Jahnel Oliver, Hoffmann Bernd, Merkel Rudolf, Bossinger Olaf, Leube Rudolf E
Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Aachen, Germany.
Institute of Complex Systems, ICS-7: Biomechanics, Jülich, Germany.
Methods Enzymol. 2016;568:681-706. doi: 10.1016/bs.mie.2015.08.030. Epub 2015 Nov 3.
It is commonly accepted that intermediate filaments have an important mechanical function. This function relies not only on intrinsic material properties but is also determined by dynamic interactions with other cytoskeletal filament systems, distinct cell adhesion sites, and cellular organelles which are fine-tuned by multiple signaling pathways. While aspects of these properties and processes can be studied in vitro, their full complexity can only be understood in a viable tissue context. Yet, suitable and easily accessible model systems for monitoring tissue mechanics at high precision are rare. We show that the dissected intestine of the genetic model organism Caenorhabditis elegans fulfills this requirement. The 20 intestinal cells, which are arranged in an invariant fashion, are characterized by a dense subapical mesh of intermediate filaments that are attached to the C. elegans apical junction. We present procedures to visualize details of the characteristic intermediate filament-junctional complex arrangement in living animals. We then report on methods to prepare intestines with a fully intact intermediate filament cytoskeleton and detail procedures to assess their viability. A dual micropipette assay is described to measure mechanical properties of the dissected intestine while monitoring the spatial arrangement of the intermediate filament system. Advantages of this approach are (i) the high reproducibility of measurements because of the uniform architecture of the intestine and (ii) the high degree of accessibility allowing not only mechanical manipulation of an intact tissue but also control of culture medium composition and addition of drugs as well as visualization of cell structures. With this method, examination of worms carrying mutations in the intermediate filament system, its interacting partners and its regulators will become feasible.
人们普遍认为中间丝具有重要的机械功能。这种功能不仅依赖于内在的材料特性，还由与其他细胞骨架丝系统、不同的细胞粘附位点以及由多种信号通路精细调节的细胞器的动态相互作用所决定。虽然这些特性和过程的某些方面可以在体外进行研究，但其全部复杂性只有在活组织环境中才能被理解。然而，用于高精度监测组织力学的合适且易于获取的模型系统却很少。我们表明，遗传模式生物秀丽隐杆线虫的解剖肠道满足了这一要求。20个以不变方式排列的肠道细胞的特征是在亚顶端有密集的中间丝网络，这些中间丝附着在秀丽隐杆线虫的顶端连接处。我们展示了在活体动物中可视化特征性中间丝 - 连接复合体排列细节的方法。然后我们报告了制备具有完全完整中间丝细胞骨架的肠道的方法，并详细介绍了评估其活力的程序。描述了一种双微量移液器测定法，用于测量解剖肠道的机械性能，同时监测中间丝系统的空间排列。这种方法的优点是：（i）由于肠道结构均匀，测量具有高重现性；（ii）高度可及性，不仅允许对完整组织进行机械操作，还能控制培养基成分、添加药物以及可视化细胞结构。通过这种方法，对中间丝系统、其相互作用伙伴及其调节因子携带突变的线虫进行检测将变得可行。
