Title: Assembly mechanisms of ciliary cytoskeletal motors
Funder: The Academy of Medical Sciences

Abstract: Motile cilia are cellular protrusions that perform vital biological functions ranging from cell motility, embryonic patterning to organ homeostasis throughout life. Cilia motion is powered by cytoskeletal motors called axonemal dyneins. Defects in the assembly of axonemal dyneins lead to the severe and incurable human disease – Primary Ciliary Dyskinesia (PCD) which is characterised by infertility, left-right body axis defects, increased risk of ectopic pregnancies and chronic respiratory disorders. Here, I propose to elucidate the axonemal dynein assembly pathway by integrating proteomic, biochemical and structural techniques. A set of ten dynein axonemal assembly factors (DNAAFs), aids dynein motor assembly. Genetic mutations in DNAAFs directly impact the stability of dynein motors suggesting they cooperate with molecular chaperones to facilitate dynein assembly. The precise molecular mechanisms of DNAAFs are unknown. I will use the unicellular ciliate Tetrahymena thermophila as it is the best model system currently available to biochemically and structurally investigate the DNAAFs. Firstly, I will identify DNAAF interaction partners during active dynein assembly using affinity purification and mass spectrometry. This will reveal the assembly complexes that the DNAAFs participate in. Secondly, I will biochemically isolate the assembly complexes from large scale Tetrahymena cultures and visualize their structures using negative stain electron microscopy. Lastly, I will immunostain DNAAFs to study their sub-cellular properties during the assembly process. This work will inform future cryo-EM and correlative light and electron microscopy studies and ultimately reveal the molecular mechanisms of DNAAFs, specifically advancing the development of therapies for motile cilia-related respiratory disorders.

Lay abstract: Biological motion is a key attribute of life. In our own bodies and in some single-celled organisms this motion is observed in slender hair-like structures called cilia which move to help propel swimming sperm towards the egg or brush out mucus from our airways. Cilia motion is powered by motor proteins called dyneins which are complex machines with many parts that need to be assembled together. A failure in dynein assembly results in cilia which get stalled. Genetic defects which cause dyneins to mis-assemble lie at the heart of a debilitating lung condition that affects new-borns who are unable to clear their lungs due to their stalled cilia. Here, I propose to understand the dynein assembly process. Ten assembly factors build dynein motors similar to the way car mechanics assemble motor engines that power cars. To understand the assembly process, first I need to know how the assembly factors bind different parts of the dynein and make them fit together. I will use protein interaction studies which can reveal how one protein binds to another. Additionally, to get a visual blueprint of how the different parts of dyneins are linked together by the assembly factors, I will use electron microscopes to obtain detailed snapshots of the motors in various stages of assembly. This work will provide a more complete picture of how motor proteins are assembled and inform on new ways in which we can correct problems that prevent their assembly for the benefit of patients.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE