Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, China.
Cell Cycle. 2011 Nov 15;10(22):3929-37. doi: 10.4161/cc.10.22.18106.
Microtubule-binding proteins are a group of molecules that associate with microtubules, regulate the structural properties of microtubules, and thereby participate in diverse microtubule-mediated cellular activities. A recent mass spectrometry-based proteomic study has identified microtubule-associated protein 7 (MAP7) domain-containing 3 (Mdp3) as a potential microtubule-binding protein. However, its subcellular localization and functional importance are not characterized. In this study, by GST-pulldown assays, we found that Mdp3 interacted with tubulin both in cells and in vitro. Immunofluorescence microscopy and microtubule cosedimentation assays revealed that Mdp3 also associated with microtubules. Serial deletion experiments showed that the two coiled coil motifs of Mdp3 were critical for its interaction with tubulin and microtubules. Cold recovery and nocodazole washout assays further demonstrated an important role for Mdp3 in regulating cellular microtubule assembly. Our data also showed that Mdp3 significantly enhanced the stability of cellular microtubules. By tubulin turbidity assay, we found that Mdp3 could promote microtubule assembly and stability in the purified system. In addition, we found that Mdp3 expression varied during the cell cycle and in primary tissues. These findings thus establish Mdp3 as a novel microtubule-binding protein that regulates microtubule assembly and stability.
微管结合蛋白是一类与微管结合、调节微管结构特性的分子，从而参与多种微管介导的细胞活动。最近一项基于质谱的蛋白质组学研究鉴定了微管相关蛋白 7（MAP7）结构域包含 3 号（Mdp3）作为一种潜在的微管结合蛋白。然而，其亚细胞定位和功能重要性尚未确定。在这项研究中，通过 GST 下拉实验，我们发现 Mdp3 既在细胞内又在体外与微管蛋白相互作用。免疫荧光显微镜和微管共沉淀实验显示 Mdp3 也与微管结合。串联缺失实验表明 Mdp3 的两个卷曲螺旋基序对于其与微管蛋白和微管的相互作用至关重要。冷恢复和诺考达唑洗脱实验进一步证明了 Mdp3 在调节细胞微管组装中的重要作用。我们的数据还表明，Mdp3 显著增强了细胞微管的稳定性。通过微管浊度测定，我们发现 Mdp3 可以在纯化系统中促进微管组装和稳定性。此外，我们发现 Mdp3 的表达在细胞周期和原组织中发生变化。这些发现因此确立了 Mdp3 作为一种新的微管结合蛋白，调节微管组装和稳定性。
