Assessing the flexibility of intermediate filaments by atomic force microscopy
Assessing the flexibility of intermediate filaments by atomic force microscopy
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DOI：
10.1016/j.jmb.2003.11.038
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发表时间：
2004-01-30
影响因子：
5.6
通讯作者：
Langowski, J
中科院分区：
文献类型：
--
作者：
Mücke, N;Kreplak, L;Langowski, J
真核细胞包含三种细胞骨架细丝系统，它们具有非常不同的组装特性、超分子结构、动力学行为和机械特性。微管和微丝是相对刚性的极性结构，其组装由结合的核苷酸的水解状态调节。相比之下，中间丝（IF）是更灵活的非极性结构，由类似于45 nm长的卷曲螺旋二聚体作为基本构建块组装而成。通过比较负染色脱水丝的电子显微照片和通过各种物理方法直接测量F-肌动蛋白丝（类似于3 - 10 μ m）和微管（类似于1 - 8 mm）的持久长度，定性地描述了三种丝系统之间存在的柔韧性差异。然而，关于持续长度的IFs的定量数据仍然missing.Toward这一目标，我们已经进行了原子力显微镜（AFM）在生理缓冲液中，以确定单个波形蛋白IFs吸附到不同的固体支持物的形态。此外，我们将这些图像与通过负染色脱水细丝的透射电子显微镜（TEM）获得的图像进行了比较。对于每个支持，我们可以准确地测量的长丝的表观持久性长度，产生的值范围在0.3妈妈和1妈妈。对吸附机制进行简单的假设，我们可以估计IF在稀溶液中的持久长度近似于1 μ m，这表明较低的测量值反映了由相应支撑物上的细丝的吸附过程引起的约束。我们推断，与F-肌动蛋白丝相比，IF的持久长度较低是由于卷曲螺旋二聚体内存在柔性接头区域以及假定IF内二聚体之间发生轴向滑动所致。(C)2003爱思唯尔有限公司。保留所有权利。
Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a similar to 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (similar to3 - 10 mum) and microtubules (similar to 1 - 8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing.Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 mum and 1 mum. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be similar to 1 mum, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support.Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within lFs. (C) 2003 Elsevier Ltd. All rights reserved.