Chapter 49 Preparation of Ciliary and Flagellar Remnants

Abstract

Publisher Summary Gentle heating selectively depolymerizes or “melts” the microtubules of axonemes in vivo and in vitro. In both cases, considerable mass remains after thermal treatment. When the proper conditions are chosen, the tubulin derived from thermal fractionation is polymerization competent, suggesting that selective tubulin removal is not simply a denaturation artifact. The ninefold symmetry of the organelle is retained, as is its full length, from basal body to tapering tip, after solubilization of most of the tubulin of the 9 + 2 structure. This “ciliary remnant” retains most of the structural or architectural (nontubulin, nondynein) proteins of the axoneme, perhaps most significant among them being the tektins, the integral microtubule proteins that form the A-B junctional protofilaments of the outer doublets. In the case of flagella, the ninefold cylindrical structure is generally not retained, although under low-shear conditions, sheets of nine doublets will fractionate to sheets of nine singlets and further fractionation will yield insoluble long, fibrous material that will retain the same kinds of proteins as in cilia but having no ordered cylindrical structure. There is a “centriolar rim” structure that retained the basic ninefold configuration after removal of the triplet microtubules from isolated centrioles by either high ionic strength or extremes of pH. Considering that both the ninefold symmetry and the final length may be a function of the proteins that form these remnants or skeletons, study of remnants may provide some insight into the mechanism of centriole or basal body formation, ciliogenesis, and length regulation. Identification of remnant-specific proteins is useful for exploring pathways for their differential incorporation into growing cilia. This chapter presents the experiment on remnant fractionation. In cilia, the A-tubule quickly follows but the A-tubule depolymerization is generally slower in flagella. The flagellar B-subfiber is far more stable than the ciliary B-subfiber to low-ionic-strength dialysis.