Investigating the molecular mechanisms of ciliary dynein motor assembly

Biological motion is a key attribute of life. An entire branch of the eukaryotic tree of life relies on slender hair-like moving structures called cilia to orchestrate motion. Motor proteins called dyneins power ciliary motion and drive fundamental processes such as the swimming of sperm or the clearance of mucus out of lungs by moving the tiny cilia lining our respiratory tracts. Dynein motors are comprised of many parts which must be assembled. A failure in assembly results in motors that do not function resulting in stalled cilia. This mis-assembly lies at the heart of a debilitating lung condition called Primary Ciliary Dyskinesia (PCD) that affects new-borns who are unable to clear their lungs due to static cilia. Here, I propose to understand the dynein assembly process. Nineteen assembly factors build dynein motors in cellular compartments called assembly factories in the same way in which car mechanics might assemble motor engines that power cars in a factory. To fully understand the assembly process, I need to know three things. 1) How do the assembly factors work together to meld different parts of the dynein motor and make them fit together? I will use protein-protein interaction studies as well as biochemical assays to study how one DNAAF groups with another DNAAF as well as identify other binding partners which might form larger groups to assemble dyneins. 2) What do these groups of assembly factors look like? Directly looking at the 3D structure of the assembly factors is the most straightforward way of understanding how they work. However, because proteins are a billion times smaller than a human being, taking ultra-high-resolution pictures of proteins requires the use of a powerful instrument called a cryo-electron microscope (cryo-EM). I will use a cryo-EM to snap several thousand pictures of DNAAF complexes and combine these in a computer to generate their 3D models. These 3D reconstructions will provide me with a sufficient level of detail to understand how DNAAFs work together to assemble the various parts of the dynein motors and more importantly, how PCD causing mutations prevent groups of DNAAFs from forming. 3) How do the assembly factories work inside a cell? In a healthy situation, assembly factories function smoothly but when assembly gets blocked due to PCD mutations in the assembly factors, this leads to a pile-up of mis-assembled motors inside cells which can be harmful. How assembly factories operate under normal conditions is unclear and it is important to understand this first before trying to resolve the pileups in a diseased condition. To gain deeper insights, I will use imaging techniques to directly observe assembly factories in healthy cells and compare these to diseased cells. This work will point to new ways to disentangle the build-up of motors and restore smooth functioning of assembly factories in patient cells. Overall, this study will provide exciting new insights into how the DNAAF proteins work, helping us understand how cells build biological motors to power the essential movement of cilia in our lungs as well as help sperm cells swim. This work could lead to new ways to kick-start the assembly process to make stalled cilia move again to cure PCD patients.