Chapter 51 Methods for the Isolation of Tektins and Sarkosyl-Insoluble Protofilament Ribbons

This chapter discusses the methods of fractionating ciliary and flagellar microtubules into a stable subset of protofilaments (known as pf-ribbons) and to subfractionate these pf-ribbons into filaments composed of the proteins tektins. Flagellar doublet microtubules from Chlamydomonas reinhardtii could be fractionated by Sarkosyl detergent into stable ribbons of three protofilaments. Similar observations were reported for sea urchin sperm flagellar microtubules. Stability of the pf-ribbons is because of their polypeptide composition. Based on the synergistic effects of detergents and urea to disrupt protein-protein interactions, a Sarkosyl-urea extraction was devised that provided a remarkably clean fractionation of the pf-ribbons. Under optimal conditions of 0.5% Sarkosyl and 2 M urea, flagellar microtubules could be fractionated into 2- to 3-nm-diameter filaments composed almost exclusively of equimolar amounts of three proteins named tektins A (∼55 kDa), B (∼51 kDa), and C (∼47 kDa). It has become possible to isolate filaments composed of only tektins A and B. Methods for isolating pf-ribbons and tektin filaments are presented in the chapter, followed by a discussion of the characterization of tektins. These methods were developed for sperm flagellar axonemes from the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus. The methods are isolation and purification of pf-ribbons and tektin filaments, to prepare tektin filaments from pf-ribbons, and glycerination. The tektin polypeptide chains are predicted to be α-helical, and evidence indicates that tektins exist as longitudinal, heterodimeric protofilaments in the pf-ribbon and thus in flagellar A-tubules. Tektin heterodimers are linear, rodlike molecules, measuring∼48 nm long, giving the tektin filament the potential to interact with and stabilize adjacent tubulin protofilaments, as well as providing longitudinal binding sites for axonemal components with periodicities that are multiples of the 8-nm tubulin dimer and the 48-nm tektin spacing. Studies of cilia and flagella have contributed to understanding microtubule motility, structure, polarity, and assembly.