Saoudi Y, Paintrand I, Multigner L, Job D
INSERM Unité 366, Centre d'Etudes Nucléaires de Grenoble, France.
J Cell Sci. 1995 Jan;108 ( Pt 1):357-67. doi: 10.1242/jcs.108.1.357.
The acidic carboxy-terminal regions of alpha- and beta-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 alpha-tubulin and cleaved beta-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca(2+)-induced disassembly. Microtubules composed of cleaved alpha- and beta-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca(2+)-induced disassembly. Polymers composed of normal alpha-tubulin and cleaved beta-tubulin or of cleaved alpha- and beta-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 alpha- and beta-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 alpha- and beta-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.
目前认为，α-和β-微管蛋白亚基的酸性羧基末端区域在微管稳定性以及微管与多种蛋白质（微管相关蛋白，如MAP2和tau蛋白）的结合中起核心作用。在此，将纯微管蛋白微管暴露于枯草杆菌蛋白酶，以产生由缺乏羧基末端的裂解微管蛋白亚基组成的聚合物。在与微管稳定性的进一步测试兼容的缓冲条件下，使聚合物暴露于枯草杆菌蛋白酶。由正常α-微管蛋白和裂解的β-微管蛋白组成的微管在对稀释诱导的解聚、低温诱导的解聚和Ca(2+)诱导的解聚的抗性方面与对照微管没有区别。由裂解的α-和β-微管蛋白组成的微管对稀释诱导的解聚和低温诱导的解聚表现出正常的敏感性，但对Ca(2+)诱导的解聚具有明显的抗性。由正常α-微管蛋白和裂解的β-微管蛋白或裂解的α-和β-微管蛋白组成的聚合物在添加MAP2、髓鞘碱性蛋白和组蛋白H1的情况下得以稳定。微管蛋白羧基末端的裂解极大地增强了tau蛋白对微管的稳定作用。我们表明，这种聚合物稳定性的增强可归因于tau诱导的微管成束。在我们的工作条件下，与tau蛋白结合时的这种成束仅发生在由裂解的α-和β-微管蛋白组成的微管的情况下，并在成束的聚合物之间引发明显的微管交叉稳定。这些结果以及直接显示枯草杆菌蛋白酶处理的微管与微管相关蛋白之间相互作用的免疫荧光分析表明，α-和β-微管蛋白的羧基末端并非主要参与微管相关蛋白与微管的结合。然而，微管蛋白羧基末端与微管相关蛋白之间的相互作用仍然可能存在，并且可能参与微管相关蛋白诱导的微管成束的调节。
