PROJECT SUMMARY Basal bodies (BBs) are cellular nanomachines that position and anchor cilia. Defects in BBs contribute to a broad spectrum of diseases including cancers and ciliopathies. Ciliopathies are a general class of human maladies that include birth defects, polydactyly, blindness, respiratory illness, hydrocephaly, and infertility. However, the molecular mechanics for how BBs organize cilia so that, when defective, they contribute to the above pathologies are not well understood. Motile cilia produce large-scale hydrodynamic fluid flow in the respiratory tract, brain ventricles and oviduct. Loss of directional fluid flow causes respiratory illness, hydrocephalus and infertility. While study of such human diseases reveals the importance of BBs and cilia, fundamental cell biology research into the mechanisms by which BBs position and anchor cilia to promote normal ciliary beating is in part limited due to a need for genetically modifiable model systems in which human disease mutations can be mimicked and where ciliary beating is accessible. Here we build on our prior funding cycle where we developed Tetrahymena as a model system to visualize ciliary forces and identified proteins and post-translational modifications responsible for normal ciliary positioning and anchoring. Our proposed funding will capitalize on these prior advances to quantify the forces that asymmetric cilia beating imposes on BBs. Such forces are predicted from computational modeling, but the direct BB movements that arise from these forces have never been studied. To ensure that BBs and cilia are correctly positioned within ciliary arrays, BBs possess BB-associated accessory structures. We will determine how these accessory structures dynamically respond to ciliary forces to preserve BB and ciliary structure and positioning to maintain proper hydrodynamic flow. Next, we will study the BB domains that ensure proper positioning and anchorage at the cell surface during ciliary beating. Ciliary forces induce asymmetric disruption of specific triplet microtubules in BB mutants. We will establish how asymmetrically localized BB stability factors define distinct BB domains and determine whether they stabilize particular BB triplet microtubules. These studies will be carried out by a collaborative group of researchers that bridges a broad research spectrum from fundamental biology, genetics, live and fixed cell microscopy, polymer engineering, computational modeling to structural studies. Excellent training opportunities exist for undergraduate and graduate students and postdoctoral researchers in the lab. Collaborations with other labs that specialize in light and electron microscopy, computational modeling, structural biology and polymer engineering, expand the innovation and impact of our studies. In summary, this collaborative proposal will illuminate how BBs position cilia and withstand the forces generated by cilia so that coherent hydrodynamic forces are generated and maintained.

R01GM099820

2012-09-24

2021-07-31