Fujimoto T, Kogo H, Nomura R, Une T
Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
J Cell Sci. 2000 Oct;113 Pt 19:3509-17. doi: 10.1242/jcs.113.19.3509.
The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.
研究了小窝蛋白-1的α和β亚型与小窝超微结构之间的关系。通过对人成纤维细胞进行免疫荧光显微镜观察，小窝被视为小窝蛋白-1阳性的点状结构，但许多被识别两种亚型的抗体（抗αβ）标记的点状结构并未被另一种针对α亚型的特异性抗体（抗α）标记。对冷冻断裂复制品进行免疫金电子显微镜观察发现了不同深度的小窝，并表明抗α优先标记深小窝而非浅小窝，而抗αβ以相同的频率和强度标记两种形式。通过标记表位标签化的β-小窝蛋白证实了深小窝中存在β亚型。当在缺乏内源性小窝蛋白的HepG2细胞中表达时，α亚型能有效地形成小窝凹陷，但β亚型几乎不能。在表达两种亚型的细胞中也形成了小窝，但其频率在同一克隆的细胞中存在差异。与单独的小窝蛋白-1相比，小窝蛋白-1和小窝蛋白-2的共表达导致深小窝的形成更有效。结果表明，小窝蛋白-1的两种亚型在形成小窝结构方面具有不同的潜力，更重要的是，深小窝和浅小窝在分子组成上可能存在差异。
