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

  1. Miriam S Weber
  2. Matthias Eibauer
  3. Suganya Sivagurunathan
  4. Thomas M Magin
  5. Robert D Goldman
  6. Ohad Medalia  Is a corresponding author
  1. University of Zürich, Switzerland
  2. Northwestern University Feinberg School of Medicine, United States
  3. University of Leipzig, Germany

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.

Data availability

Representative cryo-ET data have been deposited in the Electron Microscopy Data Bank under accession codes EMD-12958 and EMD-12959. In addition, data was uploaded to https://doi.org/10.5061/dryad.gqnk98sn4.

The following data sets were generated

Article and author information

Author details

  1. Miriam S Weber

    University of Zürich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  2. Matthias Eibauer

    University of Zürich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  3. Suganya Sivagurunathan

    Northwestern University Feinberg School of Medicine, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Thomas M Magin

    Institute of Biology and Translational Center for Regenerative Medicine, University of Leipzig, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Robert D Goldman

    Northwestern University Feinberg School of Medicine, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ohad Medalia

    University of Zürich, Zurich, Switzerland
    For correspondence
    omedalia@bioc.uzh.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0994-2937

Funding

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_179418)

  • Ohad Medalia

NIH Office of the Director (5PO1 GM096971)

  • Robert D Goldman

NIH Office of the Director (RO1GM140108)

  • Robert D Goldman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Weber et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,132
    views
  • 295
    downloads
  • 25
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

Share this article

https://doi.org/10.7554/eLife.70307

Further reading

    1. Cell Biology
    2. Evolutionary Biology
    Paul Richard J Yulo, Nicolas Desprat ... Heather L Hendrickson
    Research Article

    Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

    1. Cell Biology
    Kaima Tsukada, Rikiya Imamura ... Mikio Shimada
    Research Article

    Polynucleotide kinase phosphatase (PNKP) has enzymatic activities as 3′-phosphatase and 5′-kinase of DNA ends to promote DNA ligation and repair. Here, we show that cyclin-dependent kinases (CDKs) regulate the phosphorylation of threonine 118 (T118) in PNKP. This phosphorylation allows recruitment to the gapped DNA structure found in single-strand DNA (ssDNA) nicks and/or gaps between Okazaki fragments (OFs) during DNA replication. T118A (alanine)-substituted PNKP-expressing cells exhibited an accumulation of ssDNA gaps in S phase and accelerated replication fork progression. Furthermore, PNKP is involved in poly (ADP-ribose) polymerase 1 (PARP1)-dependent replication gap filling as part of a backup pathway in the absence of OFs ligation. Altogether, our data suggest that CDK-mediated PNKP phosphorylation at T118 is important for its recruitment to ssDNA gaps to proceed with OFs ligation and its backup repairs via the gap-filling pathway to maintain genome stability.