Motile cilia are hair-like protrusions from the cell surface that beat in a sinusoidal waveform to produce movement. This movement is responsible for the flow of mucus through the respiratory tract, organ left-right asymmetry, and sperm motility. Each cilium is composed of nine rigid tubular structures called microtubule (MT) doublets arranged in a circle around a single central pair of MTs. Microtubule motors called outer arm dyneins (OADs) slide the MT doublets relative to one another while connectors between doublets convert the sliding into bending. The wave motion is generated as OADs on opposite sides of the cilia alternate activity down the length of the cilium. Previous studies have shown that coordination persists in the absence of external cues. Models propose that OAD coordination can be achieved by responding to changes in MT curvature and interdoublet spacing during beating. Alternatively, MT sliding can regulate motor activity by generating self-organized oscillations. However, the mechanism of OAD motility and force generation remains poorly understood in comparison to the cytoplasmic isoform of dynein. Therefore, it remains unclear which model(s) apply to ciliary bending The recent development of the recombinant expression of Tetrahymena OAD allows us to study its mechanics for the first time. Thus far, many of the model predictions have been tested by using cytoplasmic dynein which is structurally distinct from OAD. In contrast to cytoplasmic dynein, a homodimer, OAD forms a heterotrimer of (?, ?, and ?) heavy chains and is not processive at physiological ATP. To understand the mechanism of dynein self-regulation in cilia, I will characterize the motility and force generation characteristics of single OAD motors, and test how MT curvature and sliding impact OAD activity in vitro. The goal of this study is to directly test the specific biophysical predictions made by the models through three core aims: First, we will test the molecular properties of OADs, such as coordination of motor stepping along MTs and force-induced MT attachment/detachment of dynein from the MT. Second, we will construct in vitro assays that mimic the geometries of dynein/MT interactions in a beating cilium. Third, we will use solved structures for axonemal dynein and cytoplasmic dynein to make directed mutations to identify the structural components of OAD that gives rise to its nonprocessive motility, curvature sensing, and oscillatory behavior. This work will establish an experimental and theoretical framework for the study of the molecular mechanism of OAD and enable us to determine the minimum requirements for self-coordinated oscillation of motile cilia.
Motile cilia are highly regulated hair-like organelles that generate forces involved in the flow of bodily fluids and mediate cell signaling; and ciliary defects are linked to an array of human diseases known as ciliopathies. I aim to study the mechanism of that powers self-coordinated ciliary beating and improve our understanding of how specific mutations in dynein leads to male infertility and primary ciliary dyskinesia. This study will advance the discovery of dynein-specific therapeutics for the treatment of these diseases.
