Moudjou M, Bordes N, Paintrand M, Bornens M
Institut Curie, Section Recherche UMR 144, Paris, France.
J Cell Sci. 1996 Apr;109 ( Pt 4):875-87. doi: 10.1242/jcs.109.4.875.
The centrosome is one of the cellular organelles for which the mechanism by which it operates still remains to be unlavelled. The finding of the association with the centrosome of gamma-tubulin, a protein which belongs to the tubulin superfamily, has provided a long sought after biochemical tool with which to address centrosome function. We have generated a specific anti-gamma-tubulin polyclonal antibody to study the biochemical properties and the cellular distribution of the human lymphoblastic gamma-tubulin. Using cell fractionation and mass isolation of centrosomes, we observed that in contrast to the figures suggested by immunofluorescence, a minimum figure of 80% of total gamma-tubulin exists as a cytosolic form. The centrosomal form, for which at least half is not strongly associated with the centrosome, behaves in two-dimensional gel electrophoresis identically to the soluble form (as at least two spots of a pI of around 6). Post-embedding immunolocalization reveals that gamma-tubulin is distributed in the pericentriolar matrix but is also closely associated with centrioles. Using a combination of gel filtration, ion exchange chromatography, equilibrium sucrose gradient centrifugation and immunoprecipitation, we show that the major part of cytosolic gamma-tubulin might be involved in complexes heavier than the Tcp1 particle. We further demonstrate, by co-immunoprecipitation of gamma-tubulin and Tcp1 with either anti-Tcp1 or anti-gamma-tubulin antibodies, that a small part of gamma-tubulin participates in Tcp1-gamma-tubulin particles. Interestingly, the soluble form of gamma-tubulin co-purifies with taxol-stabilized microtubules and its association with microtubules resisted salt, ATP and GTP treatments. The existence of a centrosomal form and a large pool of cytosolic gamma-tubulin-containing complexes in somatic cells suggests that the overall gamma-tubulin cellular distribution does not seem to be as straightforward as it was drawn earlier.
中心体是一种细胞器，其运作机制仍有待阐明。γ-微管蛋白是微管蛋白超家族的一种蛋白质，它与中心体的关联被发现，这为研究中心体功能提供了一种长期以来梦寐以求的生化工具。我们制备了一种特异性抗γ-微管蛋白多克隆抗体，以研究人淋巴细胞γ-微管蛋白的生化特性和细胞分布。通过细胞分级分离和中心体的大量分离，我们观察到，与免疫荧光所显示的情况相反，至少80%的总γ-微管蛋白以胞质形式存在。中心体形式的γ-微管蛋白，其中至少一半与中心体的结合并不紧密，在二维凝胶电泳中的行为与可溶性形式相同（至少有两个等电点约为6的斑点）。包埋后免疫定位显示，γ-微管蛋白分布在中心粒周围基质中，但也与中心粒紧密相关。通过凝胶过滤、离子交换色谱、平衡蔗糖梯度离心和免疫沉淀相结合的方法，我们表明胞质γ-微管蛋白的主要部分可能参与了比Tcp1颗粒更重的复合物。我们进一步通过用抗Tcp1或抗γ-微管蛋白抗体对γ-微管蛋白和Tcp1进行共免疫沉淀证明，一小部分γ-微管蛋白参与了Tcp1-γ-微管蛋白颗粒。有趣的是，γ-微管蛋白的可溶性形式与紫杉醇稳定的微管共纯化，并且其与微管的结合能抵抗盐、ATP和GTP处理。体细胞中存在中心体形式和大量含γ-微管蛋白的胞质复合物，这表明γ-微管蛋白在细胞中的整体分布似乎不像之前所描述的那么简单直接。
