González M, Cambiazo V, Maccioni R B
International Center for Cancer and Developmental Biology (ICC), Santiago/Chile.
Eur J Cell Biol. 1995 Jun;67(2):158-69.
The interaction of different protein systems with microtubules is a critical step in the cellular function of these organelles. The family of microtube-associated proteins (MAPs) together with a set of motor proteins such as kinesin, cytosolic dynein and dynamin are among the most clear examples of microtubule-interacting proteins. In addition, an increasing number of recently discovered proteins have been shown to interact with microtubules, even though they do not remain associated after cycles of assembly and disassembly. By using affinity columns of agarose derivatized with peptides from the C-terminal regulatory domain on tubulin, we found a 90 kDa protein that interacts with tubulin and microtubules. This protein, here designated as Mip-90, was isolated from neuroblastoma N2A and HeLa cells. It was also identified in high-speed supernatants of the neuroblastoma N-115, and non-neuronal cell lines NIH 3T3, Huh-7, HTB-145 and SW-13 vim+. Mip-90 was able to specifically bind to affinity columns of the agarose-bound beta-II(422-434) and beta-II(434-443) tubulin peptides, containing the sequences of MAP binding domains on beta-II-tubulin. Specific antibodies to Mip-90 along with an anti-beta-tubulin antibody used in double immunofluorescence experiments revealed a striking colocalization of this protein with the microtubule network. Nocodazole-treated cells showed significant changes in Mip-90 distribution as correlated to disruption of the microtubule cytoskeleton. On the other hand, Mip-90 colocalized with microtubule bundles with a perinuclear distribution in HeLa cells treated with taxol. The binding of Mip-90 to microtubules was confirmed by cosedimentation experiments. This protein also exhibited a strong affinity for a calmodulin-agarose affinity matrix, and a preparation of Mip-90 isolated by this affinity procedure was able to promote in vitro tubulin assembly into microtubules. The capacity of Mip-90 to interact with microtubules and with calmodulin suggested functional similarities to tau proteins. However, Western blot analysis using a polyclonal antibody against this protein revealed no cross-reactivity of Mip-90 with tau components. In addition, the 90 kDa protein is a thermosensitive protein. On the other hand, site-directed antibodies that recognize a repetitive binding domain on tau, MAP-2 and MAP-4 failed to react with Mip-90. The studies suggest that Mip-90, a microtubule-interacting protein incorporates into microtubules in vitro, and may play a role in modulating microtubule assembly and organization in non-neuronal cells, thus contributing to the regulation of the dynamics of the cytoskeletal network.
不同蛋白质系统与微管的相互作用是这些细胞器细胞功能中的关键步骤。微管相关蛋白（MAPs）家族以及一组运动蛋白，如驱动蛋白、胞质动力蛋白和发动蛋白，是与微管相互作用蛋白中最典型的例子。此外，越来越多最近发现的蛋白质已被证明可与微管相互作用，尽管它们在组装和解聚循环后不会保持结合状态。通过使用用来自微管蛋白C末端调节域的肽衍生化的琼脂糖亲和柱，我们发现了一种与微管蛋白和微管相互作用的90 kDa蛋白质。这种蛋白质，在这里命名为Mip - 90，是从神经母细胞瘤N2A和HeLa细胞中分离出来的。它也在神经母细胞瘤N - 115以及非神经元细胞系NIH 3T3、Huh - 7、HTB - 145和SW - 13 vim +的高速上清液中被鉴定出来。Mip - 90能够特异性结合到结合了琼脂糖的β - II（422 - 434）和β - II（434 - 443）微管蛋白肽的亲和柱上，这些肽包含β - II - 微管蛋白上的MAP结合域序列。在双重免疫荧光实验中，针对Mip - 90的特异性抗体与抗β - 微管蛋白抗体一起显示出该蛋白质与微管网络有显著的共定位。用诺考达唑处理的细胞显示Mip - 90分布有显著变化，这与微管细胞骨架的破坏相关。另一方面，在用紫杉醇处理的HeLa细胞中，Mip - 90与具有核周分布的微管束共定位。通过共沉降实验证实了Mip - 90与微管的结合。这种蛋白质还对钙调蛋白 - 琼脂糖亲和基质表现出强烈亲和力，通过这种亲和方法分离得到的Mip - 90制剂能够促进体外微管蛋白组装成微管。Mip - 90与微管和钙调蛋白相互作用的能力表明它与tau蛋白在功能上有相似之处。然而，使用针对该蛋白质的多克隆抗体进行的蛋白质印迹分析显示Mip - 90与tau成分没有交叉反应。此外，这种90 kDa的蛋白质是一种热敏蛋白。另一方面，识别tau、MAP - 2和MAP - 4上重复结合域的定点抗体未能与Mip - 90反应。这些研究表明，Mip - 90作为一种与微管相互作用的蛋白质，在体外可整合到微管中，并可能在调节非神经元细胞中的微管组装和组织中发挥作用，从而有助于调节细胞骨架网络的动态变化。
