The in situ three-dimensional organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level.
Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription. Here, we use cryo-electron tomography (cryo-ET) to determine the 3D arrangement of nuclear macromolecular complexes, including nucleosomes, in frozenhydrated Schizosaccharomyces pombe cells. Using 3D classification analysis, we did not find evidence that nucleosomes resembling the crystal structure are abundant. This observation and those from other groups support the notion that a subset of fission yeast nucleosomes may be partially unwrapped in vivo. In both interphase and mitotic cells, there is also no evidence of monolithic structures the size of Hi-C domains. The chromatin is mingled with two features: pockets, which are positions free of macromolecular complexes; and "megacomplexes," which are multimegadalton globular complexes like preribosomes. Mitotic chromatin is more crowded than interphase chromatin in subtle ways. Nearestneighbor distance analyses show that mitotic chromatin is more compacted at the oligonucleosome than the dinucleosome level. Like interphase, mitotic chromosomes contain megacomplexes and pockets. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: a subset of genes is up-regulated. chromatin | condensation | cryo-ET | fission yeast C hromatin structure influences key nuclear activities such as transcription, DNA repair, and replication (1). The fundamental unit of chromatin is the nucleosome, which consists of ∼147 bp of DNA wrapped around a histone octamer (2). In mammalian cells, 2-35 nucleosomes pack into irregular "clutches" (3). More than 500 sequential nucleosomes (calculated from the nucleosome repeat length) are thought to interact as topologically associating domains (4). Likewise, in the fission yeast Schizosaccharomyces pombe, some 300-7,000 nucleosomes are thought to associate as compact globular chromatin bodies called domains (5-7). Furthermore, superresolution imaging of S. pombe revealed that the chromatin is not uniformly distributed in vivo (8). These studies suggest that chromatin higher-order structure arises from physical interactions within large groups of nucleosomes. In mitotic cells, chromosomes condense into discrete structures that can be resolved in a light microscope. The factors involved in condensation have been well characterized (9), and a number of models have been considered for the largescale organization of chromatin domains (10). However, the molecular details of chromatin reorganization are still unknown. Knowledge of how chromosomes condense in 3D at the molecular level is needed to explain the nearly global transcriptional repression that happens to the mitotic cells of most eukaryotes (11). Such a model could also explain how a subset of fission yeast genes escape this mitotic repression and get up-regulated (12)(13)(14). Some insights on in vivo chromatin organization were made possible by new methods, including chromatin-conformation capture (Hi-...
VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson’s disease–linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an ∼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER–endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo–electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.
The 30-nm fiber is commonly formed by oligonucleosome arrays in vitro but rarely found inside cells. To determine how chromatin higher-order structure is controlled, we used electron cryotomography (cryo-ET) to study the undigested natural chromatin released from two single-celled organisms in which 30-nm fibers have not been observed in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is condensed into a large mass in vitro. Rare irregular 30-nm fibers, some of which include face-to-face nucleosome interactions, do form at the periphery of this mass. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains condensed, with very few open motifs. Yeast chromatin packing is largely unchanged in the absence of linker histone and mildly decondensed when histones are more acetylated. Natural chromatin is therefore generally nonpermissive of regular motifs, even at the level of oligonucleosomes.
VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous crystallographic and cryo-EM studies of VPS13 fragments suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from in situ studies, however, is still missing. Here we have capitalized on AlphaFold predictions to complement the structural information already acquired and to generate a model of full-length human VPS13C, the Parkinson's disease-linked VPS13 paralog localized at contacts between the ER and endo/lysosomes. Such model predicts a ~30-nm rod-shaped structure with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER-endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused-ion-beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) in HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods we identified rod-like densities that span the space separating the two adjacent membranes and that fit the predicted sizes of full lengths VPS13C or of its shorter truncated mutant. Only a subset of them appeared to directly contact the ER bilayer in spite of being anchored to the ER membrane by VAP. Collectively these findings provide strong evidence for a bridge-model of lipid transport, and also suggest that direct contact of VPS13C with the ER bilayer to mediate lipid transport may be regulated.
