Reassembly of flagellar B(αβ) tubulin into singlet microtubules: Consequences for cytoplasmic microtubule structure and assembly

Abstract

B(αβ) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived B tubulin fraction (100,000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication-derived flagellar tubulin and purified vertebrate tubulin. The 'microtubules' reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single 'adlumenal component' applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in an A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.