Electron tomography of cells

Gan, Lu; Jensen, Grant J.

doi:10.1017/s0033583511000102

Cited by 154 publications

(136 citation statements)

References 184 publications

(220 reference statements)

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“…For cryosectioning, synchronized and LatA-treated S. pombe cdc25-22 rlc1-3GFP cells (OD 600 = 0.5) were harvested (7,197 × g, 10 min), and the pellet was mixed with 40% dextran (wt/vol) in YES media. The samples were transferred to brass planchettes and rapidly frozen in an HPM010 HPF machine (Bal-Tec Leica).…”

Section: Experimental Methodsmentioning

confidence: 99%

“…Here we sought to visualize the precise arrangement of F-actin within the AMR and its interface with the membrane by imaging intact cells in a cryopreserved state using electron cryotomography (ECT) (7). Because whole S. pombe cells are too thick for ECT, which is limited to specimens thinner than a few hundred nanometers, we overcame this obstacle by first rapid freezing dividing cells and then either cryosectioning them or using the recently developed method cryofocused ion beam (cryo-FIB) milling to produce thin sections or lamellae suitable for ECT analysis.…”

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confidence: 99%

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Structure of the fission yeast actomyosin ring during constriction

Swulius

Nguyen

Ladinsky

et al. 2018

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

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Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of ∼150 nm in diameter that "saddles" the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane.C ytokinesis, the final step of cell division in eukaryotic cells, is typically driven by a contractile actomyosin ring (AMR) primarily composed of actin (1) and myosin (2). Our understanding of the molecular mechanisms of cytokinesis is most detailed in the rodshaped unicellular eukaryote Schizosaccharomyces pombe (otherwise known as fission yeast), which shares a remarkably conserved set of cytokinesis genes with metazoans (3). In S. pombe, the AMR undergoes multiple phases known as assembly, maturation, constriction, and disassembly (4), with open questions in each of these four stages. Due to a lack of information about the precise arrangement of filamentous actin (F-actin) within the force-generating network of the AMR, we chose to focus on imaging the AMR during constriction.In S. pombe, glancing sections through plastic-embedded, dividing cells gave the first glimpse of actin filaments running parallel to the division plane at the front of the septum (5). Unfortunately, the study yielded limited examples and lacked 3D information for a full analysis. In an ambitious pioneering effort, Kamasaki et al. (6) produced 3D reconstructions of entire S. pombe AMRs by imaging serial sections through permeabilized cells decorated with myosin S1 fragments. The amount of F-actin and the size of the rings appeared significantly altered by the procedure used for preserving them (details in Discussion), but the continuous bundles that were reconstructed were composed of mixed polarity filaments running circumferentially around the cell.Here we sought to visualize the precise arrangement of F-actin within the AMR and its interface with the membrane by imaging intact cells in a cryopreserved state using electron cryotomography (ECT) (7). Because whole S. pombe cells are too thick for ECT, which is limited to specimens thinner than a few hundred nanometers, we overcame this obstacle by first rapid freezing dividing cells and then either cryosectioning them or using the recently developed method cryofocused ion beam (cryo-FIB) milling to ...

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Section: Experimental Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Structure of the fission yeast actomyosin ring during constriction

Swulius

Nguyen

Ladinsky

et al. 2018

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

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“…To generate SNAP-DRC3 cells, about 10 6 of the drc3 cells were transformed with linearized 1.4 g of pSNAP-DRC3 containing the marker gene aph 7Љ gene (28) as described previously. 4 Colonies on Tris acetate/phosphate plates containing hygromycin B were randomly picked and screened using immunofluorescence microscopy with anti-DRC3. Expression of SNAP-DRC3 in the selected strain was confirmed by Western blot with anti-DRC3 and anti-SNAP.…”

Section: Methodsmentioning

confidence: 99%

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Song

Awata

Tritschler

et al. 2015

Journal of Biological Chemistry

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Background: Techniques to localize proteins in situ at high resolution are important but limited. Results: Combining SNAP tag technology with cryo-electron tomography, we precisely localized proteins within the N-DRC that are important for ciliary motility. Conclusion: The developed method was applied to localize proteins with ϳ3 nm resolution without interfering with the complex function. Significance: The method is a powerful tool for studies of proteins in situ.

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“…These include the development of phase plates (Danev et al 2014;Glaeser, 2013;Murata et al 2010;Nagayama, 2014), Cc correctors (Kabius et al 2009) and cooling to liquid helium temperature (Fujiyoshi, 1998) provided that the problems of beam-induced movement can be minimised. There are also valuable publications that focus on specific issues, including map validation (Rosenthal & Rubinstein, 2015), specimen preparation Glaeser, 2016), electron cryotomography (Gan & Jensen, 2012;Medalia et al 2002) and finally three volumes of Methods in Enzymology covering a wide range of topics in more detail (Volumes 481, 482, 483, published in 2010).…”

Section: Introductionmentioning

confidence: 99%

Single particle electron cryomicroscopy: trends, issues and future perspective

Vinothkumar

Henderson

2016

Quart. Rev. Biophys.

175

115

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Abstract. There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2-to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

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Electron tomography of cells

Cited by 154 publications

References 184 publications

Structure of the fission yeast actomyosin ring during constriction

Structure of the fission yeast actomyosin ring during constriction

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Single particle electron cryomicroscopy: trends, issues and future perspective

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