Graduate Thesis Or Dissertation
 

Isolation of newt lung ciliated cell models and characterization of their motile capabilities

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  • The lack of a suitable demembranated cell model has hindered the study of the mechanisms responsible for the regulation of ciliary waveform and coordination. In this study procedures for the isolation of ciliated cells from the newt, Taricha granulosa, by trypsin dissociation, and their subsequent demembranation with Triton X-100 and reactivation with MgATP are described. Reactivation of these cell models with a high degree of mechanochemicalcoupling depends on avoidance of mechanical damage, and maintenance of optimal conditions during tyrypsinization, demembranation, and reactivation. Highly motile models were prepared from cells incubated in 0.5% trypsin for 48 hours at 4°C, treated briefly with 2 mM EDTA, separated by gentle agitation, and concentrated by centrifugation at low G forces. Optimal demembranation and reactivation conditions are similar to those described previously for isolated newt lung axonemes. Under these conditions, nearly 100% of the models can be reactivated when provided with MgATP, and 90-95% show coordinated beating in the ciliary tuft. Beat frequencies within the range measured in living cells can be obtained. The reactivated motility is stable for 30 minutes at constant MgATP. Ciliary waveform and the pattern of coordination are not different from those observed in living cells. On the basis of these results, it can be concluded that these cell models possess a high degree of mechanochemical coupling, and display motility that is characteristic of the living system. These highly coupled models were used to show that: 1) development of coordination in the ciliary tuft occurs at a higher substrate concentration range (10-25 μM) than that required to initiate motility per se (2-10 μM); 2) the outer dynein arms are essentially nonfunctional at substrate levels below 35 μM ATP, and they are not required for the coordinated beating of cilia; 3) ciliary waveforms and the type of metachrony are regulated independent of beat frequency; 4) beat frequency varies biphasically in response to MgATP, as has been shown previously for isolated, individual newt lung axonemes. Apparent Fmax and Km values of 25 Hz and 0.15 mM, and 35 Hz and 0.47 mM, respectively, have been obtained for each linear segment of the biphasic double reciprocal plot.
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