Chapin S J, Bulinski J C
Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Cell Motil Cytoskeleton. 1994;27(2):133-49. doi: 10.1002/cm.970270205.
Previous immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule-associated protein of M(r) approximately 210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802-808; DeBrabander et al. (1981) J. Cell Biol. 91:438-455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double-label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277-288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC-7), and those made by human neuroblastoma cells (IMR-32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC-7 cells, we also observed MAP4-depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity.
先前使用多种灵长类动物培养细胞系进行的免疫定位研究表明，一种分子量约为210,000的微管相关蛋白（现称为MAP4）存在于间期和有丝分裂细胞的微管全长上[布林斯基和博里西（1980年）《细胞生物学杂志》87:802 - 808；德布拉班德等人（1981年）《细胞生物学杂志》91:438 - 455]。由于MAP4被认为是一种微管稳定剂，我们询问是否所有类型的微管都含有等量的MAP4。我们使用传统的双标记免疫荧光和抗体阻断技术[舒尔茨和基尔希纳（1987年）《细胞生物学杂志》104:277 - 288]重新检查了MAP4的细胞分布，以突出缺乏或耗尽MAP4的微管。这些技术表明，从猴肾细胞（TC - 7）延伸出的细突起，以及人神经母细胞瘤细胞（IMR - 32）在视黄酸作用下形成的突起，通常缺乏MAP4免疫反应性。由于这两种类型的细胞突起都含有稳定的微管，这些微管富含去酪氨酸化（Glu）微管蛋白，我们在体外测试了MAP4与由纯Glu微管蛋白和纯酪氨酸化（Tyr）微管蛋白制成的微管结合的能力。MAP4与这两种类型的微管都结合，并且MAP4与Glu微管和Tyr微管结合的饱和水平相似，这表明与这些形式的微管蛋白的差异结合并不直接导致体内MAP4在微管上分离的机制。在TC - 7细胞中，我们还观察到单个微管、宽阔细胞质延伸的远端区域以及分裂细胞的中体上存在MAP4耗尽现象。MAP4耗尽可能反映了微管最近的快速生长，MAP4尚未与之结合，或者存在其他可能与MAP4竞争微管上结合位点的微管相关蛋白。我们认为，微管上不同水平的MAP4可能直接调节单个细胞内的微管动力学，以及其他微管功能，如涉及微管运动活性的功能。
