The in situ structures of mono-, di-, and trinucleosomes in human heterochromatin

Cai, Shujun; Böck, Desirée; Pilhofer, Martin; Gan, Lu

doi:10.1091/mbc.e18-05-0331

Cited by 83 publications

(108 citation statements)

References 66 publications

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“…In G0 cells, we did not observe chromatin packed into either ultra-compact structures or ordered fibers. This observation is consistent with all previous in situ cryo-EM and cryo-ET studies of eukaryotic cell nuclei 11,15,[47][48][49][50][51][52] . Thus far, the most compact forms of natural chromatin studied by electron microscopy are the samples that are released from lysed nuclei 11,27,53 , reconstituted arrays in the presence of divalent cations 54 , and 30-nm fibers in isolated chicken erythrocyte nuclei 55 .…”

Section: Discussionsupporting

confidence: 93%

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Macromolecular and cytological changes in fission yeast G0 nuclei

Cai

Nie

et al. 2020

Preprint

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Cells change their cytology in response to environmental cues and stress. Notably, large changes in nuclear architecture are accompanied by transcriptional reprogramming. When starved of nitrogen, Schizosaccharomyces pombe cells become rounder and they enter a quiescent "G0" state. These cells have smaller nuclei and undergo near-global transcriptional repression. Here we use electron cryotomography (cryo-ET) and cell-biology approaches to investigate the structural and biochemical changes of G0 S. pombe nuclei. We find that G0 cells have a denser nucleoplasm and fewer chromatin-associated multi-megadalton globular complexes (megacomplexes) than proliferating cells. These structural changes are correlated with mild histone deacetylation. Induced histone hyperacetylation in G0 results in cells that have larger nuclei and less condensed chromatin. However, these histone-hyperacetylated G0 cells still have repressed transcription, few megacomplexes, and a dense nucleoplasm. Like in budding yeast, S. pombe G0 nuclear phenotypes are controlled by multiple biochemical factors.

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Section: Discussionsupporting

confidence: 93%

“…Furthermore, G0 cells have such a dense nucleoplasm that their chromatin packing at the macromolecular level remains mysterious. Newer cryo-ET hardware, sample-preparation, and image-processing technologies may help reveal differences at the level of oligonucleosome clustering and folding 11,51…”

Section: Discussionmentioning

confidence: 99%

Macromolecular and cytological changes in fission yeast G0 nuclei

Cai

Nie

et al. 2020

Preprint

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“…These cell nuclei contained 10-nm-diameter nucleosome-like particles and ≥ 20-nmwide multi-megadalton-sized complexes (megacomplexes, such as pre-ribosomes) like those seen in cryo-ET studies of yeast, insect, and HeLa nuclei (Chen et al, 2016;Cai et al, 2018a;Cai et al, 2018b;Eltsov et al, 2018;Ng et al, 2019). After 2 hours in SM, a few cells had small bundles of filaments that present a trefoil motif when view along their long axis ( Fig.…”

Section: Visualization Of Scs In Situ By Cryo-etmentioning

confidence: 96%

“…To better characterize the SC's crystalline properties, we attempted to make 3-D models of bundles of triple-helical filaments. Because the remapping of subtomogram averages can reveal higher-order motifs in chromatin structure (Cai et al, 2018a;Cai et al, 2018c), we remapped the subtomogram averages of the SC triple helix segments back into the tomogram. This approach failed because the filaments appeared discontinuous; template matching and classification are imperfect, resulting in the removal of many SC triple helix segments.…”

Section: Scs Pack Into Crystalline Bundlesmentioning

confidence: 99%

“…Preliminary 3-D refinement on EW104 SC triple helix segments produced a Fourier shell correlation curve that did not drop to zero in the highest-resolution range. This artifact was caused by new "duplicate" particles that were generated by the 3-D classification translational search (Cai et al, 2018a). These new duplicates were removed by re-running template matching with PEET, using only the coordinates of the 3-D classified particles.…”

Section: Subtomogram Classificationmentioning

confidence: 99%

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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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The promise and the challenges of cryo‐electron tomography

2020

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Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This ‘divide and conquer’ approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo‐electron tomography (Cryo‐ET) combines the power of 3D molecular‐level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or ‘molecular sociology’ of cells and to discover the unexpected. Here, we review state‐of‐the‐art Cryo‐ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo‐ET.

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The in situ structures of mono-, di-, and trinucleosomes in human heterochromatin

Cited by 83 publications

References 66 publications

Macromolecular and cytological changes in fission yeast G0 nuclei

Macromolecular and cytological changes in fission yeast G0 nuclei

Meiotic budding yeast assemble bundled triple helices but not ladders

The promise and the challenges of cryo‐electron tomography

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