Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM)
Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM)
复制标题
通过全内反射荧光显微镜 (TIRFM) 解构波形蛋白组装的后期
作者：
Stefan H Winheim;Aaron R Hieb;M. Silbermann;Eva;T. Wedig;H. Herrmann;J. Langowski;N. Mücke
在组装过程的高级阶段对中间丝 (IF) 进行定量成像在技术上很困难，因为结构有几微米长，因此它们超出了许多电子 (EM) 或原子力显微镜 (AFM) 技术的视野。因此，定量研究变得极其费力和耗时。为了克服这些困难，我们制备了荧光标记的波形蛋白，用于通过全内反射荧光显微镜（TIRFM）进行可视化。为了研究标记是否影响蛋白质的组装特性，我们首先通过分析超速离心在低离子条件下确定未标记波形蛋白与增加量的标记波形蛋白混合的缔合状态。我们发现，即使一半的波形蛋白被标记，也能形成真正的四聚体复合物。此外，我们通过定量原子力显微镜和电子显微镜证明，当标记的波形蛋白的比例低于10%时，丝的形态和组装特性不受影响。使用快速帧速率，我们通过 TIRFM 实时观察到荧光标记 IF 在玻璃支撑物上的快速沉积。通过追踪它们的轮廓，我们计算出长固定波形蛋白 IF 的持续长度为 1 µm，该值与较短的未标记波形蛋白所确定的值相同。这些结果表明，长丝的结构性能并未受到染料的显着影响。此外，为了分析后期伸长阶段，我们混合了含有 Alexa 488 或 Alexa 647 标记的波形蛋白的长丝。所获得的长丝的“片状”结构明确地显示了通过对单个长丝进行直接端到端退火而实现的长 IF 的伸长。
Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several µm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 µm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The ‘patchy’ structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments.
影响因子：
5.6
作者：
Mücke, N;Wedig, T;Herrmann, H
通讯作者：
Herrmann, H
影响因子：
5.6
作者：
Rivetti, C;Guthold, M;Bustamante, C
通讯作者：
Bustamante, C
影响因子：
56.9
作者：
BONIFAS, JM;ROTHMAN, AL;EPSTEIN, EH
通讯作者：
EPSTEIN, EH