Structural investigations of intermediate filaments and their assembly by advanced electron microscopy

Abstract

Intermediate filaments (IFs) assemble to produce an extensive nanofibrillar, structural network in animal cells. IFs are found both in the cytoplasm and the nucleus and in association with cross-bridging proteins form a major component of Cytoskeletal architecture. A greater knowledge of the structural underpinnings of IFs is essential for the understanding of fundamental cellular processes and their possible involvement in malfunctions that Iead to disease. Detailed knowledge on mode of assembly, structure and structural integrity of IFs and their association with other Cytoskeletal components is limited as these have proven to be challenging issues in biology. This thesis has aimed to take the structural approach to study IFs to a new level using leading-edge chemical labelling techniques and advanced transmission electron microscopy (TEM) as applied to filaments generated from assembled human vimentin. In order to preserve the native organisation of IFs, traditional methods employing chemical fixatives were avoided. First, protocols were developed for the assembly of IFs in vitro. Frozen-hydrated specimen preparation methods were utilised with the goal to identify structural features at near physiological conditions by applying cryo-electron microscopy (cryo-EM). Secondly, site-specific labelling was used to narrow down the number of possible models for the assembly of IF precursors. Finally, state-of-the-art cryo-electron tomography was applied to IFs for the first time with the objective to obtain a detailed visualisation in three-dimensions. This work demonstrates that vimentin filaments in cross-section exhibit predominantly a four-stranded protofibrillar organisation with a right-handed supertwist and a helical pitch of approximately 96 nm. New structural features for the vimentin filaments were revealed from observations that both compact and untwisted conformations exist when prepared in the absence of chemical fixatives. Vitrified preparations often showed several hundred nanometer Iong compact, rectilinear regions interrupted by short bent segments which are partially unraveled and appeared flexible. This indicates the highly dynamic nature of IFs which may be physiologically relevant. Verification of site-specific gold labelling of cysteine residues within vimentin molecules was confirmed by applying analytical ultracentrifugation and electron microscopy. Such a labelling in assembled filaments did not appear to follow a rigid periodic pattern, suggesting that the IF subunits might themselves be capable of dynamic rearrangements within mature filaments. The vision is that through an improved knowledge of IF molecular structure in 3- dimensions, it will allow a better understanding of how this diverse family of filaments functions in the cytoskeleton of the Iiving cell. New insights which have been achieved and the methods developed in the course of this study constitute a strong basis for further studies on other types of IFs in vitro and eventually in Iive cell models, to elucidate if they follow a similar architecture. Ultimately, the findings embodied in this thesis contribute towards understanding the structural complexities underlying molecular mechanisms that cause IF related diseases, which may assist in the future development of new therapeutic strategies.