Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins

Abstract

The acidic carboxy-terminal regions of α- and β-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins. Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal α-tubulin and cleaved β-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca2+-induced disassembly. Microtubules composed of cleaved α- and β-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca2+-induced disassembly. Polymers composed of normal α-tubulin and cleaved β-tubulin or of cleaved α- and β-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working conditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved α- and β-tubulins and triggered apparent microtubule cross-stabilization among the bundled polymers. These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of α- and β-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, interactions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.