scholarly journals Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina

2019 ◽  
Author(s):  
K. Tanuj Sapra ◽  
Zhao Qin ◽  
Anna Dubrovsky-Gaupp ◽  
Ueli Aebi ◽  
Daniel J. Müller ◽  
...  
Keyword(s):  
Mechanical Response ◽  
Mechanical Stability ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Small World ◽  
Nuclear Lamina ◽  
Integrative Approach ◽  
Graph Theory Analysis ◽  
Dynamics Simulations

AbstractThe nuclear lamina – a meshwork of intermediate filaments termed lamins – functions as a mechanotransduction interface between the extracellular matrix and the nucleus via the cytoskeleton. Although lamins are primarily responsible for the mechanical stability of the nucleus in multicellular organisms, in situ characterization of lamin filaments under tension has remained elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in its three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior – they deform reversibly under a force of a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough, similar to natural silk and superior to the synthetic polymer Kevlar®. Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but the meshwork topology follows ‘small world’ properties. Our results suggest that the lamin filaments arrange to form a robust, emergent meshwork that dictates the mechanical properties of individual lamin filaments. The combined approach provides quantitative insights into the structure-function organization of lamins in situ, and implies a role of meshwork topology in laminopathies.

scholarly journals Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
K. Tanuj Sapra ◽  
Zhao Qin ◽  
Anna Dubrovsky-Gaupp ◽  
Ueli Aebi ◽  
Daniel J. Müller ◽  
...  
Keyword(s):  
Mechanical Response ◽  
Mechanical Stability ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Small World ◽  
Nuclear Lamina ◽  
Integrative Approach ◽  
Graph Theory Analysis ◽  
Dynamics Simulations ◽  
Multicellular Organisms

AbstractThe nuclear lamina—a meshwork of intermediate filaments termed lamins—is primarily responsible for the mechanical stability of the nucleus in multicellular organisms. However, structural-mechanical characterization of lamin filaments assembled in situ remains elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior – they deform reversibly at a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough similar to natural silk and superior to the synthetic polymer Kevlar®. Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but exhibits small-world properties. Our results suggest that lamin filaments arrange to form an emergent meshwork whose topology dictates the mechanical properties of individual filaments. The quantitative insights imply a role of meshwork topology in laminopathies.

Three-Dimensional Imaging of In Situ Specimens with Low-Dose Electron Tomography to Analyze Protein Conformation

2004 ◽  
Vol 2 (5) ◽  
pp. 561-567 ◽  
Author(s):  
Martina Banyay ◽  
Fredrik Gilstring ◽  
Elenor Hauzenberger ◽  
Lars-Göran Öfverstedt ◽  
Anders B. Eriksson ◽  
...  
Keyword(s):  
Protein Conformation ◽  
Low Dose ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Three Dimensional Imaging

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals Study of Pseudomonas aeruginosa PPF-1 biofilm formation using microfluidics: effect of magnesium on biofilm detachment in engineered conduits

2021 ◽  
Author(s):  
William Y. Harvey ◽  
Cynthia Gagné-Thivierge ◽  
Sepideh Fakari ◽  
Jean Barbeau ◽  
Steve Charette ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Reference Strain ◽  
Mechanical Stability ◽  
Three Dimensional ◽  
Opportunistic Pathogen ◽  
Shear Stresses ◽  
Density Maps ◽  
Biofilm Detachment ◽  
Flow Systems ◽  
Dynamics Simulations

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen in certain organisms, including humans, but can also survive and proliferate in natural and engineered water systems. Microfluidic technology can address hydrodynamic questions related to bacterial contamination of water flow systems and infrastructure. In this work, a microfluidic approach was devised to study the effect of shear stresses on biofilms from a dental unit waterline (DUWL)-isolated P. aeruginosa strain, PPF-1. During application of relevant shear stress levels to DUWLs, the response of the PPF-1 biofilm was observed and compared to a clinical P. aeruginosa reference strain, PAO1. The response measurements were repeated for biofilms exposed to additional Mg2+ ions. Using a microfluidic approach to transforming optical density maps into three-dimensional images, we applied computational fluid dynamics simulations and determined the critical shear stresses for biofilm sloughing. In the absence of Mg2+, PPF-1 biofilms showed weaker attachment than PAO1 biofilms, resulting in continuous slough/regrowth cycles triggered by applied shear stresses of 1.42 +/- 0.32 Pa. Introducing Mg2+ into the PPF-1 biofilm culture medium seemed to place the biofilm into a viscoplastic mechanical state, thereby increasing mechanical stability, which resulted in elevated tolerances to shear stresses up to a critical value of 5.43 +/- 1.52 Pa. This resulted in a propensity for less frequent but more catastrophic sloughing events like that observed for the PAO1 reference strain. This suggests that in a low ionic environment, biofilms from the PPF-1 strain can result in higher and more continuous ejection of biofilm materials, possibly leading to increased downstream colonization of engineered flow systems.

