EXPRESSION OF MUTANT KERATIN CDNAS IN EPITHELIAL-CELLS REVEALS POSSIBLE MECHANISMS FOR INITIATION AND ASSEMBLY OF INTERMEDIATE FILAMENTS
EXPRESSION OF MUTANT KERATIN CDNAS IN EPITHELIAL-CELLS REVEALS POSSIBLE MECHANISMS FOR INITIATION AND ASSEMBLY OF INTERMEDIATE FILAMENTS
复制标题
DOI：
10.1083/jcb.108.4.1477
复制
发表时间：
1989-04-01
影响因子：
7.8
通讯作者：
FUCHS, E
中科院分区：
文献类型：
--
作者：
ALBERS, K;FUCHS, E
我们删除了编码人类I型表皮角蛋白K14氨基和羧基末端部分的cDNA序列，并研究了在简单上皮细胞和鳞状细胞癌细胞系中强迫这些突变体表达的分子后果。为了跟踪我们的突变产物在转染细胞中的表达，我们用编码神经肽P物质抗原结构域的序列标记了K14编码序列的3”末端。使用DNA转染和免疫组织化学（使用针对P物质的抗体），我们定义了将K14序列纳入体内角蛋白丝网络而不破坏其结构所需的限度。我们还发现了羧基端和氨基端的主要差异。-螺旋突变会扰乱IFs的细胞骨架网络：羧基末端突变会在细胞质中产生角蛋白聚集体，而氨基末端突变则倾向于产生角蛋白聚集体，这些角蛋白聚集体似乎位于核表面。对羧基端和氨基端突变体产生的表型和转染后细胞后期行为的检查表明，一种模型表明，丝组装的起始发生在核膜上的离散位点，丝从细胞核向细胞质生长。
We have deleted cDNA sequences encoding portions of the amino-and carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3'' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have defined the limits of K14 sequence necessary to incorporate into a keratin filament network in vivo without disrupting its architecture. We have also uncovered major differences in the behavior of carboxy-and amino-terminal .alpha.-helical mutants which do perturb the cytoskeletal network of IFs: whereas carboxy terminal mutants give rise to aggregates of keratin in the cytoplasm, amino-terminal mutants tend to produce aggregates of keratins which seem to localize at the nuclear surface. An examination of the phenotypes generated by the carboxy and amino-terminal mutants and the behavior of cells at late times after transfection suggests a model whereby initiation of filament assembly occurs at discrete sites on the nuclear envelope and filaments grow from the nucleus toward the cytoplasm.