Hi-D: Nanoscale mapping of nuclear dynamics in single living cells

Shaban, Haitham A.; Barth, Roman; Bystricky, Kerstin

doi:10.1101/405969

Cited by 9 publications

(27 citation statements)

References 70 publications

(82 reference statements)

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“…To quantify the experimentally observed chromatin dynamics at the nanoscale, down to the size of one pixel (13.5 nm), we used a dense reconstruction of flow fields, Optical Flow (OF; Figure 4A, Materials and Methods), which was previously used to analyze images taken on confocal ( 19 , 26 ), and Structured Illumination Microscopes ( 13 ). We examined the suitability of OF for super-resolution based on single molecule localization images using simulations.…”

Section: Resultsmentioning

confidence: 99%

“…Overall, the spatial cross-correlation between chromatin structure and dynamics indicates that the NND between blobs and their mobility stand in a strong mutual, negative, relationship. This relationship, however, concerns chromatin density variations at the nanoscale, but not global spatial density variations such as in eu- or heterochromatin ( 19 ). These results support a model in which regions with high local chromatin density, larger blobs are more prevalent and are mobile, while small blobs are sparsely distributed and less mobile (Figure 5B).…”

Section: Resultsmentioning

confidence: 99%

“…Given the persistence at which correlations of chromatin structure and, foremost, dynamics occur in a spatio-temporal manner, we speculate that the interplay of chromatin structure and dynamics could involve a functional relationship ( 34 ): transcriptional activity is closely linked to chromatin accessibility and the epigenomic state ( 35 ). Because chromatin structure and dynamics are related, dynamics could also correlate with transcriptional activity ( 19 , 36 ). However, it is currently unknown if the structure-dynamics relationship revealed here is strictly mutual or if it may be causal.…”

Section: Discussionmentioning

confidence: 99%

“…The integration of these flow fields from super-resolution images results in trajectories displaying the local motion of bulk chromatin with temporal and high spatial resolution. Further, the trajectories are classified into various diffusion models and parameters describing the underlying motion are computed ( 19 ). Here, we use the effective diffusion coefficient D (in units of m 2 / s α ), which reflects the magnitude of displacements between successive frames (the velocity of particles or monomers in the continuous limit) and the anomalous exponent α ( 19 ).…”

Section: Methodsmentioning

confidence: 99%

“…Consisting of chromatin in close physical and genomic proximity, blobs nevertheless adopt TAD-like interaction patterns when chromatin confirmations are averaged over time. Using a combination of Deep-PALM and high-resolution dense motion reconstruction ( 19 ), we simultaneously analyzed both structural and dynamic properties of chromatin. Our analysis emphasizes the presence of spatio-temporal cross-correlations between chromatin structure and dynamics, extending several micrometers in space and tens of seconds in time.…”

Section: Introductionmentioning

confidence: 99%

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Coupling chromatin structure and dynamics by live super-resolution imaging

Barth

Bystricky

Shaban

2019

Preprint

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19Chromatin conformation regulates gene expression and thus constant remodeling of chromatin 20 structure is essential to guarantee proper cell function. To gain insight into the spatio-temporal 21 organization of the genome, we employ high-density photo-activated localization microscopy and 22 deep learning to obtain temporally resolved super-resolution images of chromatin in vivo. In 23 combination with high-resolution dense motion reconstruction, we confirm the existence of 24 elongated ~ 45 to 90 nm wide chromatin 'blobs', which appear to be dynamically associating 25 chromatin fragments in close physical and genomic proximity and adopt TAD-like interactions in 26 the time-average limit. We found the chromatin structure exhibits a spatio-temporal correlation 27 extending ~ 4 μm in space and tens of seconds in time, while chromatin dynamics are correlated 28 over ~ 6 μm and outlast 40 s. Notably, chromatin structure and dynamics are closely interrelated, 29 which may constitute a mechanism to grant access to regions with high local chromatin 30 concentration. 32 The three-dimensional organization of the eukaryotic genome plays a central role in gene regulation 33 (1-3). Its spatial organization has been prominently characterized by molecular and cellular 34 approaches including high-throughput chromosome conformation capture (Hi-C) (4) and 35 fluorescent in situ hybridization (FISH) (5). Topologically associated domains (TADs), genomic 36 regions that display a high degree of interaction, were revealed and found to be a key architectural 37 feature (6). Direct 3D localization microscopy of the chromatin fiber at the nanoscale (7) confirmed 38 the presence of TADs in single cells but also, among others, revealed great structural variation of 39 chromatin architecture (8, 9). To comprehensively resolve the spatial heterogeneity of chromatin, 40 super-resolution microscopy must be employed. Previous work showed that nucleosomes are 41 distributed as segregated, nanometer-sized accumulations throughout the nucleus (10-13) and that 42 the epigenetic state of a locus has a large impact on its folding (14, 15). However, to resolve the 43 fine structure of chromatin, high labeling densities, long acquisition times and, often, cell fixation 44 are required. This precludes capturing dynamic processes of chromatin in single live cells, yet 45 chromatin moves at different spatial and temporal scales. 46The first efforts to relate chromatin organization and its dynamics were made using a combination 47 of Photo-activated Localization Microscopy (PALM) and tracking of single nucleosomes (16). It 48 could be shown that nucleosomes mostly move coherently with their underlying domains, in 49 accordance with conventional microscopy data (17); however, a quantitative link between the 50 observed dynamics and the surrounding chromatin structure could not yet be established in real-51 time. Although it is becoming increasingly clear that chromatin motion and long-range interactions 52 are key to genome o...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Coupling chromatin structure and dynamics by live super-resolution imaging

Barth

Bystricky

Shaban

2019

Preprint

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Heterogeneous fluid-like movements of chromatin and their implications to transcription

2020

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Eukaryotic chromatin is a complex of genome DNA and associated proteins, and its structure and dynamics play a crucial role in regulating DNA functions. Chromatin takes rather irregular structures in the nucleus and exhibits heterogeneous sub-diffusive movements as polymers fluctuating in a fluid state. Using genome-wide single-nucleosome tracking data, heterogeneity of movements was statistically analyzed, which categorized chromatin into two types: slow chromatin that moves under structurally constrained environments and fast chromatin that moves with less constraints. Interactions of chromatin to various protein factors determine the motional constraints. For example, loss of the cohesin complex that bundles the chromatin chains reduces the motional constraints and increases the population of fast chromatin. Another example is the transcriptional machinery. While it was previously thought that the transcriptional activity is associated with more open and dynamic chromatin structure, recent studies suggested a more nuanced role of transcription in chromatin dynamics: dynamic association/dissociation of active RNA polymerase II (RNAPII) and other transcription factors and Mediators (TF-Meds) transiently bridges transcriptionally active DNA regions, which forms a loose network of chromatin and constrains chromatin movement, enhancing the slow chromatin population. This new view on the dynamical effects of transcription urges a reflection on the traditional model of transcription factories and invites the more recent models of condensates/phase-separated liquid droplets of RNAPII, transcription factors, and Mediators. The combined procedure of genome-wide single-nucleosome tracking and its statistical analysis would unveil heterogeneity in the chromatin movement, which should provide a key to understanding the relations among chromatin dynamics, structure, and function.

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