Salas P J
University of Miami School of Medicine, Department of Cell Biology and Anatomy, Miami, Florida 33101, USA.
J Cell Biol. 1999 Aug 9;146(3):645-58. doi: 10.1083/jcb.146.3.645.
We have previously shown that a thin ( approximately 1 microm) layer of intermediate filaments located beneath the apical membrane of a variety of simple epithelial cells participates in the organization of apical microfilaments and microtubules. Here, I confirmed the apical distribution of gamma-tubulin-containing structures (potential microtubule-organizing centers) in CACO-2 cells and demonstrated perfect colocalization of centrosomes and nearly 50% of noncentrosomal gamma-tubulin with apical intermediate filaments, but not with apical F-actin. Furthermore, the antisense-oligonucleotide-mediated downregulation of cytokeratin 19, using two different antisense sequences, was more efficient than anticytoskeletal agents to delocalize centrosomes. Electron microscopy colocalization suggests that binding occurs at the outer boundary of the pericentriolar material. Type I cytokeratins 18 and 19 present in these cells specifically coimmunoprecipitated in multi-protein fragments of the cytoskeleton with gamma-tubulin. The size and shape of the fragments, visualized at the EM level, indicate that physical trapping is an unlikely explanation for this result. Drastic changes in the extraction protocol did not affect coimmunoprecipitation. These results from three independent techniques, indicate that insoluble gamma-tubulin-containing structures are attached to apical intermediate filaments.
我们之前已经表明，位于多种单层上皮细胞顶端膜下方的一层薄薄的（约1微米）中间丝参与了顶端微丝和微管的组织。在此，我证实了CACO - 2细胞中含γ - 微管蛋白结构（潜在的微管组织中心）的顶端分布，并证明中心体和近50%的非中心体γ - 微管蛋白与顶端中间丝完美共定位，但与顶端F - 肌动蛋白不共定位。此外，使用两种不同的反义序列，通过反义寡核苷酸介导的细胞角蛋白19下调比抗细胞骨架剂更有效地使中心体脱离定位。电子显微镜共定位表明结合发生在中心粒周围物质的外边界。这些细胞中存在的I型细胞角蛋白18和19与γ - 微管蛋白在细胞骨架的多蛋白片段中特异性共免疫沉淀。在电子显微镜水平观察到的片段大小和形状表明，物理捕获不太可能解释这一结果。提取方案的剧烈变化并不影响共免疫沉淀。这三种独立技术的结果表明，不溶性含γ - 微管蛋白的结构附着于顶端中间丝。
