Solubility properties of neuronal tubulin: evidence for labile and stable microtubules

Abstract

The solubility properties of tubulin and microtubules in pure cultures of sympathetic neurons were examined by electron microscopic and biochemical techniques. For morphological analyses, neurons were extracted with Triton X-100 in the presence or absence of 1 mM CaCl2, and the resulting detergent-extracted residues were examined for microtubules. In parallel experiments, the solubility of tubulin was determined under various solution conditions. Detergent-extracted residues of neurons prepared without Ca2+ contained many microtubules. Neurite residues prepared in the presence of Ca2+ also contained microtubules, but at substantially lower numbers than in residues prepared without Ca2+. The biochemical data parallel the morphological observations. Following detergent-extraction under microtubule stabilizing conditions, 30% of the tubulin was detergent-soluble (i.e. unpolymerized), while 70% was detergent-insoluble (i.e. polymerized). A more detailed examination of the solubility properties of tubulin indicated that 62% was detergent-insoluble but soluble in buffers containing mM CaCl2, while 5-8% was detergent and Ca2+-insoluble. A variety of control experiments indicated that non-specific adsorption of tubulin onto detergent-insoluble components of the cultures, assembly of tubulin onto pre-existing microtubules, and incomplete extraction of tubulin from cells contributed minimally to the levels of Ca2+-soluble and insoluble tubulin obtained with the extraction conditions us…