The Geometry of Ciliary Coordination: How Spacing, Angular Relationships, and Power Stroke Directions Affect the Degree of Ciliary Coordination
Worral, Amber Miriam Monin
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Hypothesis . Antilaeoplectic metachrony is the defined convention of coordinated beating observed in all vertebrate respiratory tissue. This requires a fixed arrangement of cilia into 'grids' defined by geometric distances between cilia, Cartesian angles (theta), and the relationship of the power stroke to this 'grid' (deltaGamma). These characteristics are specifically optimized for coordination. Coordinated ciliary beating is an essential component of mucociliary clearance resulting in the effective expulsion of mucus and foreign bodies captured within the mucous blanket (Randell, Boucher et al. 2006). The cilia in such systems do not beat randomly. Rather, their beat cycles are highly and efficiently coordinated, in an antilaeoplectic metachronous pattern (Knight-Jones 1954; Machemer 1974). Failures in coordination that negatively affect transport can result either from genetic causes, so called primary ciliary dyskinesis (PCD), or environmental causes, called secondary ciliary dyskinesis (SCD) (Mygind, Pedersen et al. 1983; Bertrand, Collet et al. 2000; Afzelius 2004; Randell, Boucher et al. 2006). A number of approaches have been used to study ciliary coordination, including photoelectric methods, high speed imaging based methods, and even electron microscopic observations (Sanderson and Sleigh 1981; Gheber and Priel 1989; Min, Yun et al. 1998; Rhee, Min et al. 2001; Sisson, Stoner et al. 2003). These methods allow one to measure ciliary beat frequency, power stroke direction and metachronal direction and wavelength. However, none of these methods monitor individual ciliary behavior and therefore quantify the degree of coordination. In this thesis methods have been developed for this purpose using primary explant cultures of newt lung and porcine trachea combined with high extinction, high resolution Nomarski DIC optics, high speed imaging, and digital image analysis. New methods were developed for measuring the following parameters of individual cilia—beat frequency, phase, position, and power stroke direction. Phase differences were used as a starting point for determining ciliary coordination. Changes in positional parameters, the distance between cilia (d), angle of a cilium's power stroke (Gamma), positional cartesian angle (theta), were examined and correlated to decreases in coordination relationships. Several quantitative measures of ciliary coordination were developed. These measures are cumulative phase difference (mCPD) and the standard deviation of cumulative phase difference (SDCPD), both of which were used to evaluate ciliary coordination. A more generalized and accurate quantitative measure of coordination then was established using mean SDCPD and mean deltaGamma. With the tools developed here, it was found that coordinated cilia were characterized by low nearest neighbor distances and low deltaGamma values, whereas uncoordinated cilia were characterized by high values of both. These data support the proposed hypothesis. In contrast, synchrony between adjacent cilia could be found at many angles other than those predicted by antilaeoplectic metachrony. The ability to measure the degree of coordination of a group of cilia is novel. The methods developed and data acquired here provide valuable tools for experimentally studying the mechanism of ciliary coordination. In addition they potentially can be used as a means of quantitatively determining effects of drugs and other chemical or environmental agents on ciliary coordination.