Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek; Mai, Sabine; Cremer, Christoph

doi:10.1093/nar/gkw1301

Cited by 36 publications

(69 citation statements)

References 66 publications

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“…This showed the coexistence of dense chromatin fibers and nanoscale voids ( Figure 3C), consistent with recent super-resolution observations (Benke and Manley, 2012;Ricci et al, 2015;Szczurek et al, 2017). Remarkably, a strong correlation was found between the SMdM map of mEos3.2 and the SMLM image of DNA: the highest D values were consistently observed…”

Section: Diffusivity In the Mammalian Nucleus Is Spatially Heterogenesupporting

confidence: 91%

Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

Xiang¹,

Chen²,

Yan³

et al. 2019

Preprint

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Intracellular diffusion underlies vital cellular processes. However, it remains difficult to elucidate how an average-sized protein diffuses inside the cell with good spatial resolution and sensitivity. Here we introduce single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanometer-scale mapping of intracellular diffusivity through the local statistics of instantaneous displacements of freely diffusing single molecules. We thus show that diffusion in the mammalian cytoplasm and nucleus to both be spatially heterogeneous at the nanoscale, and that such variations in local diffusivity correlate well with the ultrastructure of the actin cytoskeleton and the chromosome, respectively. Moreover, we identify the net charge of the diffuser as a key determinant of diffusion rate: intriguingly, the possession of positive, but not negative, net charges significantly impedes diffusion, and the exact degree of slowdown is determined by the specific subcellular environments. We thus open a new door to understanding the physical rules that govern the intracellular interactions between biomolecules at the nanoscale.

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Section: Diffusivity In the Mammalian Nucleus Is Spatially Heterogenesupporting

confidence: 91%

Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

Xiang¹,

Chen²,

Yan³

et al. 2019

Preprint

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“…In order to test this hypothesis, it is necessary to explore the space-time compaction and accessibility of CDs. Super-resolved fluorescence microscopy, including single molecule localization microscopy (SMLM) and stochastic optical reconstruction microscopy (STORM), may become the methods of choice to measure absolute differences of DNA/chromatin compaction with spatial resolution at the nanometer scale [79, 84, 85], whereas chromatin accessibility can be probed indirectly with methods that allow to measure molecular diffusion rates [86–89].…”

Section: Discussionmentioning

confidence: 99%

Cohesin depleted cells rebuild functional nuclear compartments after endomitosis

Cremer

Brandstetter

Maiser

et al. 2019

Preprint

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43The human genome forms thousands of "contact domains", which are intervals of enhanced 44 contact frequency. Some, called "loop domains" are thought to form by cohesin-mediated loop 45 extrusion. Others, called "compartmental domains", form due to the segregation of active and 46 inactive chromatin into A and B compartments. Recently, Hi-C studies revealed that the 47 depletion of cohesin leads to the disappearance of all loop domains within a few hours, but 48 strengthens compartment structure. Here, we combine live cell microscopy, super-resolution 49 microscopy, Hi-C, and studies of replication timing to examine the longer-term consequences 50 of cohesin degradation in HCT-116 human colorectal carcinoma cells, tracking cells for up to 51 30 hours. Surprisingly, cohesin depleted cells proceed through an aberrant mitosis, yielding a 52 single postmitotic cell with a multilobulated nucleus. Hi-C reveals the continued disappearance 53 of loop domains, whereas A and B compartments are maintained. In line with Hi-C, microscopic 54 observations demonstrate the reconstitution of chromosome territories and chromatin 55 domains. An interchromatin channel system (IC) expands between chromatin domain clusters 56 and carries splicing speckles. The IC is lined by active chromatin enriched for RNA Pol II and 57 depleted in H3K27me3. Moreover, the cells exhibit typical early-, mid-, and late-DNA 58 replication timing patterns. Our observations indicate that the functional nuclear 59 compartmentalization can be maintained in cohesin depleted pre-and postmitotic cells.60 However, we find that replication foci -sites of active DNA synthesis -become physically 61 larger consistent with a model where cohesin dependent loop extrusion tends to compact 62 intervals of replicating chromatin, whereas their genomic boundaries are associated with 63 compartmentalization, and do not change.64 65 3 Abbreviations 66 3D FISH = 3D fluorescence in situ hybridization 67 3D SIM = 3D structured illumination microscopy 68 AID = auxin inducible degron 69 ANC / INC = active / inactive nuclear compartment 70 CT = chromosome territory 71 CD(C) = chromatin domain (cluster) 72 CTCF = CCCTC binding factor 73 DAPI = 4',6-diamidino-2-phenylindole 74 EdU = 5-Ethynyl-2'-deoxyuridine 75 Hi-C = chromosome conformation capturing combined with deep sequencing 76 IC = interchromatin compartment 77 MLN = multilobulated nucleus 78 NC = nucleosome cluster 79 PBS = phosphate buffered saline 80 PBST = phosphate buffered saline with 0.02% Tween 81 PR = perichromatin region 82 RD = replication domain 83 RL = replication labeling 84 TAD = topologically associating domain 85 86 109 strengthened, leading to the presence of compartment domains and even compartment loops110 (but no loop domains) in the treated cells [18]. Other studies, using different cell types and 111 approaches for cohesin elimination yielded similar results [19-21], (reviewed in [22]). 112Here, we study the longer-term consequences of cohesin depletion and its effects on 113 the higher order nuclear a...

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“…The possibility to record the precise position of very large numbers of independent blinking events in space from fluorophores conjugated to a cellular target of interest has opened the way to super‐resolved localization microscopy with PALM and STORM . Further improvements paved the way to use fluorophores conventionally used in microscopic studies of the cell nucleus . The highest light optical resolution in the analysis of nuclear genome nanostructures has been obtained so far with SMLM and is currently in the order of 20–50 nm and even better in special circumstances .…”

Section: Support Of the Anc‐inc Model With Super‐resolved Fluorescencmentioning

confidence: 99%

The Interchromatin Compartment Participates in the Structural and Functional Organization of the Cell Nucleus

et al. 2020

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This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis). IC channels are lined by less-compacted euchromatin, called the perichromatin region (PR). The PR and IC together form the active nuclear compartment (ANC). The ANC is co-aligned with the inactive nuclear compartment (INC), comprising more compacted heterochromatin. It is postulated that the INC is accessible for individual transcription factors, but inaccessible for larger macromolecular aggregates (limited accessibility hypothesis). This functional nuclear organization depends on still unexplored movements of genes and regulatory sequences between the two compartments.

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Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

Cited by 36 publications

References 66 publications

Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

Cohesin depleted cells rebuild functional nuclear compartments after endomitosis

The Interchromatin Compartment Participates in the Structural and Functional Organization of the Cell Nucleus

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