13 14 Background: Cells are powered by a large set of macromolecular complexes, which 15 work together in a crowded environment. The in situ mechanisms of these complexes 16 are unclear because their 3-D distribution, organization, and interactions are largely 17 unknown. Electron cryotomography (cryo-ET) is a key tool to address these knowledge 18 gaps because it produces cryotomograms --3-D images that reveal biological structure 19 at approximately 4-nm resolution. Cryo-ET does not involve any fixation, dehydration, 20 staining, or plastic embedment, meaning that cellular features are visualized in a life-21 like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we have 22 Conclusions: Cellular cryo-ET data can be mined to obtain new cell-biological, 36 structural, and 3-D statistical insights in situ. Because these data capture cells in a life-37 like state, they contain some structures that are either absent or not visible in traditional 38 EM data. Template matching and subtomogram averaging of known macromolecular 39 complexes can reveal their 3-D distributions and low-resolution structures. Furthermore, 40 these data can serve as testbeds for high-throughput image-analysis pipelines, as 41 training sets for feature-recognition software, for feasibility analysis when planning new 42 structural cell-biology projects, and as practice data for students who are learning 43 cellular cryo-ET. 44 45
16The 30-nm fiber is commonly found in oligonucleosome arrays in vitro but rarely 17 found in chromatin within nuclei. To determine how chromatin high-order structure is 18 controlled, we used cryo-ET to study the undigested natural chromatin released from 19 cells that do not have evidence of 30-nm fibers in vivo: picoplankton and yeast. In the 20 presence of divalent cations, most of the chromatin from both organisms is compacted 21 into a large mass. Rare irregular 30-nm fibers do form at the periphery of this mass, 22 some of which include face-to-face interactions. In the absence of divalent cations, 23 picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin 24 mostly remains compact with looser nucleosome packing, even after treatment with 25 histone-deacetylase inhibitor. The 3-D configuration of natural chromatin is therefore 26 sensitive to the local environment, but generally nonpermissive of regular motifs, even 27 at the level of oligonucleosomes.28 70 erythrocytes (Scheffer et al., 2011). These and other studies challenge whether 30-nm 71 fibers is the best model of chromatin within somatic cells (Hansen, 2012;; Nishino et al., 72 2012;; van Holde and Zlatanova, 1995).73 5 The different conclusions between in vitro and in vivo chromatin studies raise the 74 question: can the chromatin of cells that do not show evidence of 30-nm fibers in vivo 75 be made to adopt a 30-nm-fiber structure in vitro? To address this question, we used 76 cryo-ET to study the 3-D organization of undigested natural chromatin from 77 picoplankton and yeast. In the presence of even traces of divalent cations, most of the 78 chromatin in both organisms compacts into large masses. Some of the chromatin does 79 form 30-nm fibers, predominantly of the irregular variety. In the absence of divalent 80 cations, picoplankton chromatin decondenses into open zigzags, but yeast chromatin 81 remains either in a mass or folded as irregular 30-nm fibers. To better understand how 82these nucleosomes interact, we classified the chromatin in both 2-D and 3-D, and found 83 that there is no dominant higher-order packing motif, but the orientation of the DNA at 84 the core-particle entry/exit points is not too variable. Therefore, the higher-order 85 structure of natural chromatin is sensitive to environmental conditions, but this 86 sensitivity varies by species. 87 6 RESULTS 88 89Release of natural picoplankton chromatin 90 To study the higher-order structure of natural picoplankton chromatin in vitro, we 91 lysed cells in hypotonic buffer on ice either with or without divalent cations (Fig. 1B). 92This treatment released all cellular contents, including the chromatin, and is expected to 93 produce thinner plunge-frozen samples that generate higher-contrast cryotomograms. 94Compared to intact cells (Fig. 1C), the lysed cells' contents spread over a much larger 95 area and indeed allowed the ice to be thinner, resulting in higher-contrast tomograms 96 (Fig. 1D). The majority of the densities came from remnants of...
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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