scholarly journals Microstructural deformation observed by Mueller polarimetry during traction assay on myocardium samples

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicole Tueni ◽  
Jérémy Vizet ◽  
Martin Genet ◽  
Angelo Pierangelo ◽  
Jean-Marc Allain
Keyword(s):  
Mechanical Response ◽  
Three Dimensional ◽  
Full Field ◽  
Tissue Scattering ◽  
Traction Device ◽  
First Time ◽  
Heart Wall ◽  
Mechanical Traction ◽  
Collagen Layer

AbstractDespite recent advances, the myocardial microstructure remains imperfectly understood. In particular, bundles of cardiomyocytes have been observed but their three-dimensional organisation remains debated and the associated mechanical consequences unknown. One of the major challenges remains to perform multiscale observations of the mechanical response of the heart wall. For this purpose, in this study, a full-field Mueller polarimetric imager (MPI) was combined, for the first time, with an in-situ traction device. The full-field MPI enables to obtain a macroscopic image of the explored tissue, while providing detailed information about its structure on a microscopic scale. Specifically it exploits the polarization of the light to determine various biophysical quantities related to the tissue scattering or anisotropy properties. Combined with a mechanical traction device, the full-field MPI allows to measure the evolution of such biophysical quantities during tissue stretch. We observe separation lines on the tissue, which are associated with a fast variation of the fiber orientation, and have the size of cardiomyocyte bundles. Thus, we hypothesize that these lines are the perimysium, the collagen layer surrounding these bundles. During the mechanical traction, we observe two mechanisms simultaneously. On one hand, the azimuth shows an affine behavior, meaning the orientation changes according to the tissue deformation, and showing coherence in the tissue. On the other hand, the separation lines appear to be resistant in shear and compression but weak against traction, with a forming of gaps in the tissue.

Zonal Poisson Effect on the Chondron Deformation in Articular Cartilage under Compression

2007 ◽  
Vol 342-343 ◽  
pp. 133-136
Author(s):  
Jae Bong Choi
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Mechanical Response ◽  
Three Dimensional ◽  
Pericellular Matrix ◽  
Poisson Effect ◽  
Type Vi Collagen ◽  
Confocal Images ◽  
Three Dimensional Models

The objective of this study was to quantify the zonal difference of the in situ chondron’s Poisson effect under different magnitudes of compression. Fluorescence immunolabeling for type VI collagen was used to identify the pericellular matrix (PCM) and chondron, and a series of fluorescent confocal images were recorded and reconstructed to form quantitative three-dimensional models. The zonal variations in the mechanical response of the chondron do not appear to be due to zonal differences in PCM properties, but rather seem to result from significant inhomogeneities in relative stiffnesses of the extracellular matrix (ECM) and PCM with depth.

Three-dimensional nano-structure of in situ silica in natural rubber as revealed by 3D-TEM/electron tomography

Polymer ◽  
2005 ◽  
Vol 46 (12) ◽  
pp. 4440-4446 ◽  
Author(s):  
Shinzo Kohjiya ◽  
Astushi Katoh ◽  
Junichi Shimanuki ◽  
Toshinori Hasegawa ◽  
Yuko Ikeda
Keyword(s):  
Natural Rubber ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nano Structure ◽  
In Situ Silica

scholarly journals Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

2008 ◽  
Vol 183 (5) ◽  
pp. 923-932 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Molecular Architecture ◽  
Heavy Chains ◽  
Distal Side ◽  
Cryo Electron Tomography ◽  
Dynein Arms

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.

scholarly journals In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges

2020 ◽  
Author(s):  
Beata Turoňová ◽  
Mateusz Sikora ◽  
Christoph Schürmann ◽  
Wim J. H. Hagen ◽  
Sonja Welsch ◽  
...  
Keyword(s):  
Large Scale ◽  
Vaccine Development ◽  
Electron Tomography ◽  
Structural Features ◽  
Globular Domain ◽  
Data Set ◽  
Host Cell Surface ◽  
Subtomogram Averaging ◽  
Dynamics Simulations

AbstractThe spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 Ångstrom, and is publicly available at EMPIAR-10453.

scholarly journals In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Swetha Vijayakrishnan ◽  
Marion McElwee ◽  
Colin Loney ◽  
Frazer Rixon ◽  
David Bhella
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Cell Nuclei ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Cryo Electron Tomography

Abstract Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.

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