Cell cycle changes in A-type lamin associations detected in human dermal fibroblasts using monoclonal antibodies
Cell cycle changes in A-type lamin associations detected in human dermal fibroblasts using monoclonal antibodies
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DOI：
10.1023/a:1018496309156
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发表时间：
1997-09-01
影响因子：
2.6
通讯作者：
Hutchison, CJ
中科院分区：
文献类型：
--
作者：
Dyer, JA;Kill, IR;Hutchison, CJ
产生了一组新的抗A型纤层蛋白单克隆抗体。通过免疫印迹对GST-lamin融合肽进行表位定位。用双间接免疫荧光法比较了人真皮成纤维细胞（HDFs）增殖和静止培养中A型层粘胶蛋白的分布与增殖标志物Ki67的分布。抗体被定位到核纤层蛋白C尾部的一个区域，对增殖细胞和静止细胞的核膜进行了同样明亮的染色。相比之下，识别层粘连蛋白A和C的头结构域和杆结构域以及层粘连蛋白A的尾结构域的表位的抗体对静止细胞的核膜染色强烈，但与增殖细胞的核膜反应差或根本不反应。免疫印迹法未检测到层粘连蛋白A和层粘连蛋白C表达水平的变化。然而，表位掩蔽被揭示，这是通过两种不同的机制发生的。层粘连蛋白A和C头部结构域的表位掩蔽是蛋白磷酸化的结果。层状蛋白A和C的棒结构域和层状蛋白A的尾结构域的表位掩蔽是通过层状蛋白与染色质和/或其他核蛋白之间的物理联系发生的。在血清饥饿后重新刺激的HDFs中，研究了表位掩蔽的细胞周期定时。在再刺激的细胞中，广泛的表位掩蔽只发生在细胞经过有丝分裂后。这些结果与假设一致，即当细胞从静止状态发展到增殖状态时，a型层蛋白丝的重排导致了层间关系的改变。
A new panel of anti-A-type lamin monoclonal antibodies was generated. Epitope mapping was performed by immunoblotting against GST-lamin fusion peptides. Epitopes were mapped to four different regions of human lamin A and three different regions of human lamin, C. The distribution of A-type lamins was compared with the distribution of the proliferation marker Ki67 in proliferating and quiescent cultures of human dermal fibroblasts (HDFs) using a double indirect immunofluorescence assay. Antibodies that had been mapped to a region of the lamin C tail stained the nuclear envelope of proliferating and quiescent cells equally brightly. In contrast, antibodies recognizing epitopes in the head domain and rod domain of lamins A and C and the tail domain of lamin A stained the nuclear envelope of quiescent cells strongly but reacted poorly or not at all with the nuclear envelope of proliferating cells. Changes in the level of expression of lamins A and C were not detected in immunoblotting assays. However, epitope masking was revealed, and this occurred by two distinct mechanisms. Epitope masking in the head domain of lamins A and C occurred as a result of protein phosphorylation. Epitope masking in the rod domain of lamins A and C and in the tail domain of lamin A occurred through a physical association between the lamin and chromatin and/or other nuclear proteins. The cell cycle timing of epitope masking was investigated in HDFs that had been restimulated after serum starvation. Extensive epitope masking in restimulated cells only occurred after cells had passed through mitosis. These results ape consistent with the hypothesis that rearrangement of A-type lamin filaments, as cells progress from a quiescent to a proliferating state, results in altered lamina associations.