Investigating the molecular mechanisms of ciliary dynein motor assembly
Lead Research Organisation:
University of Oxford
Department Name: Sir William Dunn Sch of Pathology
Abstract
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.
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.
Technical Summary
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. Before functioning in the cilia, these multi-subunit motors undergo an elaborate assembly pathway in the cytoplasm. Defects in this pathway cause the severe and incurable human lung disease - Primary Ciliary Dyskinesia (PCD). Here, I propose to elucidate the dynein assembly pathway by integrating biochemical, structural, and cellular imaging techniques.
19 axonemal dynein assembly factors (DNAAFs) build dyneins in ill-defined sub-cellular compartments (referred as assembly factories in this proposal). Loss of DNAAFs destabilizes dynein subunits suggesting they chaperone subunits till they assemble into larger stable complexes. The molecular mechanisms by which DNAAFs achieve this are unknown because 1) how DNAAFs interact with their substrates and other binding partners is unknown and 2) because DNAAF complexes have not been structurally investigated to inform on their functions. To address this, I will use the unicellular model ciliate Tetrahymena thermophila to biochemically purify and proteomically map DNAAF interactions and define the composition of DNAAF complexes involved in dynein assembly for further structural studies. I will focus on obtaining cryo-EM structures of three key DNAAF complexes (based on preliminary data) and map mutations on them to understand how they cause PCD.
Lastly, I will combine live cell imaging and quantitative correlative light and electron microscopy (CLEM) studies to track fluorescently tagged DNAAFs with dynein subunits in assembly factories inside healthy and diseased cells actively assembling dyneins. This work will discover the functions of assembly factories within which DNAAFs operate. Overall, this proposal will reveal the mechanisms of DNAAFs at a cellular, molecular and atomic level.
19 axonemal dynein assembly factors (DNAAFs) build dyneins in ill-defined sub-cellular compartments (referred as assembly factories in this proposal). Loss of DNAAFs destabilizes dynein subunits suggesting they chaperone subunits till they assemble into larger stable complexes. The molecular mechanisms by which DNAAFs achieve this are unknown because 1) how DNAAFs interact with their substrates and other binding partners is unknown and 2) because DNAAF complexes have not been structurally investigated to inform on their functions. To address this, I will use the unicellular model ciliate Tetrahymena thermophila to biochemically purify and proteomically map DNAAF interactions and define the composition of DNAAF complexes involved in dynein assembly for further structural studies. I will focus on obtaining cryo-EM structures of three key DNAAF complexes (based on preliminary data) and map mutations on them to understand how they cause PCD.
Lastly, I will combine live cell imaging and quantitative correlative light and electron microscopy (CLEM) studies to track fluorescently tagged DNAAFs with dynein subunits in assembly factories inside healthy and diseased cells actively assembling dyneins. This work will discover the functions of assembly factories within which DNAAFs operate. Overall, this proposal will reveal the mechanisms of DNAAFs at a cellular, molecular and atomic level.
