Chapter 50 In Vitro Polymerization of Tubulin from Echinoderm Sperm Flagellar Microtubules

Abstract

Publisher Summary The purpose of this chapter is to outline two basic methods for the purification and in vitro polymerization of tubulin from echinoderm sperm flagellar microtubules and to summarize the uses and advantages of this system. Methods were developed for the purification and assembly of tubulin and microtubule-associated proteins (MAPS), by depolymerization at 0°C and repolymerization at 37°C, in Mg 2+ /GTP-containing buffers. Relative to the basal body (minus) and the distal (plus) ends of cilia and flagella, microtubules were defined kinetically as having plus and minus ends; the plus end corresponds to the fast-growing end with a higher on rate for tubulin assembly and it is distal relative to the cell center, whereas the minus end is a slow-growing end generally associated with the centrosomes/spindle poles in both interphase and mitotic cells. Flagellar doublet microtubules could be solubilized by sonication in Mg 2+ /GTP-containing buffers at 0°C and repolymerized by elevating the temperature to 37°C; the conditions and kinetic parameters of flagellar tubulin assembly in vitro were essentially identical to those for mammalian brain tubulin. A method developed by combining the fractionation procedure used to selectively solubilize tubulin from the B-subfiber and the need to protect the GTP binding site during solubilization to answer the question of how tubulin isoforms might influence tubulin assembly and microtubule structure. The methods described in the chapter are solubilization of doublet tubules by sonication, thermal fractionation of doublet tubules into crude, soluble B-Tubulin, and cycled polymerization and depolymerization of soluble doublet tubulin. Native doublet microtubules are complex structures consisting of an A-tubule with 13 protofilaments and an incomplete B-tubule with 10 protofilaments that are attached to the A-tubule in a specific pattern. Soluble doublet tubulin polymerizes in vitro to give singlet microtubules with variable numbers of protofilaments and these microtubules are cold labile.