Traub P, Bauer C, Hartig R, Grüb S, Stahl J
Max-Planck-Institut für Zellbiologie, Ladenburg/Heidelberg, Germany.
Biol Cell. 1998 Jul;90(4):319-37.
Previous experiments have revealed a relatively weak electrostatic binding capacity of in vitro reconstituted intermediate filaments (IFs) as well as of natural IFs of whole cell mount preparations for purified ribosomal particles of mammalian origin. In order to demonstrate that such associations also occur in vivo, intact cells were subjected to double immunofluorescence microscopy using antibodies directed against vimentin and ribosomal protein S17. Since in proliferating cells the majority of the ribosomal particles are assembled into polyribosomes and these are to a great extent associated with microfilaments, in vitro cultured mouse embryo skin fibroblasts (MSF cells) were treated with puromycin to allow the formation of single ribosomes. Employing confocal laser scanning microscopy, the ribosomes were detected in colocalization with vimentin IFs. Disassembly of polyribosomes was also achieved by serum starvation of cultured cells. In this case, MSF cells of a low passage attained an extended and flattened appearance with the vimentin IFs being directly associated with the cell nuclei, radiating into the peripheral areas of the cells or showing a stress fiber-like distribution. In both cases, considerable quantities of ribosomal material were seen in close neighborhood to vimentin IFs. Frequently, these ribosome-IF associations were coaligned with microtubules and they also surrounded myosin I-decorated stress fibers. Double labeling with the vital, RNA-specific fluorochrome SYTO 14 produced a fluorescence pattern largely superimposable on that of ribosomal protein S17. Treatment of the starved cells with either demecolcine or cytochalasin D had an only moderately disturbing effect on vimentin IF distribution and the ribosomes stayed in contact with the vimentin IFs. On the basis of these results, it is conceivable that IFs play a role in the storage of ribonucleoprotein particles in general and non-translating ribosomes in particular in the cytoplasm of animal cells. In addition, the often seen coalignment of IFs with microtubules and microfilaments might serve facilitated and directional transport of ribonucleoprotein particles from the nucleus to peripheral areas of the cell.
先前的实验表明，体外重构的中间丝（IFs）以及全细胞装片制备物中的天然IFs对哺乳动物来源的纯化核糖体颗粒的静电结合能力相对较弱。为了证明这种结合也发生在体内，使用针对波形蛋白和核糖体蛋白S17的抗体对完整细胞进行双重免疫荧光显微镜检查。由于在增殖细胞中，大多数核糖体颗粒组装成多核糖体，并且这些多核糖体在很大程度上与微丝相关，因此用嘌呤霉素处理体外培养的小鼠胚胎皮肤成纤维细胞（MSF细胞），以允许形成单个核糖体。利用共聚焦激光扫描显微镜，检测到核糖体与波形蛋白IFs共定位。通过培养细胞的血清饥饿也可实现多核糖体的解体。在这种情况下，低传代的MSF细胞呈现出延长和平坦的外观，波形蛋白IFs直接与细胞核相关联，辐射到细胞的周边区域或呈现应力纤维样分布。在这两种情况下，都可以看到大量核糖体物质紧邻波形蛋白IFs。这些核糖体-IF结合常常与微管共线，并且它们还围绕着肌球蛋白I标记的应力纤维。用活性RNA特异性荧光染料SYTO 14进行双重标记产生的荧光模式与核糖体蛋白S17的荧光模式基本重叠。用秋水仙碱或细胞松弛素D处理饥饿细胞对波形蛋白IF分布只有适度的干扰作用，核糖体仍与波形蛋白IFs保持接触。基于这些结果，可以设想IFs在动物细胞细胞质中一般的核糖核蛋白颗粒储存，特别是非翻译核糖体的储存中发挥作用。此外，IFs与微管和微丝经常共线可能有助于核糖核蛋白颗粒从细胞核到细胞周边区域的定向运输。
