Reconstructing adhesion structures in tissues by cryo-electron tomography of vitrified frozen sections

Bokstad, Melanie; Sabanay, Helena; Dahan, Idit; Geiger, Benjamin; Medalia, Ohad

doi:10.1016/j.jsb.2011.10.013

Cited by 18 publications

(10 citation statements)

References 52 publications

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“…An interesting observation is that adhesions similar to those found in 2D can be observed at the edge (but not at the centre) of the 3D culture dish, where the anchorage of collagen bundles to the culture dish increases the rigidity of the matrix and thus the tension experienced by cells (Fraley et al, 2011). Accordingly, cells plated on soft 2D substrates show irregular and unstable focal adhesions (Pelham and Wang, 1997) whereas adhesions similar in structure and molecular composition to those found in 2D can be detected in vivo in cells submitted to high tensile forces (Bokstad et al, 2012;Ralphs et al, 2002). These observations suggest that the formation and maturation of focal adhesions is sensitive to cellular tension and substrate stiffness (Kuo, 2013;Parsons et al, 2010).…”

Section: Box 1 Front-to-back Polarity In a Migrating Cellmentioning

confidence: 95%

Cell migration: from tissue culture to embryos

2014

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Cell migration is a fundamental process that occurs during embryo development. Classic studies using in vitro culture systems have been instrumental in dissecting the principles of cell motility and highlighting how cells make use of topographical features of the substrate, cell-cell contacts, and chemical and physical environmental signals to direct their locomotion. Here, we review the guidance principles of in vitro cell locomotion and examine how they control directed cell migration in vivo during development. We focus on developmental examples in which individual guidance mechanisms have been clearly dissected, and for which the interactions among guidance cues have been explored. We also discuss how the migratory behaviours elicited by guidance mechanisms generate the stereotypical patterns of migration that shape tissues in the developing embryo.

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Section: Box 1 Front-to-back Polarity In a Migrating Cellmentioning

confidence: 95%

Cell migration: from tissue culture to embryos

2014

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“…The Tokuyasu method has been previously used to produce sections that were subsequently vitrified for cryo-EM imaging 10 . However, the use of vitrified frozen sections (VFS) has thus far been limited to imaging tissue sections by cryo-EM and cryo-ET [10][11][12] .…”

Section: Discussionmentioning

confidence: 99%

“…The method is relatively high-throughput; many sections may be prepared at once, with a greater likelihood of successful imaging. To our knowledge however, use of this method has thus far been confined to 3D imaging of tissue specimens [10][11][12][13] .…”

Section: Introductionmentioning

confidence: 99%

In situstructure determination of virus capsids imaged within cell nuclei by correlative light and cryo-electron tomography

Víjayakríshnan

McElwee

Loney

et al. 2020

Preprint

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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 at 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 equipment of limited availability. 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 reveal that the capsid associated tegument complex is present on capsids prior to nuclear egress. We show that this approach to cryogenic imaging of cells is suited to both correlative light/electron microscopy and 3D structure determination.

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“…Cryo-sectioning was carried out at a speed of 0.4-0.6 mm/s at À 105 1C using a sectioning knife (Cryo immuno 351, Diatome). Sections of 200 nm were collected from the cutting knife as five-section ribbons, picked up with a droplet of 2.3 M sucrose, applied to plasma-cleaned holey carbon-coated copper grids (Quantifoil), and left rehydrating in a pool of water for 2 h. Standard gold markers (BSA gold tracers, 10 nm, Aurion, EMS) were applied to the grids, followed by plunging them into liquid-nitrogen-cooled liquid ethane and transferring them to be stored (Bokstad et al, 2012).…”

Section: Cryo-sectioning Of Ce-lamin Microfibersmentioning

confidence: 99%

The assembly of C. elegans lamins into macroscopic fibers

Zingerman-Koladko

Khayat

Harapin

et al. 2016

Journal of the Mechanical Behavior of Biomedical Materials

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Intermediate filament (IF) proteins are known mainly by their propensity to form viscoelastic filamentous networks within cells. In addition, IF-proteins are essential parts of various biological materials, such as horn and hagfish slime threads, which exhibit a range of mechanical properties from hard to elastic. These properties and their self-assembly nature made IF-proteins attractive building blocks for biomimetic and biological materials in diverse applications. Here we show that a type V IF-protein, the Caenorhabditis elegans nuclear lamin (Ce-lamin), is a promising building block for protein-based fibers. Electron cryo-tomography of vitrified sections enabled us to depict the higher ordered assembly of the Ce-lamin into macroscopic fibers through the creation of paracrystalline fibers, which are prominent in vitro structures of lamins. The lamin fibers respond to tensile force as other IF-protein-based fibers, i.e., hagfish slime threads, and possess unique mechanical properties that may potentially be used in certain applications. The self-assembly nature of lamin proteins into a filamentous structure, which is further assembled into a complex network, can be easily modulated. This knowledge may lead to a better understanding of the relationship in IF-proteins-based fibers and materials, between their hierarchical structures and their mechanical properties.

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Reconstructing adhesion structures in tissues by cryo-electron tomography of vitrified frozen sections

Cited by 18 publications

References 52 publications

Cell migration: from tissue culture to embryos

Cell migration: from tissue culture to embryos

In situstructure determination of virus capsids imaged within cell nuclei by correlative light and cryo-electron tomography

The assembly of C. elegans lamins into macroscopic fibers

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