Chapter 14 Isolation of Respiratory Cilia

Abstract

The membrane that surrounds the ciliary axoneme of mammals is active in interaction with the external environment. This activity may result in the alteration of ciliary function. Zwitterionic 3-[(3-cholamidopropyI) dimethylammonio]-1-propanesulfonate (CHAPS) detergent at 0.1% is effective combined with brushing the epithelium. This injury multiplies the yield of cilia approximately 10-fold compared with nontraumatic extraction, but also increases cellular debris. For CHAPS at 0.5% without brushing, there is release ciliary embranes into solution during axoneme isolation. A different approach homogenizes epithelial cells scraped off the trachea, followed by MgCl 2 aggregation and ouabain affinity chromatography to remove intracellular and basolateral membranes from the apical membrane preparation; however, this method results in a mixture of epithelial cells other than just ciliated ones. The method given in this chapter specifically yields isolated intact cilia, derives membranes from the isolated cilia, and identifies apical membrane surface components by prior labeling of the epithelium with biotin, as similarly applied to Chlamydomonas. Solubilizing ciliary membranes releases the dense matrix proteins from within the axoneme. Reconstitution of membrane vesicles after detergent removal separates integral membrane components from soluble matrix proteins. Use of Triton X-114 in a study of Tetrahymena ciliary membrane proteins facilitates further partitioning into aqueous and detergent phases. The chapter discusses the materials and their sourcing, and the procedure involved. Standard transmission electron microscopy (TEM) techniques are employed in embedment, sectioning, staining, and examination. Presence of membranes is ascertained by TEM that also detects a well-camouflaged Gram-negative bacterium, the cilia-associated respiratory bacillus. This microbe is morphologically indistinguishable from cilia by light microscopy unless immunostained with specific antisera.