Investigating eukaryotic cells with cryo-ET

Ng, Cai Tong; Gan, Lu

doi:10.1091/mbc.e18-05-0329

Cited by 25 publications

(16 citation statements)

References 156 publications

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“…Meiotic nuclei have been studied for decades by traditional EM [11, 12], but the 3-D macromolecular organization of meiosis-specific complexes remains unknown in situ because traditional EM has fixation, dehydration, and staining artifacts. Cryo-ET can reveal 3-D nanoscale structural details of cellular structures in a life-like state because the samples are kept unfixed, unstained, and frozen-hydrated during all stages of sample preparation and imaging [15]. We use cryo-ET to visualize the organization of meiotic yeast nuclei throughout meiosis I, focusing on pachytene.…”

Section: Introductionmentioning

confidence: 99%

Meiotic budding yeast assemble bundled triple helices but not ladders

Olivia

Chong

et al. 2019

Preprint

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The synaptonemal complex (SC) is the large, conserved, proteinaceous scaffold that assembles between and holds together homologous chromosomes in meiotic prophase. Knowledge of the native structure of this complex is needed to evaluate how the SC carries out its functions. Traditional electron microscopy and superresolution light microscopy have revealed that in many organisms, the SC has a ladder-like structure: two rail-like lateral elements are bridged by a set of rung-like transverse filaments. The transverse filaments are connected along their centers by a central element. To determine the 3-D architecture of the SC in situ, we studied frozen-hydrated meiotic yeast cell cryosections by Volta phase-contrast electron cryotomography and subtomogram analysis. We find the SC is built from triplehelical filaments that pack into dense polycrystalline bundles. These structures are also abundant in the polycomplexes of pachytene-arrested cells. Dissolution by 1,6hexanediol treatment suggests that these triple-helical filaments belong to the central region of the SCs. Subtomogram averaging revealed that the SC's triple-helical filaments are up to 12-nm thick and have a 5-nm rise and 130-nm pitch. Single triplehelices and polymers thinner than the triple helix, such as single or double strands, were not detected, consistent with the strong self-oligomerization properties of SC proteins. The dense packing of SC subunits supports the notion that the SC's mechanical properties help coordinate the rapid end-to-end communication across synapsed chromosomes.

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Section: Introductionmentioning

confidence: 99%

Meiotic budding yeast assemble bundled triple helices but not ladders

Olivia

Chong

et al. 2019

Preprint

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“…One advantage of cryoET is the ability to directly visualize the cellular landscape and determine the structural dynamics and spatial organization of biomolecules and macromolecular machines from various organisms within their native environment [ 20 , 21 , 22 ]. Advances in cryoET technology and subtomogram averaging have enabled structural exploration and detailed characterization of cellular processes in yeast cells [ 23 , 24 , 25 ]. Here, we apply cryoET and subtomogram analysis to determine the structure and spatial distribution of the putative GS complex in C. glabrata .…”

Section: Introductionmentioning

confidence: 99%

Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins

Jiménez‐Ortigosa

Jiang

Chen

et al. 2021

JoF

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Echinocandin drugs have become a front-line therapy against Candida spp. infections due to the increased incidence of infections by species with elevated azole resistance, such as Candida glabrata. Echinocandins target the fungal-specific enzyme ß-(1,3)-glucan synthase (GS), which is located in the plasma membrane and catalyzes the biosynthesis of ß-(1,3)-glucan, the major component of the fungal cell wall. However, resistance to echinocandin drugs, which results from hotspot mutations in the catalytic subunits of GS, is an emerging problem. Little structural information on GS is currently available because, thus far, the GS enzyme complex has resisted homogenous purification, limiting our understanding of GS as a major biosynthetic apparatus for cell wall assembly and an important therapeutic drug target. Here, by applying cryo-electron tomography (cryo-ET) and subtomogram analysis, we provide a preliminary structure of the putative C. glabrata GS complex as clusters of hexamers, each subunit with two notable cytosolic domains, the N-terminal and central catalytic domains. This study lays the foundation for structural and functional studies of this elusive protein complex, which will provide insight into fungal cell wall synthesis and the development of more efficacious antifungal therapeutics.

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“…The absence of canonical nucleosome class averages could have either a technical or a biological cause. Because complexes within cryosections are affected by compression forces [4,17,18], it is possible that canonical nucleosomes were distorted in these samples. While GFP does not perturb nucleosome structure in vitro, we cannot dismiss the possibility that it perturbs nucleosome in situ.…”

Section: Cryo-fib Milled Yeast Also Lack Canonical Nucleosome Classesmentioning

confidence: 99%

Heterogeneous non-canonical nucleosomes predominate in yeast cellsin situ

Cai

Noble

et al. 2021

Preprint

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Nuclear processes depend heavily on the organization of chromatin, whose subunits are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes in situ resemble the canonical structure determined in vitro 24 years ago. The structure of nucleosomes in situ is otherwise poorly understood. Here we use cryo-ET and 3-D classification analysis to study the structure of yeast nucleosomes both in vitro and in situ. We show that the class averages of GFP-tagged yeast nucleosomes in vitro resemble canonical nucleosomes, with additional GFP densities. In contrast, none of the class averages of nucleosome-like particles in situ (inside cells) resemble canonical nucleosomes. The heterogeneous nature of the in situ class averages suggests that the intranuclear environment favors multiple conformations. Using the structural observations here and the results of previous genomics and biochemical studies, we propose a model in which the average yeast nucleosome's DNA is partially detached in situ.

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Investigating eukaryotic cells with cryo-ET

Cited by 25 publications

References 156 publications

Meiotic budding yeast assemble bundled triple helices but not ladders

Meiotic budding yeast assemble bundled triple helices but not ladders

Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins

Heterogeneous non-canonical nucleosomes predominate in yeast cellsin situ

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