Motile cilia are microtubule based, cellular appendages that asymmetrically undulate to generate directed fluid flow. This fluid flow is vital for the conserved functions of cell motility and respiratory airway mucus clearance. Disruption of motile cilia contributes to pathologies such as chronic obstructive pulmonary disease (COPD), asthma and primary ciliary dyskinesia (PCD). Motile cilia are directly anchored to the cell by basal bodies (BB). BB are radially symmetric, cylinder shaped structures made up of nine-triplet microtubule blades. BBs must both resist and transmit mechanical forces from beating cilia to the cell for effective fluid flow. The Pearson lab identified proteins and the microtubule post-translational modification (PTM), glutamylation, to stabilize BBs against ciliary forces. While BBs are radially symmetric, the forces received by cilia are asymmetric. We find that BB PTM glutamylation localizes asymmetrically to BB regions predicted to experience the most mechanical force from cilia. Microtubule glycylation and glutamylation are competitive PTMs that modify the same residues of tubulin. PTMs are known to regulate microtubules by intrinsically controlling physical characteristic like bending or the binding of microtubule associated proteins. Moreover, tubulin glutamylation levels directly control protein activity. This gives rise to the fascinating possibility that BB stabilization is responsive to mechanical forces received from cilia. It is not known whether BB glycylation stabilizes BBs against ciliary forces. It is also unclear whether the competition between BB microtubule glutamylation and glycylation regulate BB stability.
In Aim 1, I will use quantitative light microscopy to determine how PTM levels impact BB stability in response to ciliary stress. BB glutamylation asymmetrically localizes to BB domains that experience the greatest mechanical force from cilia. How this asymmetry is established and whether BB glutamylation and glycylation respond to changes in ciliary forces is unknown.
In Aim 2, I will determine how BB PTMs asymmetrically localize and whether they respond to forces from ciliary beating. BB glutamylation stabilizes BBs against ciliary forces. Whether this stabilization is achieved through intrinsic BB microtubule regulation like bending or through BB stabilizing or destabilizing binding proteins is unknown.
In Aim 3, I will determine whether microtubule PTMs affect BB bending and the localization of BB stabilizing or destabilizing proteins. My project will provide a mechanistic perspective on how cell structures interact with physical forces. I will measure how BBs are stabilized against forces from ciliary beating by employing quantitative imaging, genetic and molecular manipulation. By using multi-disciplinary approaches and quantitative analyses, I will hone skills that directly translate into my aspirations of being an independent investigator and teacher.
Cilia are cellular appendages that undulate to produce directed fluid flow vital for mucus clearance from respiratory airways. Basal bodies anchor cilia and must be stabilized against the physical forces they receive from undulating cilia. The goal of this study is to define how basal bodies are stabilized against physical forces from cilia to provide an understanding of how cell structures respond to disease states with impaired respiratory airway mucus clearance.
