Wang X M, Peloquin J G, Zhai Y, Bulinski J C, Borisy G G
Laboratory of Molecular Biology, University of Wisconsin-Madison 53706, USA.
J Cell Biol. 1996 Feb;132(3):345-57. doi: 10.1083/jcb.132.3.345.
Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.
微管相关蛋白4（MAP4）在体外促进微管组装，且在体内定位于微管上。这些结果以及MAP4是非神经元细胞中的主要微管相关蛋白这一事实表明，MAP4的正常功能可能包括原位稳定微管。为了解MAP4在体内的功能，我们制备了一种阻断抗体（Ab）以防止MAP4与微管结合。MAP4的COOH末端微管结合结构域在大肠杆菌中作为谷胱甘肽转移酶融合蛋白表达，并注射到兔子体内以产生抗血清，然后对其进行亲和纯化，结果表明该抗血清对MAP4具有单特异性。在体外试验中，这种抗体阻断了>95%的MAP4与微管的结合。用针对MAP4 NH2末端突出结构域的大鼠多克隆抗体检测，将亲和纯化的抗体显微注射到人成纤维细胞和猴上皮细胞中，消除了MAP4与微管的结合。MAP4从微管上移除的同时，它被隔离到可见的MAP4-Ab免疫复合物中。然而，微管网络看起来正常。微管蛋白光活化和诺考达唑敏感性试验表明，从微管上移除MAP4后，微管动力学没有明显改变。在没有MAP4与微管结合的情况下，细胞能够进入有丝分裂，纺锤体形态正常。从微管上耗尽MAP4也不影响微管蛋白亚基的翻译后修饰状态。此外，未检测到微管依赖性细胞器分布的扰动。我们得出结论，MAP4与微管的结合对于微管组装或我们能够检测的培养细胞中基于微管的功能并非必不可少。然而，这些结果并不排除MAP4具有重要作用，因为MAP4可能是功能冗余系统的一个组成部分。
