Coordination of respiratory cilia: Exploring the role of gamma-tubulin associated with the basal apparatus using a new correlative imaging method

Primary ciliary dyskinesia (PCD) is a disease characterized by a diminished if not totally inhibited ability to effect mucociliary transport, a phenomenon especially important in the respiratory system, where it serves as a disease preventative. This process relies on coordinated ciliary beating which is conspicuously absent in PCD. Solving the underlying basis of coordinated ciliary beating is paramount for better understanding the etiology of PCD. We hypothesize that the presence of gamma-tubulin, associated with the basal foot of the ciliary basal body, is responsible, at least in part, for the maintenance of the integrity of the ciliary array, which in turn is necessary to effect coordinated beating. Because gamma-tubulin may be partially responsible for basal microtubule organization, we have demonstrated a general correlation between the amount of gamma-tubulin and ciliary coordination using live cell imaging combined with gamma-tubulin immunofluorescence. A better correlation will depend upon a better method for quantifying coordination. In the present study, we also provide the initial results of a newly developed method known as "correlative live cell imaging and indirect immunofluorescence" (CLIF). We show that beat frequency, phase, and beat direction can be determined independently for each component of a ciliary tuft.