Domínguez J E, Buendia B, López-Otín C, Antony C, Karsenti E, Avila J
Centro de Biología Molecular (CSIC-UAM), Fac. Ciencias UAM Cantoblanco, Madrid, Spain.
J Cell Sci. 1994 Feb;107 ( Pt 2):601-11.
The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubules nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immunofluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps. These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericentriolar material.
中心体是哺乳动物细胞主要的微管组织中心。从结构上看，它由一对中心粒组成，周围环绕着一种纤维颗粒物质（中心粒周围物质），微管由此成核。然而，参与微管成核的中心体分子的性质仍不清楚。由于脑微管相关蛋白（MAPs）在体外能降低微管蛋白溶液中微管成核所需的临界微管蛋白浓度，我们研究了它们与中心体的可能关联。通过免疫荧光法，针对MAP1B产生的单克隆抗体和多克隆抗体能使培养细胞以及纯化的中心体中的中心体染色，而针对MAP2产生的抗体则给出完全阴性反应。免疫电子显微镜显示，与MAP1B相关的抗原定位于中心粒周围物质。在聚丙烯酰胺凝胶上分析的纯化中心体制剂中，有一种迁移行为与脑MAP1B相同的蛋白质。转移到硝酸纤维素膜上后，它能被抗MAP1B抗体识别，并且该蛋白质水解片段的氨基酸序列与脑MAP1B相似。此外，脑MAP1B及其中心体对应物具有相同的磷酸化特征和相似的肽图。这些数据有力地表明，中心体中存在一种与MAP1B同源的蛋白质，它很可能参与中心粒周围物质的成核活性。
