Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: implications of crowding

Grünewald, Kay; Medalia, Ohad; Gross, Ariane; Steven, Alasdair C.; Baumeister, Wolfgang

doi:10.1016/s0301-4622(02)00307-1

Cited by 102 publications

(60 citation statements)

References 86 publications

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“…To determine the architecture of the thylakoid membranes of the chloroplast, we used dual-axis electron microscope tomography, which provides a powerful means to obtain high-resolution details in three dimensions for cellular structures and organelles (Mastronarde, 1997;Grunewald et al, 2003). To preserve the ultrastructure of the chloroplasts, leaf samples were cryoimmobilized by high-pressure freezing, followed by freeze substitution and resin embedding.…”

Section: Resultsmentioning

confidence: 99%

Three-Dimensional Organization of Higher-Plant Chloroplast Thylakoid Membranes Revealed by Electron Tomography

et al. 2005

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The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dualaxis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.

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Section: Resultsmentioning

confidence: 99%

Three-Dimensional Organization of Higher-Plant Chloroplast Thylakoid Membranes Revealed by Electron Tomography

et al. 2005

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“…Cell thickness is a limiting factor in such investigations, and studies have used small cells (16,17), the edge of frozen-hydrated cells (18)(19)(20), or vitreous sections (21,22). Here we report electron cryomicroscopy of whole-mount human umbilical vein endothelial cells (HUVECs) that are vitrified by rapid plunge-freezing, providing an outstanding view of WPBs and their surrounding architecture at the thin edge of the cell without the fixation, dehydration, embedding, staining, and sectioning used in earlier studies employing conventional plastic sections (23) or high pressure freezing/freeze substitution (24).…”

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confidence: 99%

Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

Berriman

Hewlett³

et al. 2009

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

140

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In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules.electron cryomicroscopy ͉ paracrystal ͉ von Willebrand factor ͉ tomography E ndothelial cells line the inner surfaces of blood vessels and play important roles in hemostasis, thrombosis, and inflammation. Some of these roles are achieved by secretion of the large, multimeric blood glycoprotein von Willebrand factor (VWF). VWF has multiple ligands and on acute release functions as an adhesive protein to bind platelets to sites of vascular injury. VWF circulating in the bloodstream also functions as a carrier for coagulation Factor VIII, increasing its lifetime. Defects in VWF and its storage are responsible for bleeding disorders including von Willebrand's disease (1).VWF is synthesized as a 350-kDa precursor (proVWF) that forms disulfide-linked dimers in the ER through its C-terminal cysteine knot domain. Proteolytic cleavage of proVWF in the Golgi gives rise to the N-terminal propolypeptide (a 100-kDa protein called proregion) and to mature VWF dimers that form large homo-oligomers through disulfide-links near each of its mature N-termini, a process catalyzed by proregion (2, 3). VWF and proregion remain non-covalently associated and are stored together in specialized secretory organelles called WeibelPalade bodies (WPBs), first identified by EM of fixed tissue sections as rod-shaped organelles containing fine tubules (4). Secretagogues stimulate WPB exocytosis, releasing VWF and other low molecular weight molecules such as cytokines and chemokines into the bloodstream (5), although mature VWF and its proregion account for greater than 95% of the protein in the granule (6). On release, VWF multimers are able to unfurl to strings up to 100 m long and associate with multiple ligands on platelet and endothelial cell surfaces at the site of vascular injury to help form a platelet plug. Mechanical shear exposes ligand binding sites on VWF as well as sites for cleavage by the protease ADAMTS13, which regulates the length of VWF multimers in the bloodstream (7).Like most other secretory granules, WPBs are thought to form at the trans-Golgi network (TGN) in a pH-dependent process. P-selectin is also recru...

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“…Because of the heterogeneity described for many bunyaviruses and to preserve structural details of RVFV particles, flash freezing of unfixed virus suspensions in a cryogen was performed (8,11). Cryo-ET allows for 3D reconstruction of asymmetric and/or structurally heterogeneous macromolecules and their complexes, as well as cellular organelles, at a moderate resolution (3 to 6 nm) (1,16,24,29,46). RVFV MP-12 particles in our tomograms were similar in diameter and overall organization, and their different orientations allowed particle averaging to improve resolution (40) and to restore missing information due to the limited tilt range of the sample in EM.…”

mentioning

confidence: 99%

Three-Dimensional Organization of Rift Valley Fever Virus Revealed by Cryoelectron Tomography

Freiberg¹,

Sherman

Morais

et al. 2008

J Virol

121

126

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Rift Valley fever virus (RVFV) is a member of the Bunyaviridae virus family (genus Phlebovirus)and is considered to be one of the most important pathogens in Africa, causing viral zoonoses in livestock and humans. Here, we report the characterization of the three-dimensional structural organization of RVFV vaccine strain MP-12 by cryoelectron tomography. Vitrified-hydrated virions were found to be spherical, with an average diameter of 100 nm. The virus glycoproteins formed cylindrical hollow spikes that clustered into distinct capsomeres. In contrast to previous assertions that RVFV is pleomorphic, the structure of RVFV MP-12 was found to be highly ordered. The three-dimensional map was resolved to a resolution of 6.1 nm, and capsomeres were observed to be arranged on the virus surface in an icosahedral lattice with clear T‫21؍‬ quasisymmetry. All icosahedral symmetry axes were visible in self-rotation functions calculated using the Fourier transform of the RVFV MP-12 tomogram. To the best of our knowledge, a triangulation number of 12 had previously been reported only for Uukuniemi virus, a bunyavirus also within the Phlebovirus genus. The results presented in this study demonstrate that RVFV MP-12 possesses T‫21؍‬ icosahedral symmetry and suggest that other members of the Phlebovirus genus, as well as of the Bunyaviridae family, may adopt icosahedral symmetry. Knowledge of the virus architecture may provide a structural template to develop vaccines and diagnostics, since no effective anti-RVFV treatments are available for human use.

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Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: implications of crowding

Cited by 102 publications

References 86 publications

Three-Dimensional Organization of Higher-Plant Chloroplast Thylakoid Membranes Revealed by Electron Tomography

Three-Dimensional Organization of Higher-Plant Chloroplast Thylakoid Membranes Revealed by Electron Tomography

Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

Three-Dimensional Organization of Rift Valley Fever Virus Revealed by Cryoelectron Tomography

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