Wang X, Traub P
Max-Planck-Institut für Zellbiologie, Ladenburg bei Heidelberg, Federal Republic of Germany.
J Cell Sci. 1991 Jan;98 ( Pt 1):107-22. doi: 10.1242/jcs.98.1.107.
The karyo-cytoskeleton of cells cultured in vitro was investigated employing resinless section immunogold electron microscopy. Cells were entrapped in low-melting agarose, sequentially extracted with various buffers and digested with nucleases to obtain karyo-cytoskeletal frameworks and reacted with specific primary and gold-conjugated secondary antibodies or gold-conjugated protein A to decorate structural elements of these frameworks. Following embedment of the gold-labeled residual cell structures in diethylene glycol distearate and their sectioning, the embedding material was removed with organic solvent and the sections were finally subjected to CO2 critical point drying. When this technique was applied to mouse skin fibroblasts (MSF), it revealed a dense and salt-stable intranuclear network of fibrogranular material. Antibodies directed against vimentin and lamin B detected a cytoplasmic meshwork of intermediate filaments (IFs) and a nuclear lamina, respectively; the latter, however, only after removal of chromatin from nuclei by nuclease digestion of DNA. Intranuclear filaments free of adhering globular material were morphologically very similar to cytoplasmic vimentin filaments. By contrast, mouse plasmacytoma MPC-11 cells lacking detectable amounts of cytoplasmic IF proteins and lamins A and C were devoid of a salt-stable internal nuclear matrix. The same holds true for MPC-11 cells that had been treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to induce vimentin synthesis and establish a cytoplasmically extended IF network. These findings were in accordance with the biochemical behavior of Triton X-100-treated MSF and MPC-11 cells and their appearance in immunofluorescence microscopy upon extraction with high ionic strength buffer. While the chromatin was quantitatively retained in the residual cell structures derived from MSF cells, in those obtained from MPC-11 cells the nuclear lamina was disrupted and the chromatin was released from the nuclei, suggesting that MPC-11 cells lack the salt-stable nuclear scaffold to which chromatin is normally anchored.
采用无树脂切片免疫金电子显微镜技术对体外培养细胞的核细胞骨架进行了研究。将细胞包埋于低熔点琼脂糖中，依次用各种缓冲液进行抽提，并用核酸酶消化，以获得核细胞骨架结构，然后与特异性一抗和金标记二抗或金标记蛋白A反应，对这些结构的结构成分进行标记。将金标记的残余细胞结构包埋于二乙二醇二硬脂酸酯中并进行切片后，用有机溶剂去除包埋材料，最后对切片进行二氧化碳临界点干燥处理。当将该技术应用于小鼠皮肤成纤维细胞（MSF）时，发现了一种致密且对盐稳定的核内纤维颗粒物质网络。针对波形蛋白和核纤层蛋白B的抗体分别检测到中间丝（IFs）的细胞质网络和核纤层；然而，只有在通过DNA核酸酶消化从细胞核中去除染色质后才能检测到核纤层。不含附着球状物质的核内细丝在形态上与细胞质波形蛋白细丝非常相似。相比之下，缺乏可检测量的细胞质IF蛋白以及核纤层蛋白A和C的小鼠浆细胞瘤MPC-11细胞没有盐稳定的核内基质。用佛波酯12-O-十四酰佛波醇-13-乙酸酯处理以诱导波形蛋白合成并建立细胞质延伸的IF网络的MPC-11细胞也是如此。这些发现与用Triton X-100处理的MSF和MPC-11细胞的生化行为及其在高离子强度缓冲液抽提后的免疫荧光显微镜下的外观一致。虽然染色质在源自MSF细胞的残余细胞结构中被定量保留，但在源自MPC-11细胞的残余细胞结构中，核纤层被破坏，染色质从细胞核中释放出来，这表明MPC-11细胞缺乏染色质通常锚定的盐稳定核支架。
