Intranucleus Single-Molecule Imaging in Living Cells

Shao, Shuang; Bai, Xue; Sun, Yujie

doi:10.1016/j.bpj.2018.05.017

Cited by 24 publications

(20 citation statements)

References 73 publications

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“…Indeed, it is currently the preferred choice for live-cell single molecule imaging. Combined with the development of Cas9-mediated genome-editing (Ran et al, 2013), endogenous Halotagging of proteins has thus become the gold standard (Chong et al, 2018;Hansen et al, 2017;Komatsubara et al, 2019;Rhodes et al, 2017aRhodes et al, , 2017bStevens et al, 2017;Teves et al, 2016aTeves et al, , 2018Youmans et al, 2018), because it avoids the now well-established limitations and potential artifacts associated with protein overexpression (Hansen et al, 2017;Shao et al, 2018;Teves et al, 2016a). Now that we have determined the absolute abundance of CTCF in U2OS cells and cross-validated it using FCS-calibrated confocal imaging ( Figure 1A-D, F),…”

Section: A Simple General Methods For Determining the Abundance Of Halmentioning

confidence: 91%

Determining cellular CTCF and cohesin abundances to constrain 3D genome models

Cattoglio

Pustova

Walther

et al. 2018

Preprint

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Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes. RESULTS Determining the number of CTCF and cohesin proteins per cell

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Section: A Simple General Methods For Determining the Abundance Of Halmentioning

confidence: 91%

Determining cellular CTCF and cohesin abundances to constrain 3D genome models

Cattoglio

Pustova

Walther

et al. 2018

Preprint

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“…What is least understood at present is the dynamic nature of nuclear structure and function, which implies a preferred location of chromatin loops and chromatin domains or even entire CTs for the proper execution of functional tasks, including a variable course of channels depending on functional needs. To solve these problems, it will be mandatory to observe movements of DNA sequences and entire CDs between the ANC and INC directly in live cell studies …”

Section: Concluding Remarks and Future Perspectivesmentioning

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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“…Chromatin dynamics is intimately related to the hierarchical chromatin structure and nuclear functions (Shao et al, 2018). Previous studies have shown that the diffusion dynamics of genomic loci are not mere Brownian motion driven by thermal fluctuation but are often convoluted with active processes such as transcription and DNA repair (Chuang et al, 2006;Dimitrova et al, 2008;Cho et al, 2014;Ochiai et al, 2015;Gu et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells

Chao

Shao

et al. 2020

Protein Cell

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The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.

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Intranucleus Single-Molecule Imaging in Living Cells

Cited by 24 publications

References 73 publications

Determining cellular CTCF and cohesin abundances to constrain 3D genome models

Determining cellular CTCF and cohesin abundances to constrain 3D genome models

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

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells

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