Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly

Abstract

B(.alpha..beta.) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin [Strongylocentrotus purpuratus] sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble 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 differ 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). Some possess a single adlumenal component applied to their inside walls, regardless of the number of protofilaments. Following the 1st cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAP remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between 2 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 3-dimensional organization of microtubule-associated components.