Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80S yeast ribosomes appear unaffected

Pierson, Jason; Ziese, Ulrike; Sani, Musa; Peters, Peter J.

doi:10.1016/j.jsb.2010.09.017

Cited by 39 publications

(27 citation statements)

References 40 publications

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“…A 25° cryo-immuno diamond knife (Diatome, Biel, Switzerland) with a clearance angle of 6° was used to obtain ribbons of cryosections at a nominal cutting feed of 50 nm and at cutting speeds of 10 mm/s as described (Han et al, 2008). Ribbons of cryo-sections were attached to pre-cooled 600 mesh EM grids with an eyelash using electrostatic charging with the Leica EM CRION (Leica Microsystems, Vienna, Austria) as described by (Pierson et al, 2010).…”

Section: Cemovis Of Bacillus Subtilis Sporesmentioning

confidence: 99%

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

Schertel

Snaidero

Hong-mei

et al. 2013

Journal of Structural Biology

174

113

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Volume microscopy at high resolution is increasingly required to better understand cellular functions in the context of three-dimensional assemblies. Focused ion beam (FIB) milling for serial block face imaging in the scanning electron microscope (SEM)is an efficient and fast method to generate such volume data for 3D analysis. Here, we apply this technique at cryo-conditions to image fully hydrated frozen specimen of mouse optic nerves and Bacillus subtilis spores obtained by high-pressure freezing (HPF). We established imaging conditions to directly visualize the ultrastructure in the block face at −150°C by using an in-lens secondary electron (SE) detector. By serial sectioning with a focused ion beam and block face imaging of the optic nerve we obtained a volume as large as X=7.72 µm, Y=5.79 µm and Z=3.81 µm with a lateral pixel size of 7.5 nm and a slice thickness of 30 nm in Z. The intrinsic contrast of membranes was sufficient to distinguish structures like Golgi cisternae, vesicles, endoplasmic reticulum and cristae within mitochondria and allowed for a threedimensional reconstruction of different types of mitochondria within an oligodendrocyte and an astrocytic process. Applying this technique to dormant Bacillus subtilis spores we obtained volumes containing numerous spores and discovered a bright signal in the core, which can not be related to any known structure so far. In summary, we describe the use of cryo FIB-SEM as a tool for direct and fast 3D cryo-imaging of large native frozen samples including tissues.

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Section: Cemovis Of Bacillus Subtilis Sporesmentioning

confidence: 99%

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

Schertel

Snaidero

Hong-mei

et al. 2013

Journal of Structural Biology

174

113

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“…The cutting-induced compression is an inhomogeneous volumetric deformation and thus cannot be corrected in the tomographic reconstructions. A recent report has shown that compression has an effect mainly at cellular level and has no effect on macromolecular complexes such as ribosomes (14). In addition to these problems, sections are usually not flat and thus are poorly attached to the support film.…”

mentioning

confidence: 99%

The three-dimensional molecular structure of the desmosomal plaque

Al‐Amoudi

Castaño-Díez

Devos

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a lowdensity layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.vitreous sectioning | image processing | genetic algorithms | multi-protein fitting C ell junctions are widely distributed in animal tissues and are most abundant in tissues that are subjected to considerable mechanical stress, such as heart, epidermis, and muscle. These junctions are composed of two main classes of proteins: transmembrane adhesion proteins with an extracellular domain that interacts with either the ECM (e.g., focal adhesion and hemidesmosome) or the extracellular domains of specific transmembrane adhesion proteins from the juxtaposed cell (e.g., adherens junctions and desmosomes); and a cytoplasmic tail that binds to one or more intracellular anchor proteins. These form a distinct plaque on the cytoplasmic face of the plasma membrane and connect the junctional complexes to actin filaments or intermediate filaments (IFs). When they have been assembled, these junctions play essential roles in tissue morphogenesis and maintenance; plaque disruption is a hallmark of many blistering and cancer diseases (1, 2). Biochemical and mutagenesis studies have revealed many interactions between the plaque components and their connections to the extracellular space (3-8). However, the organization and interactions of the constituent proteins in the context of the assembled junctions remain poorly understood. For example, whether or how actin filaments bind in situ to the adherens junctions and how the anchoring proteins in hemidesmosome plaque connect integrin to the IFs is still unclear.In the current study, we present the 3D cytoplasmic surface of the desmosome-the desmosomal plaque-fr...

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“…The representative area from (b) appears well vitrified, which is confirmed by the accompanying select-area diffraction pattern (b 0 ). [19,20]; all these contribute to producing ribbons of vitreous cryo-sections more quickly and in a more reproducible manner. Figure 2 displays the results of using such an equipment to produce long ribbons of vitreous cryo-sections ( Fig.…”

Section: Methods For Cryo-immoblizationmentioning

confidence: 99%

“…However, images of ribosomes [19,20] ( Fig. 3), microtubules [23] and other cellular components appear to resist compression at the given resolution, which may imply that compression is inhomogeneous.…”

Section: Methods For Cryo-ultramicrotomymentioning

confidence: 99%

“…In the best cases, using the current methods, molecular resolution can be obtained, and multi-resolution maps of macromolecular complexes that enhance the structural interpretation of such protein complexes can often be constructed [2]. Figure 4 shows one such multiresolution map from an 80S yeast ribosome constructed from semi-automated particle selection and volumetric averaging from electron cryotomograms of vitreous cryo-sections [19,20]. The measurements are presented in the graph (i), which displays the relationship between dimensions measured parallel (blue) and perpendicular (red) to the cutting direction.…”

Section: Electron Cryo-imaging Strategiesmentioning

confidence: 99%

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Perspectives on electron cryo-tomography of vitreous cryo-sections

et al. 2011

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A major objective of modern structural biology is to appreciate the cellular organization by elucidating the spatial arrangement of macromolecular complexes within a cell. Cryogenic sample preparation, combined with cryo-ultramicrotomy, enables large cells and pieces of biological tissues to be thinned for electron cryo-tomography, which provides a three-dimensional view of the biological sample. There are, however, limitations associated with the technique that must be realized, addressed and overcome for the procedure to become mainstream. Here, we provide perspectives on the continued advancements in cryogenic sample preparation for vitreous cryo-sectioning, image collection and post-image processing that have expanded the attainable information limit within the three-dimensional reconstructions of cells and pieces of biological tissues.

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Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80S yeast ribosomes appear unaffected

Cited by 39 publications

References 40 publications

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

The three-dimensional molecular structure of the desmosomal plaque

Perspectives on electron cryo-tomography of vitreous cryo-sections

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