STABILIZATION AND BUNDLING OF SUBTILISIN-TREATED MICROTUBULES INDUCEDBY MICROTUBULE-ASSOCIATED PROTEINS

The acidic carboxy-terminal regions of alpha- and beta-tubulin subunit s are currently thought to be centrally involved in microtubule stabil ity and in microtubule association with a variety of proteins (MAPs) s uch as MAP2 and tan proteins. Here, pure tubulin microtubules were exp osed to subtilisin to produce polymers composed of cleaved tubulin sub units lacking carboxy termini. Polymer exposure to subtilisin was achi eved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal alpha-tubulin and cleaved beta-tubulin were indistinguishable from control microtubules with reg ard to resistance to dilution-induced disassembly, to cold temperature -induced disassembly and to Ca2+-induced disassembly. Microtubules com posed of cleaved alpha- and beta-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassemb ly, but marked resistance to Ca2+-induced disassembly. Polymers compos ed of normal alpha-tubulin and cleaved beta-tubulin or of cleaved alph a- and beta-tubulins were stabilized in the presence of added MAP2, my elin basic protein and histone H1, Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We sho w that this potentiation of polymer stabilization can be ascribed to t au-induced microtubule bundling. In our working conditions, such bundl ing upon association with tau proteins occurred only in the case of mi crotubules composed of cleaved alpha- and beta-tubulins and triggered apparent microtubule cross-stabilization among the bundled polymers. T hese results, as well as immunofluorescence analysis, which directly s howed interactions between subtilisin-treated microtubules and MAPs, s uggest that the carboxy termini of alpha- and beta-tubulins are not pr imarily involved in the binding of MAPs onto microtubules. However, in teractions between tubulin carboxy termini and MAPs remain possible an d might be involved in the regulation of MAP-induced microtubule bundl ing.