Miller R K, Vikstrom K, Goldman R D
Northwestern University Medical School, Department of Cell, Molecular, and Structural Biology, Chicago, Illinois 60611.
J Cell Biol. 1991 May;113(4):843-55. doi: 10.1083/jcb.113.4.843.
The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.
在对生物素化角蛋白进行显微注射后，研究了体内含角蛋白的中间丝（IF）网络的特性。角蛋白中间丝被生物素化、拆解，并通过离子交换色谱法分离为I型和II型蛋白。这些衍生化的I型和II型角蛋白重新组合形成了直径为10纳米的中间丝。将I型角蛋白显微注射到上皮细胞中，并通过免疫荧光显微镜观察。注射后15 - 20分钟，在整个细胞质中发现了富含生物素的斑点。注射后30 - 45分钟可见短的生物素化纤维结构，其中大部分与内源性中间丝束（张力丝）共定位。显微注射后1.5至2小时，明显可见广泛的生物素化角蛋白中间丝样网络。这些网络与整个细胞内的内源性张力丝高度重合，包括桥粒连接处的张力丝。这些结果表明，沿着内源性张力丝束的长度存在多个位点的相对快速的亚基掺入机制。这些观察结果支持角蛋白中间丝是动态细胞骨架成分的观点。
