Cueille N, Blanc C Tallichet, Popa-Nita S, Kasas S, Catsicas S, Dietler G, Riederer B M
Département de Biologie Cellulaire et de Morphologie, Université de Lausanne, 9 rue du bugnon, CH-1005 Lausanne, Switzerland.
Brain Res Bull. 2007 Mar 30;71(6):610-8. doi: 10.1016/j.brainresbull.2006.12.003. Epub 2007 Jan 3.
Microtubule-associated protein 1B is an essential protein during brain development and neurite outgrowth and was studied by several assays to further characterize actin as a major interacting partner. Tubulin and actin co-immunoprecipitated with MAP1B at similar ratios throughout development. Their identity was identified by mass spectrometry and was confirmed by Western blots. In contrast to previous reports, the MAP1B-actin interaction was not dependent on the MAP1B phosphorylation state, since actin was precipitated from brain tissue throughout development at similar ratios and equal amounts were precipitated before and after dephosphorylation with alkaline phosphatase. MAP1B heavy chain was able to bind actin directly and therefore the N-terminal part of MAP1B heavy chain must also contain an actin-binding site. The binding force of this interaction was measured by atomic force microscopy and values were in the same range as those of MAP1B binding to tubulin or that measured in MAP1B self-aggregation. Aggregation was confirmed by negative staining and electron microscopy. Experiments including COS-7 cells, PC12 cells, cytochalasin D and immunocytochemistry with subsequent confocal laser microscopy, suggested that MAP1B may bind to actin but has no obvious microfilament stabilizing effect. We conclude, that the MAP1B heavy chain has a microtubule-stabilization effect, and contains an actin-binding site that may play a role in the crosslinking of actin and microtubules, a function that may be important in neurite elongation.
微管相关蛋白1B是大脑发育和神经突生长过程中的一种重要蛋白质，通过多种检测方法对其进行了研究，以进一步确定肌动蛋白是其主要的相互作用伙伴。在整个发育过程中，微管蛋白和肌动蛋白与MAP1B共免疫沉淀的比例相似。通过质谱鉴定了它们的身份，并通过蛋白质免疫印迹法进行了确认。与之前的报道不同，MAP1B与肌动蛋白的相互作用不依赖于MAP1B的磷酸化状态，因为在整个发育过程中，肌动蛋白从脑组织中沉淀的比例相似，并且在用碱性磷酸酶去磷酸化前后沉淀的量相等。MAP1B重链能够直接结合肌动蛋白，因此MAP1B重链的N端部分也必须包含一个肌动蛋白结合位点。通过原子力显微镜测量了这种相互作用的结合力，其值与MAP1B与微管蛋白结合或MAP1B自聚集测量的值在同一范围内。通过负染色和电子显微镜证实了聚集现象。包括COS-7细胞、PC12细胞、细胞松弛素D以及随后的共聚焦激光显微镜免疫细胞化学在内的实验表明，MAP1B可能与肌动蛋白结合，但没有明显的微丝稳定作用。我们得出结论，MAP1B重链具有微管稳定作用，并含有一个肌动蛋白结合位点，该位点可能在肌动蛋白和微管的交联中发挥作用，这一功能在神经突伸长中可能很重要。
