Dr. Joanne Conover

Field of Study

Physiology and Neurobiology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Motile cilia and flagella are essential to many biological functions that require cellular or fluid movement. The motility of cilia and flagella in turn is dependent upon the action of large protein complexes that serve as molecular motors. Such axonemal motors are composed of multiple dyneins assembled in the cytoplasm of cells with motile cilia. The mechanisms underlying this assembly are not fully understood. Fortuitously, we found that genetic deletion of exons 2–4 of Dyx1c1 in mice caused a phenotype resembling Primary Ciliary Dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. Ultrastructural and immunofluorescence analyses of Dyx1c1-mutant motile cilia in mice showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). Dyx1c1 localizes to the cytoplasm of respiratory epithelial cells. Its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic axonemal motor assembly factor DNAAF2 (KTU) as well as transition zone protein Septin2. Additionally, a missense mutation in DYX1C1 found to lead to PCD in humans exhibits a weaker interaction of DYX1C1 with Sept2 and KTU and has reduced activity in promoting association of dyneins. These findings suggest that Dyx1c1 (DNAAF4) plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects. Dyx1c1 interacts with intermediate and light chain subunits of the dynein arm complex in the cytoplasm of tracheal epithelial cells. Light and intermediate chains fail to form complexes in the cytoplasm of tracheal epithelial cells of Dyx1c1 mutants. Dyx1c1 increases by four fold the association of light chain subunits Dnal1 and Dnal4 in heterologously expressing HEK 293-T cells. Finally, a missense mutation in Dyx1c1 (p.L19Q) which cause PCD in humans is impaired in its ability to assembly Dnal1 and Dnal4. In conclusion, we identify a step in axonemal dynein pre-assembly in which Dyx1c1 facilitates formation of light and intermediate chain dynein complexes.

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