Unveiling the polarity of actin filaments by cryo-electron tomography

Martins, Bruno; Sorrentino, Simona; Chung, Wen-Lu; Tatli, Meltem; Medalia, Ohad; Eibauer, Matthias

doi:10.1016/j.str.2020.12.014

Cited by 40 publications

(49 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To understand how these structural features are organized at the level of long keratin filaments, it was important to determine how the class averages are arranged along keratin filaments which are up to several hundreds of nanometers in length. For this purpose, we utilized a back-mapping strategy that permits the computational reconstitution of the original filament out of 2D class averages ( Figure 2—figure supplement 1A,B ; Kronenberg-Tenga et al, 2021 ; Martins et al, 2021 ). Therefore, every segment was represented by its corresponding 2D class image, which was inversely transformed, so that it matched the original orientation of the raw segment.…”

Section: Resultsmentioning

confidence: 99%

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

Weber

Eibauer

Sivagurunathan

et al. 2021

eLife

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

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

Weber

Eibauer

Sivagurunathan

et al. 2021

eLife

Self Cite

View full text Add to dashboard Cite

show abstract

“…It is therefore essential researchers increase the resolution of cryo-TEM and -ET technologies (e.g., TITAN Krios cryo-TEM) in order to correctly assess the arrangement and polarity of actin filament bundles within TNTs. The application of novel computational approaches elegantly developed by Medalia and colleagues to study actin filament polarity in cryo-EM datasets will assist in this endeavor (Martins et al, 2021).…”

Section: Cytoskeletal Architecturementioning

confidence: 99%

Peering into tunneling nanotubes—The path forward

Cervantes

Zurzolo

2021

The EMBO Journal

View full text Add to dashboard Cite

The identification of Tunneling Nanotubes (TNTs) and TNT‐like structures signified a critical turning point in the field of cell‐cell communication. With hypothesized roles in development and disease progression, TNTs’ ability to transport biological cargo between distant cells has elevated these structures to a unique and privileged position among other mechanisms of intercellular communication. However, the field faces numerous challenges—some of the most pressing issues being the demonstration of TNTs in vivo and understanding how they form and function. Another stumbling block is represented by the vast disparity in structures classified as TNTs. In order to address this ambiguity, we propose a clear nomenclature and provide a comprehensive overview of the existing knowledge concerning TNTs. We also discuss their structure, formation‐related pathways, biological function, as well as their proposed role in disease. Furthermore, we pinpoint gaps and dichotomies found across the field and highlight unexplored research avenues. Lastly, we review the methods employed to date and suggest the application of new technologies to better understand these elusive biological structures.

show abstract

“…Using a convolutional neural network 44 , we identified and extracted 390,297 segments of VIFs with a box size of 65 x 65 x 65 nm 3 . These segments were projected along the direction of the electron beam and subsequently subjected to a 2D classification procedure 45,46 (Supplementary Fig. 1c).…”

Section: Cryo-et Of Vifsmentioning

confidence: 99%

“…Resolving the structural fluctuations along the VIFs requires the reconstruction of extended polymer stretches 45,53 . Therefore, we extracted smaller segments (38 x 38 nm 2 ) and decreased the distance separating them, in order to increase the number of samples to more accurately track the natural bending of the VIFs.…”

Section: Long-range Tracing Of Vifsmentioning

confidence: 99%

The molecular architecture of vimentin filaments

Eibauer

Weber

Turgay

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Intermediate filaments are integral components of the cytoskeleton in metazoan cells. Due to their specific viscoelastic properties they are principal contributors to flexibility and tear strength of cells and tissues. Vimentin, an intermediate filament protein expressed in fibroblasts and endothelial cells, assembles into ~11 nm thick biopolymers, that are involved in a wide variety of cellular functions in health and disease. Here, we reveal the structure of in-situ polymerized vimentin filaments to a subnanometer resolution by applying cryo-electron tomography to mouse embryonic fibroblasts grown on electron microscopy grids. We show that vimentin filaments are tube-like assemblies with a well-defined helical symmetry. Their structure is comprised of five octameric, spring-like protofibrils harboring 40 vimentin polypeptide chains in cross-section. The protofibrils are connected by the intrinsically disordered head and helix 1A domains of vimentin. Individual filaments display two polymerization states characterized by either the presence or absence of a luminal density along the helical axis. The structure of vimentin filaments unveils the generic building plan of the intermediate filament superfamily in molecular details.

show abstract

Unveiling the polarity of actin filaments by cryo-electron tomography

Abstract: Highlights d Determining the polarity of individual actin filaments inside cells d Reconstruction of actin networks from cryo-tomograms d The polarity of actin changes from mixed to uniform along focal adhesions

Cited by 40 publications

References 59 publications

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

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

Peering into tunneling nanotubes—The path forward

The molecular architecture of vimentin filaments

Contact Info

Product

Resources

About