Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy

Abstract

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures.

A cryo-ET survey of microtubules and intracellular compartments in mammalian axons

“…The intermediate filaments we found have less density in their core, supporting the idea that they are tubular ( Parry and Steinert, 1999 ; Watts et al, 2002 ). During the revision of this paper, two cryo-ET studies reported that the nonneuronal intermediate filaments vimentin and keratin are also tubular ( Eibauer et al, 2021 Preprint ; Weber et al, 2021 ). This architecture is therefore likely a common feature of these filaments.…”

Molecular Interactions Driving Intermediate Filament Assembly

“…Here also, their heterogeneity is a major obstacle. In a recent cryoEM study [ 61 ], in vivo assembled keratin networks were shown to feature significant diameter fluctuations even along a single filament. Moreover, the 10 nm cytoplasmic filaments appeared quite smooth and lack specific surface features.…”

“…At the same time, the filaments isolated from living cells are obviously presenting a very attractive study object. However, heterogeneity was also observed for such filaments [ 61 ]. Several post-translation modifications are known to affect the assembly process of IF proteins [ 22 ].…”

Filament assembly of the C. elegans lamin in the absence of helix 1A

“…This was possible because each extracted particle was saved with its original coordinates. The filaments are reconstituted in silico [ 11 , 35 ]. To properly analyze reconstituted filaments, we also applied an unbending algorithm from ImageJ [ 36 ].…”