Yoon K H, Yoon M, Moir R D, Khuon S, Flitney F W, Goldman R D
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
J Cell Biol. 2001 Apr 30;153(3):503-16. doi: 10.1083/jcb.153.3.503.
The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t(1/2) of approximately 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 microm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.
在用绿色荧光蛋白（GFP）标记的K18和/或K8（I/II型中间丝蛋白）转染后，对角蛋白中间丝（IFs）的特性进行了研究。GFP-K8和-K18被整合到张力原纤维中，张力原纤维由角蛋白IFs束组成。这些张力原纤维表现出范围广泛的运动和动态活动。光漂白后荧光恢复（FRAP）分析表明，它们以约100分钟的恢复半衰期（t(1/2)）缓慢恢复荧光。恢复过程中漂白区域的移动表明，紧密间隔的张力原纤维（间距<1微米）通常以不同速率和不同方向移动。单个张力原纤维经常改变其形状，在某些情况下，这些变化表现为沿其长轴传播的波形。此外，在细胞周边可见短纤维，称为角蛋白卷曲，它们主要向细胞中心移动。还将角蛋白IFs的运动特性与PtK2细胞中III型IFs（波形蛋白）的运动特性进行了比较。有趣的是，在同一细胞质区域内，角蛋白张力原纤维和卷曲的动态特性与波形蛋白纤维和卷曲的动态特性有显著差异。这表明在同一细胞质区域内，存在不同的因素调节不同类型IFs的动态特性。
