Norlén Lars, Masich Sergej, Goldie Kenneth N, Hoenger Andreas
Medical Nobel Institute, Department of Cellular and Molecular Biology (CMB), Karolinska Institute, and Dermatology Clinic, Karolinska University Hospital, Stockholm, Sweden.
Exp Cell Res. 2007 Jun 10;313(10):2217-27. doi: 10.1016/j.yexcr.2007.03.037. Epub 2007 Apr 11.
Intermediate filaments are a large and structurally diverse group of cellular filaments that are classified into five different groups. They are referred to as intermediate filaments (IFs) because they are intermediate in diameter between the two other cytoskeletal filament systems that is filamentous actin and microtubules. The basic building block of IFs is a predominantly alpha-helical rod with variable length globular N- and C-terminal domains. On the ultra-structural level there are two major differences between IFs and microtubules or actin filaments: IFs are non-polar, and they do not exhibit large globular domains. IF molecules associate via a coiled-coil interaction into dimers and higher oligomers. Structural investigations into the molecular building plan of IFs have been performed with a variety of biophysical and imaging methods such as negative staining and metal-shadowing electron microscopy (EM), mass determination by scanning transmission EM, X-ray crystallography on fragments of the IF stalk and low-angle X-ray scattering. The actual packing of IF dimers into a long filament varies between the different families. Typically the dimers form so called protofibrils that further assemble into a filament. Here we introduce new cryo-imaging methods for structural investigations of IFs in vitro and in vivo, i.e., cryo-electron microscopy and cryo-electron tomography, as well as associated techniques such as the preparation and handling of vitrified sections of cellular specimens.
中间丝是一大类结构多样的细胞丝，可分为五个不同的组。它们被称为中间丝（IFs），是因为其直径介于另外两种细胞骨架丝系统（即丝状肌动蛋白和微管）之间。中间丝的基本构建单元是一个主要为α螺旋的杆状结构，其N端和C端结构域为可变长度的球状结构。在超微结构层面，中间丝与微管或肌动蛋白丝有两个主要区别：中间丝是非极性的，且不呈现大的球状结构域。中间丝分子通过卷曲螺旋相互作用形成二聚体和更高阶的寡聚体。人们已运用多种生物物理和成像方法对中间丝的分子构建方案进行了结构研究，如负染色和金属投影电子显微镜（EM）、扫描透射EM进行质量测定、对中间丝杆状片段进行X射线晶体学分析以及低角度X射线散射分析。不同家族中，中间丝二聚体实际组装成长丝的方式有所不同。通常，二聚体形成所谓的原纤维，原纤维进一步组装成丝。在此，我们介绍用于体外和体内中间丝结构研究的新低温成像方法，即低温电子显微镜和低温电子断层扫描，以及相关技术，如细胞标本玻璃化切片的制备和处理。
